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

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A MORPHOLOGICAL AMD CYTOCHEMICAL ANALYSIS OF BUD INITIATION AND DEVELOPMENT IN THE FILAMENTOUS BROWN ALGA SPHACELARIA FURCIGERA by ALAN ROBERT BURNS B . S c , The U n i v e r s i t y of B r i t i s h Co lumbia , 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Depa rtrnent of Botany We accept t h i s t h e s i s as conforming to the r equ i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA October 1981 (c)Alan Burns In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 6 A b s t r a c t A s t u d y o n t h e p r o c e s s o f b u d i n i t i a t i o n a n d d e v e l o p m e n t i n t h e f i l a m e n t o u s b r o w n a l g a S p h a c e l a r i a  f u r c i g e r a ( K u t z ) was c a r r i e d o u t u s i n g t e c h n i q u e s o f l i g h t a n d e l e c t r o n m i c r o s c o p y , a s w e l l a s c y t o c h e m i s t r y . A l o c a l i z e d t h i c k e n i n g a n d s u b s e q u e n t p r o t r u s i o n o f t h e o u t e r c e l l w a l l o f t h e a x i a l m o t h e r c e l l c h a r a c t e r i z e s t h e e a r l i e s t d e t e c t a b l e s t a g e o f b u d i n i t i a t i o n . T h i s p r o t r u s i o n f o r m s t h r o u g h t h e c o m b i n e d d e p o s i t i o n o f n e w l y s y n t h e s i z e d m i c r o f i b r i l s t o g e t h e r w i t h t h e p a r t i a l l y s i s / l o o s e n i n g o f t h e e x i s t i n g c e l l w a l l . E v i d e n c e i s p r e s e n t e d t h a t o f t h e t h r e e e n z y m e a c t i v i t i e s l o c a l i z e d , p e r o x i d a s e , a d e n o s i n e t r i p h o s p h a t a s e a n d a c i d p h o s p h a t a s e a c t i v i t y , o n l y p e r o x i d a s e a c t i v i t y i s r e l a t e d t o t h e l y s i s / l o o s e n i n g o f t h e c e l l w a l l d u r i n g t h e e a r l y d e v e l o p m e n t o f t h e b u d i n i t i a l . C o n t i n u e d i n c o r p o r a t i o n o f new c e l l w a l l m a t e r i a l i n t o t h e o u t e r c e l l w a l l m a i n t a i n s i t s s t r u c t u r a l i n t e g r i t y . H o w e v e r , t h e r e i s a c h a n g e i n t h e l a y e r e d a p p e a r a n c e o f t h e c e l l w a l l m i c r o f i b r i l s . T h e c e l l w a l l o f t h e b u d i n i t i a l i s c h a r a c t e r i z e d b y two c e l l w a l l l a y e r s i n s t e a d o f t h e f o u r f o u n d i n t h e p r e - e x i s t i n g c e l l w a l 1 o f t h e a x i a l m o t h e r c e l l . T h i s o r i g i n a l c e l l w a l l i s c o m p o s e d o f a n o u t e r - m o s t f u c a n l a y e r , o v e r l y i n g a n a l g i n a t e l a y e r , w h i c h i n t u r n o v e r l i e s a n o t h e r f u c a n l a y e r a n d f i n a l l y t e r m i n a t e s i n a n i n n e r - m o s t a l g i n a t e l a y e r . I n c o n t r a s t t h e b u d i n i t i a l ' s c e l l w a l l h a s o n l y a t h i n o u t e r f u c a n l a y e r a n d a t h i c k i n n e r a l g i n a t e l a y e r . C o n c o m m i t a n t i i w i t h the f o r m a t i o n of the c e l l w a l l p r o t r u s i o n , t h e r e i s a l o s s of c y t o p l a s m i c v a c u o l e s , an i n c r e a s e i n c y t o p l a s m i c mass and d e n s i t y and an i n c r e a s e i n the number of o r g a n e l l e s . The endomembrane system ( 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 s and the d e r i v a t i v e v e s i c l e s ) a l s o p r o l i f e r a t e s . O r g a n e l l e m i g r a t i o n i n t o the bud p r o t r u s i o n keeps pace w i t h bud e x p a n s i o n . The movement of the n u c l e u s , however, l a g s b e h i n d and i t m i g r a t e s towards the bud p r o t r u s i o n o n l y a f t e r a " v a c u o l e f r e e " c y t o p l a s m becomes e s t a b l i s h e d . As the n u c l e u s a p p r o a c h e s a m e d i a l p o s i t i o n between the base of the ax.ial mother c e l l and the t i p of the bud p r o t r u s i o n , c y t o p l a s m i c v a c u o l e s r e - a p p e a r . They a r e c o n f i n e d , however, to the b a s a l r e g i o n of the a x i a l mother c e l l . A f t e r k a r y o k i n e s i s , a c r o s s w a l l i s d e p o s i t e d between the two d a u g h t e r n u c l e i r e s u l t i n g i n the f o r m a t i o n of a bud c e l l and a s i s t e r a x i a l c e l l . The s i s t e r a x i a l c e l l i s h i g h l y v a c u o l a t e d and s t r u c t u r a l l y r e s e m b l e s the a d j a c e n t q u i e s c e n t a x i a l c e l l s . The bud c e l l i s dense and non-v a c u o l a t e d , a f e a t u r e c h a r a c t e r i s t i c of a m e f i s t e m a t i c c e l l . i i i TABLE OF CONTENTS A b s t r a c t i i TABLE OF CONTENTS i v LIS T OF FIGURES v i LIS T OF TABLES x i ACKNOWLEDGEMENTS x i i I n t r o d u c t i o n 1 M a t e r i a l s and Methods 4 T a b l e 1 9 R e s u l t s 10 P a r t 1 M o r p h o l o g y . 10 L i g h t m i c r o s c o p y 10 E l e c t r o n m i c r o s c o p y 11 Q u i e s c e n t a x i a l c e l l 11 Stage I 12 Stage I I 13 Stage I I I 14 Stage IV 15 Stage V 17 Stage VI 18 Stage V I I 19 P a r t 2 C y t o c h e m i s t r y 21 L i g h t m i c r o s c o p e C y t o c h e m i s t r y 21 P o l a r i z e d l i g h t m i c r o s c o p y 23 C e l l w a l l e x t r a c t i o n s & C a l c o f l u o r f l u o r e s c e n c e ... 23 U l t r a s t r u c t u r a l l o c a l i z a t i o n of enzyme a c t i v i t y ... 24 P e r o x i d a s e 24 i v A d e n o s i n e t r i - p h o s p h a t a s e ( A T P ase) 25 A c i d p h o s p h a t a s e ( A c P a s e ) 25 D i s c u s s i o n 27 P a r t 1 M o r p h o l o g y 27 N u c l e u s 27 C h l o r o p l a s t s and M i t o c h o n d r i a 30 Endomembrane system 31 V a c u o l e s 32 C e l l W a l l 33 P a r t 2 C y t o c h e m i s t r y 36 L i g h t m i c r o s c o p e C y t o c h e m i s t r y 36 P o l a r i z e d l i g h t m i c r o s c o p y 41 C e l l w a l l e x t r a c t i o n s & C a l c o f l u o r f l u o r e s c e n c e ... 42 U l t r a s t r u c t u r a l l o c a l i z a t i o n of enzyme a c t i v i t y ... 44 P e r o x i d a s e 44 ATPase 45 AcPase 47 Summary 4 9 Phase I 49 Phase I I 51 Phase I I I 52 T a b l e 2 .. 5 4 KEY FOR FIGURES 55 FIGURES 56 L i t e r a t u r e C i t e d 69 v L I S T OF FIGURES F i g u r e 1 L i g h t m i c r o g r a p h ( N o m a r s k i ) of q u i e s c e n t a x i a l c e l l 56 F i g u r e 2 L i g h t m i c r o g r a p h ( N o m a r s k i ) of s t a g e I bud 56 F i g u r e 3 L i g h t m i c r o g r a p h ( N o m a r s k i ) of s t a g e I I bud 56 F i g u r e 4 L i g h t m i c r o g r a p h ( N o m a r s k i ) of s t a g e I I I bud 56 F i g u r e 5 L i g h t m i c r o g r a p h ( N o m a r s k i ) of s t a g e IV bud 56 F i g u r e 6 L i g h t m i c r o g r a p h ( N o m a r s k i ) of s t a g e V bud 56 F i g u r e 7 L i g h t m i c r o g r a p h ( N o m a r s k i ) of s t a g e VI bud 56 F i g u r e 8 L i g h t m i c r o g r a p h ( N o m a r s k i ) of s t a g e V I I bud 56 F i g u r e 9 E l e c t r o n m i c r o g r a p h of q u i e s c e n t a x i a l c e l l 57 F i g u r e 10 E l e c t r o n m i c r o g r a p h of s t a g e I bud 57 F i g u r e 11 E l e c t r o n m i c r o g r a p h of c e l l w a l l of s t a g e 1 57 F i g u r e 12 E l e c t r o n m i c r o g r a p h of s t a g e I I bud 58 F i g u r e 13 E l e c t r o n m i c r o g r a p h of s t a g e I I I bud 58 v i F i g u r e 14 E l e c t r o n m i c r o g r a p h of c e l l w a l l of s t a g e I I I bud 58 F i g u r e 15 E l e c t r o n m i c r o g r a p h of s t a g e IV bud 59 F i g u r e 16 E l e c t r o n m i c r o g r a p h of c e l l w a l l of s t a g e IV bud 59 F i g u r e 17 L i g h t m i c r o g r a p h ( b r i g h t f i e l d ) of s t a g e IV bud 59 F i g u r e 18 L i g h t m i c r o g r a p h ( C a l c o f l u o r White f l u o r e s c e n c e ) of s t a g e IV bud 59 F i g u r e 19 E l e c t r o n m i c r o g r a p h of c e l l w a l l of s t a g e IV bud 59 F i g u r e 20 E l e c t r o n m i c r o g r a p h of s t a g e V bud 60 F i g u r e 21 E l e c t r o n m i c r o g r a p h of f i r s t s e r i a l s e c t i o n t h r o u g h n u c l e u s and c e n t r i o l e of s t a g e VI bud 60 F i g u r e 22 E l e c t r o n m i c r o g r a p h of second s e r i a l s e c t i o n t h r o u g h n u c l e u s and c e n t r i o l e of s t a g e VI bud 61 F i g u r e 23 E l e c t r o n m i c r o g r a p h of c e n t r i o l e of s t a g e VI bud 61 F i g u r e 24 E l e c t r o n m i c r o g r a p h of c e n t r i o l e and m i c r o t u b u l e s of s t a g e VI bud 61 F i g u r e 25 E l e c t r o n m i c r o g r a p h of s t a g e VI bud 61 v i i F i g u r e 26 E l e c t r o n m i c r o g r a p h of s t a g e V I I bud 6 2 F i g u r e 27 E l e c t r o n m i c r o g r a p h of c r o s s w a l l of s t a g e V I I bud 62 F i g u r e 28 S c h e m a t i c d r a w i n g of s t a g e I bud 63 F i g u r e 29 S c h e m a t i c d r a w i n g of s t a g e I I I bud 63 F i g u r e 30 S c h e m a t i c d r a w i n g of s t a g e VI bud 63 F i g u r e 31 S c h e m a t i c d r a w i n g of s t a g e V I I bud 63 F i g u r e 32 S c h e m a t i c d r a w i n g of c h e m i c a l c o m p o s i t i o n of e a r l y bud i n i t i a l c e l l w a l l 64 F i g u r e 33 S c h e m a t i c d r a w i n g of c h e m i c a l c o m p o s i t i o n of w e l l advanced bud i n i t i a l c e l l w a l l 64 F i g u r e 34 L i g h t m i c r o g r a p h of bud a f t e r TBO pH 6 . 8 s t a i n i n g 65 F i g u r e 35 L i g h t m i c r o g r a p h of bud a f t e r TBO pH 0 . 5 s t a i n i n g 65 F i g u r e 36 L i g h t m i c r o g r a p h of bud a f t e r m e t h y l a t i o n and S a f r a n i n 0 s t a i n i n g 6 5 F i g u r e 37 L i g h t m i c r o g r a p h of bud a f t e r PAS s t a i n i n g 6 5 F i g u r e 38 L i g h t m i c r o g r a p h of bud a f t e r F a s t G r e e n FCF s t a i n i n g 65 F i g u r e 39 L i g h t m i c r o g r a p h of bud a f t e r A n i l i n e Blue B l a c k s t a i n i n g . 65 v i l l F i g u r e 40 L i g h t m i c r o g r a p h of bud a f t e r Ruthenium Red s t a i n i n g 65 F i g u r e 41 L i g h t m i c r o g r a p h of bud a f t e r I K I / H 2 S 0 4 s t a i n i n g . 6 5 F i g u r e 42 E l e c t r o n m i c r o g r a p h of p e r o x i d a s e a c t i v i t y of e a r l y bud i n i t i a l 66 F i g u r e 43 E l e c t r o n m i c r o g r a p h of p e r o x i d a s e a c t i v i t y of advanced bud i n i t i a l 66 F i g u r e 44 E l e c t r o n m i c r o g r a p h of p e r o x i d a s e c o n t r o l 66 F i g u r e 45 E l e c t r o n m i c r o g r a p h of p e r o x i d a s e a c i t i v i t y of s t a g e V I I bud 66 F i g u r e 46 E l e c t r o n m i c r o g r a p h of g e n e r a l i n t r a c e l l u l a r s i t e s of ATPase a c t i v i t y 67 F i g u r e 47 E l e c t r o n m i c r o g r a p h of ATPase a c t i v i t y w i t h i n a d i c t y o s o m e 67 F i g u r e 48 E l e c t r o n m i c r o g r a p h of ATPase a c t i v i t y of the plasma membrane 67 F i g u r e 49 E l e c t r o n m i c r o g r a p h of ATPase a c t i v i t y w i t h i n a m i t o c h o n d r i o n 67 F i g u r e 50 E l e c t r o n m i c r o g r a p h of ATPase c o n t r o l ( s u b s t r a t e o m i s s i o n ) 67 F i g u r e 51 E l e c t r o n m i c r o g r a p h of ATPase c o n t r o l ( b o i l i n g ) 67 F i g u r e 52 E l e c t r o n m i c r o g r a p h of AcPase a c t i v i t y p lasma membrane 68 i x F i g u r e 53 E l e c t r o n m i c r o g r a p h of AcPase a c t i v i t y of a m i t o c h o n d r i o n 68 F i g u r e 54 E l e c t r o n m i c r o g r a p h of AcPase a c t i v i t y of bud c e l l 68 F i g u r e 55 E l e c t r o n m i c r o g r a p h of AcPase c o n t r o l 68 F i g u r e 56 E l e c t r o n m i c r o g r a p h of AcPase a c t i v i t y i n c y t o p l a s m i c v e s i c l e s 68 F i g u r e 57 E l e c t r o n m i c r o g r a p h of AcPase a c t i v i t y i n a d d i t i o n a l c y t o p l a s m i c v e s i c l e s to t h o s e of F i g . 5 6 68 x L I S T OF TABLES T a b l e 1 Summary o f l i g h t m i c r o s c o p e c y t o c h e m i s t r y 9 T a b l e 2 Summary of a l l major e v e n t s o c c u r r i n g d u r i n g bud i n i t i a t i o n and d e v e l o p m e n t 54 x i ACKNOWLEDGEMENTS I f i r s t w ish to e x p r e s s g r a t i t u d e to two s p e c i a l p e o p l e . My s u p e r v i s o r , D r . T. B i s a l p u t r a , f o r g i v i n g me the chance to l e a r n the a r t of e l e c t r o n m i c r o s c o p y , f o r p r o v i d i n g me w i t h f i n a n c i a l s u p p o r t when I needed i t and f o r n e v e r p r e s s u r i n g me i n t o c o m p l e t i n g my t h e s i s . H i s s u g g e s t i o n s and i n t e r p r e t a t i o n s of my work were i n v a l u a b l e i n p r e p a r i n g t h i s t h e s i s and i t was always an e n j o y a b l e e x p e r i e n c e w o r k i n g i n h i s l a b o r a t o r y . Thanks a r e a l s o e x t e n d e d to D r . L. O l i v e i r a f o r h i s i n f i n i t e s u g g e s t i o n s , f o r h i s e n d l e s s r e a d i n g s of my t h e s i s d r a f t s and most of a l l f o r h i s mo r a l s u p p o r t . F i n a l l y , I am s i n c e r e l y g r a t e f u l f o r the h e l p f u l c r i t i c i s m t h a t I r e c e i v e d from my committee members, D r . K. C o l e , D r . P . J . H a r r i s o n and D r . R. DeWreede. f - x i i I n t r o d u c t i o n S p h a c e l a r i a i s a f i l a m e n t o u s brown a l g a t h a t d i s p l a y s a p i c a l growth and i s u s u a l l y c o p i o u s l y b r a n c h e d ( F r i t s c h 1 9 5 9 ) . Each a x i s p o s s e s s e s a p r o m i n e n t a p i c a l c e l l t h a t upon d i v i s i o n p r o d u c e s p r i m a r y segment c e l l s p r o x i m a l l y . These p r i m a r y segment c e l l s e l o n g a t e and t h e n undergo a t r a n s v e r s e d i v i s i o n w h i c h r e s u l t s i n two s e c o n d a r y segment c e l l s , one ' s u p e r i o r ' and one ' i n f e r i o r ' . Once formed t h e s e do n o t expand i n s i z e ( B o l d and Wynne 1 9 7 8 ) . I n S.  f u r c i g e r a ( K u t z ) , the s p e c i e s used i n t h i s r e p o r t , the s e c o n d a r y segment c e l l s may t h e n p r o c e e d to d i v i d e l o n g i t u d i n a l l y p r o d u c i n g a m u l t i s e r i a t e f i l a m e n t ( F r i t s c h 1 9 5 9 ) . The p r o d u c t i o n of b r a n c h e s ( l a t e r a l s ) i n S p h a c e l a r i a i n v a r i a b l y a r i s e s from s u p e r i o r ( F r i t s c h 1959) and r a r e l y from i n f e r i o r segment c e l l s ( D u c r e ux 1977). The r e g u l a r i t y w i t h w h i c h S . f u r c i g e r a u n d e r g o e s t h i s p r o c e s s has been n o t e d by o t h e r a u t h o r s ( D o w o r e t s k y e t a l . 1 9 8 0 ) . The u n d e r l y i n g p r o c e s s of b r a n c h f o r m a t i o n i s w e l l c h a r a c t e r i z e d i n f u n g i ( s e e B u r n e t t and T r i n c i 1979 f o r a r e v i e w ) . In t h i s r e s p e c t , the l i t e r a t u r e i s b i a s e d towards t h e s e o r g a n i s m s j u d g i n g by the v i r t u a l absence of any non-f u n g a l s t u d i e s . S t u d i e s on the u l t r a s t r u c t u r a l p r o c e s s of b r a n c h f o r m a t i o n i n n o n - f u n g a l o r g a n i s m s have been m o s t l y c o n f i n e d to the moss Funar i a ( S c m i e d e l and S c h n e p f 1 979a, 1979b), the b l a d d e r w o r t U t r i c u l a r i a ( F i n e r a n 1 9 8 0 ) , the r e d a l g a Batrachospermum ( A g h a j a n i a n and Hommersand 1980), and 1 the g r e e n a l g a e B u l b o c h a e t e ( P i c k e t t - H e a p s 1 9 7 4 ) , O e d o c l a d ium ( P i c k e t t - H e a p s 1 97 7 ) and Chara ( D u c r e u x 1 9 7 9 ) . Even though the number of s t u d i e s i s s m a l l , i t i s a p p a r e n t t h a t the p r o c e s s of b r a n c h i n g i s common to a wide range of d i f f e r e n t o r g a n i s m s and hence t h e r e i s a need f o r a b e t t e r u n d e r s t a n d i n g of t h i s m o r p h o g e n e t i c p r o c e s s . The p u r p o s e of t h i s s t u d y i s to combine 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 w i t h c y t o c h e m i c a l t e c h n i q u e s . In d o i n g so, s i g n i f i c a n t i n f o r m a t i o n c o n c e r n i n g v a r i a t i o n s i n the c h e m i c a l c o m p o s i t i o n o f the c e l l w a l l a s s o c i a t e d w i t h m o r p h o l o g i c a l a l t e r a t i o n s t a k i n g p l a c e d u r i n g the p r o c e s s of bud i n i t i a t i o n and d e v e l o p m e n t can be o b s e r v e d . C y t o c h e m i c a l s t u d i e s of brown a l g a e u s i n g l i g h t m i c r o s c o p y a r e numerous ( M c C u l l y 1 9 6 6 , 1 9 6 8 , 1 9 7 0 , Novotny and Forman 1 975 , T o t h 1 9 7 6 ) . Most of t h e s e s t u d i e s have d e a l t w i t h the c h e m i c a l d e t e c t i o n of the two major brown a l g a l c e l l w a l l p o l y s a c c h a r i d e s , " f u c a n s " and " a l g i n a t e s " . The c e l l w a l l has been p o s t u l a t e d t o p l a y a two f o l d r o l e i n bud e x p a n s i o n ; i t must y i e l d to the d r i v i n g t u r g o r f o r c e and a t the same time 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 ( B a r t i n i c k i - G a r c i a and Lippman 1 9 7 2 , B a r t i n i c k i - G a r c i a 1 9 7 3 , Werz 1 9 7 4 , Novotny and Forman 1 9 7 5 , P i c k e t t - H e a p s 1 9 7 7 , Gooday 1 9 7 9 , Gooday and T r i n c i 1 9 8 0 ) . I t i s , t h e r e f o r e , i m p o r t a n t to m o n i t o r the b e h a v i o r of f u c a n s and a l g i n a t e s d u r i n g the c o u r s e of bud i n i t i a t i o n and d e v e l o p m e n t . The l i t e r a t u r e a l s o s u g g e s t s t h a t t h r e e enzymes, p e r o x i d a s e , a d e n o s i n e t r i p h o s p h a t a s e and a c i d 2 p h o s p h a t a s e a r e i n v o l v e d i n c e l l w a l l d e g r a d a t i o n and/or s y n t h e s i s ( H a l p e r i n 1969, Henry 1979, Coupe and D'Auzac 1980, G i o r d a n i 1 9 8 0 ) . C y t o c h e m i c a l t e c h n i q u e s e x i s t w hich p e r m i t the u l t r a s t r u c t u r a l d e t e c t i o n of t h e s e e n z y m a t i c a c t i v i t i e s w i t h i n the c e l l . Thus, i n f o r m a t i o n c o n c e r n i n g the r o l e , i f any, of enzymes i n c e l l w a l l r e s t r u c t u r i n g i s a l s o p e r t i n e n t to u n d e r s t a n d i n g bud i n i t i a t i o n and d e v e l o p m e n t . S p h a c e l a r i a f u r c i g e r a i s an e x c e l l e n t system f o r s t u d y i n g t h i s d e v e l o p m e n t a l p r o c e s s b e c a u s e a) i t p o s s e s s e s a r e g u l a r b r a n c h i n g p a t t e r n and b) i t i s an o r g a n i s m w h i c h has n o t been e x t e n s i v e l y i n v e s t i g a t e d u 1 t r a s t r u c t u r a l 1 y or c y t o c h e m i c a l l y . 3 M a t e r i a l s and Methods S p h a c e l a r i a sp. ( P h a e o p h y t a , S p h a c e l a r i a c e a e ) was o b t a i n e d from the C u l t u r e C o l l e c t i o n of A l g a e , I n d i a n a U n i v e r s i t y (now l o c a t e d a t the U n i v e r s i t y of Texas a t A u s t i n ) ( s t o c k c u l t u r e # LB 8 0 0 ) . The d e v e l o p m e n t of p r o p a g u l e s , h a i r s and u n i l o c u l a r s p o r a n g i a has p e r m i t t e d i t s i d e n t i f i c a t i o n as f u r c i g e r a . The c u l t u r e s were m a i n t a i n e d i n C h i h a r a ' s m a r i n e medium ( C h i h a r a 1968) a t 20°C and i l l u m i n a t e d by ' V i t a L i t e ' ( T M ) f l u o r e s c e n t tubes f o r 1 2hr/day a t an i n t e n s i t y of 9.1 watts/m^. The s p e c t r a l q u a l i t i e s of t h e s e f l u o r e s c e n t l i g h t s mimic v e r y c l o s e l y t h o s e of n a t u r a l s u n l i g h t . L i g h t m i c r o s c o p y o f l i v e m a t e r i a l : O b s e r v a t i o n s o f bud i n i t i a t i o n u s i n g l i v e m a t e r i a l were made w i t h N o m a r s k i d i f f e r e n t i a l i n t e r f e r e n c e c o n t r a s t o p t i c s u s i n g a Z e i s s P h o t o m i c r o s c o p e I I and w i t h b r i g h t f i e l d or r e f l e c t e d l i g h t f l u o r e s c e n c e m i c r o s c o p y u s i n g a L e i t z D i a l u x 20 EB m i c r o s c o p e . F o r the l a t t e r an HBO 50W s u p e r p r e s s u r e m e r c u r y lamp w h i c h p r o v i d e d the i l l u m i n a t i o n and was used i n c o n j u n c t i o n w i t h a BP 340-380 e x c i t i n g f i l t e r , an RKP 400 beam s p l i t t i n g m i r r o r , and an LP 43 0 s u p p r e s s i o n f i l t e r . The f l u o r e s c e n c e s t u d y employed a m o d i f i e d v e r s i o n o f the f l u o r e s c e n t b r i g h t e n e r and v i t a l dye C a l c o f l u o r White ST* s t a i n i n g method (Waaland and Waaland 1975). In t h i s s t u d y l i v e p r o p a g u l e s a t a s i m i l a r s t a g e of 4 d e v e l o p m e n t were p l a c e d f o r 30 m i n u t e s i n a 0.005% v/v dye s o l u t i o n i n c u l t u r e medium, washed 3x f o r 10 m i n u t e s each i n f r e s h c u l t u r e medium, o b s e r v e d and p h o t o g r a p h e d . Some p r o p a g u l e s were r e t u r n e d to the i n c u b a t o r and a l l o w e d to grow f o r a p e r i o d of up to 2 weeks. D u r i n g t h i s time i n -t e r v a l p r o p a g u l e s were removed and examined f o r s i g n s of bud i n i t i a t i o n . E l e c t r o n m i c r o s c o p y : C o p i o u s l y b r a n c h e d a d u l t p l a n t s used f o r 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 were f i x e d f o r 2 1/2 hr s a t room t e m p e r a t u r e i n 1.6% v/v g 1 ut a r a l de hyde i n a 0.05M sodium c a c o d y l a t e b u f f e r a t pH 7.0 which c o n t a i n e d 1% w/v c a f f e i n e ( M u e l l e r and Greenwood 1 9 7 8 ) . T h i s was f o l l o w e d by a wash i n the same b u f f e r p l u s c a f f e i n e . P o s t -f i x a t i o n was c a r r i e d out u s i n g 1% v/v OsO^ j _ n c a f f e i n e f r e e sodium c a c o d y l a t e b u f f e r c o n t a i n i n g 0.1% w/v r u t h e n i u m r e d (Sigma R-2751 p r a c t i c a l g r a d e ) f o r 16hrs a t 4°C i n d a r k n e s s (Colombo and R a s c i o 1 9 7 7 ) . A f t e r s e v e r a l washes i n b u f f e r and d e h y d r a t i o n t h r o u g h a m e t h a n o l s e r i e s , the m a t e r i a l was embedded i n Epon ( L u f t 1961) . S e c t i o n s were c u t on a R e i c h e r t 0mU3 u 1 t r a m i c r o t o m e , c o l l e c t e d on s i n g l e s l o t (2x0.5mm) c o p p e r g r i d s c o a t e d w i t h Formvar and p o s t - s t a i n e d w i t h 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 o l d s 1 9 6 3 ) . O b s e r v a t i o n s were c a r r i e d out w i t h e i t h e r a Z e i s s EM 9S or a Z e i s s EM 10 e l e c t r o n m i c r o s c o p e . L i g h t m i c r o s c o p y o f s e c t i o n e d m a t e r i a l : C o p i o u s l y b r a n c h e d 5 a d u l t p l a n t s were f i x e d i n an i d e n t i c a l manner to t h o s e p r o c e s s e d f o r e l e c t r o n m i c r o s c o p y w i t h the o m i s s i o n of a p o s t - f i x a t i o n i n OsO^ a n d r u t h e n i u m r e d . D e h y d r a t i o n t h r o u g h a m e t h a n o l s e r i e s was f o l l o w e d by embedment i n g l y c o l m e t h a c r y l a t e or J.B.4 embedding media ( P o 1 y s c i e n c e , W a r r i n g t o n , P e n n s y l v a n i a ) . One to two m i c r o n t h i c k s e c t i o n s were c u t on a Du Pont S o r v a l J.B.4 microtome u s i n g g l a s s k n i v e s . S e c t i o n s were f l o a t e d o n t o d i s t i l l e d w a t e r on c l e a n m i c r o s c o p e s l i d e s . The s l i d e s were t h e n a i r d r i e d and s t a i n e d . S t a i n i n g p r o c e d u r e s a r e summarized i n T a b l e 1. O b s e r v a t i o n s were made w i t h a L e i t z D i a l u x 20 EB m i c r o s c o p e . Enzyme l o c a l i z a t i o n a t the u 1 t r a s t r u e t u r a 1 l e v e l : C o p i o u s l y b r a n c h e d a d u l t p l a n t s were b r i e f l y f i x e d a t room t e m p e r a t u r e i n 1.6% v/v g l u t a r a l d e h y d e i n 0.05 M sodium c a c o d y l a t e b u f f e r a t pH 7.0 w h i c h c o n t a i n e d 1% c a f f e i n e ( M u e l l e r and Greenwood 1978). The m a t e r i a l was t h e n washed i n the same b u f f e r p l u s 1% c a f f e i n e and s t o r e d o v e r n i g h t a t 4°C i n 0.05 M t r i s - m a l e a t e b u f f e r a t pH 7.0. The c o m p o s i t i o n of the p r e - i n c u b a t i o n medium was: P e r o x i d a s e : 50 mM t r i s - m a l e a t e , pH 7.0 2 mM a m i n o t r i a z o l e 1 mM d i a m i n o b e n z i d i n e 6 Adenos i n e 50 mM t r i s - m a 1 e a t e , pH 7.0 t r i p h o s p h a t a s e : 1 mM MgSO^ 0.3 mM P b ( N 0 3 ) A c i d 50 mM t r i s - m a l e a t e , pH 5.0 p h o s p h a t a s e : 3 mM P b C N O ^ j A l l pr e - i n c u b a t i o n s were c a r r i e d out i n the d a r k f o r 30 m i n u t e s a t room t e m p e r a t u r e . The c o m p o s i t i o n of the i n c u b a t i o n medium was: P e r o x i d a s e : P r e - i n c u b a t i o n medium p l u s 0.3% l ^ c ^ . ( m o d i f i c a t i o n of H a l l and S e x t o n 1972) Adenos i n e P r e - i n c u b a t i o n medium p l u s 3 mM ATP. t r i p h o s p h a t a s e : ( m o d i f i c a t i o n of G i l d e r and Cronshaw 1973) Ac i d P r e - i n c u b a t i o n medium p l u s 8 mM sodium p h o s p h a t a s e : B e t a - g 1 y c e r o p h o s p h a t e . ( m o d i f i c a t i o n o f Bar k a and A n d e r s o n 1962) A l l i n c u b a t i o n s were c a r r i e d out i n t h e d a r k f o r 60 m i n u t e s a t room t e m p e r a t u r e . A f t e r i n c u b a t i o n , a l l m a t e r i a l was washed i n 0.05 M sodium c a c o d y l a t e b u f f e r a t pH 7.0, r e - f i x e d f o r 2 h r s a t room t e m p e r a t u r e i n 1.6% v/v g l u t a r a l d e h y d e i n a 0.05 M sodium c a c o d y l a t e b u f f e r a t pH 7.0. P o s t - f i x a t i o n o v e r n i g h t a t 4°C f o l l o w e d i n 1% OsO^ i n 0.05 M sodium c a c o d y l a t e a t pH 7.0. The m a t e r i a l was t h e n r o u t i n e l y p r o c e s s e d f o r 7 e l e c t r o n m i c r o s c o p y . C o n t o l p r e p a r a t i o n s were as f o l l o w s : P e r o x i d a s e : M a t e r i a l was i n c u b a t e d i n the absence of H 2 0 2 • A d e n o s i n e M a t e r i a l was b o i l e d p r i o r to i n c u b a t i o n t r i p h o s p h a t a s e : f o r 5 m i n u t e s i n 50 mM t r i s - m a l e a t e b u f f e r a t pH 7.0. Some m a t e r i a l was a l s o i n c u b a t e d i n the absence of ATP. Ac i d M a t e r i a l was i n c u b a t e d i n the p r e s e n c e p h o s p h a t a s e : of 10 mM sodium f l u o r i d e . 1 Donated by Cynamid Canada I n c . , B a i e d ' U r f e , P.Q. 8 T a b l e 1 C y t o c h c n i c e t T e c h n i q u e s I n d i c a t i c r. F o u r C c l I W a t I L a y e r e d P a t t e r n o f A x i a l . C e l Is cwi cwa cwa cv/4 T w o C e l l W a l l L a y e r e d P a t t e r n o f £ u d I n i t i a l C H O CVI S o u r c e C o r b o x y I 6 SUI(3 ZC g r o u p s P u + . P i + P u P i + + P L P i + E v e n s & H o L I i g o n 1 9 7 2 , 1 3 7 6 T 8 0 p i ! Q . 5 s u l f a t e g r o u p s P u + P i + P u -+ — P c + P i + M c C u l l y 1 S 7 0 S o f r o n i n 0 p K D . 5 s u l f a t e g r o u p s ++ • ++ + + S p i c e r 1 9 6 0 A l c i a n B l u e C C S p l i 2 . 4 c a r t o x y t G s u l f a t e C r o u p s ++ + ++ - S p i c e r 1 9 6 0 A L c i a n G l u e -. G G S pK C . 5 s u l f a t a g r o u p s + + — . + - S t e e d n a n 1 9 5 0 S a f r a n i n 0 a f t e r c o r -b o x y I n e t h -y l a t i o n s u l f a t o g r o u p s + + + C o o k 1 S 7 7 * A l c i a n B I u a 8 C S p H 2 . 4 a f t e r COP-b o x y L n o t h -y l a t l o n s u t f a t e g r o u p s + + ' + C o o k 1 3 7 7 A c r i d i n o 0 r a n g e p l i 7 . 4 a d d n u c i n s ++ + ++ + H i c k s & H a t t h a e i 1 9 5 0 A c r i d i n e 0 r a n g e p H 0 . 5 s u L f a t e g r o u p s + 4- - .+ C o o k 1 9 7 7 P e r i o d i c A c i d S c h i f f s R e s e t i o n 1 , 2 - g l y c o l g r o u p s ++ + + + + ++ F o d e r L O ' G r i e n 1 E E G e e l l u l o s e - + + - - + J e n s e n 1 9 6 2 P c l a r i z ad L i g h t c e L I u l o s e £ a l g i n a t e s — + - + A n d c r s e n 1 0 5 6 C a l c c f l u o r tthite E T b c t o - 1 , 3 £*, 1 , 4 L i n k e d p o l y -s a c c h a r i d e s + H e s l e p -H a r r i s o n £. H e & l o p -H a r r i s o n 1 9 6 1 ' F u c a n * o x t r a c t i o n f c I t o n e d b y C c I c o f l u o r . W h i t e S T a l g i n a t e s +• - - + X V / h y t c o t a l 1 2 8 1 • A l c j l n c t e ' e x t r a c t i o n f c I l o w e d b y C c I c o f I u o r W h i t e C T f u c a n s r o -n o i n S, c r c u n s t o i n e d + - + X y M o v o t n y £ F o r r . o n 1 9 7 0 R u t h c n i u n R e o . 0 2 2 a c i d i c p o l y -s a c c h o r 1 d e c ++ + + + + ++ C u r r 1 E G 5 A n i l i n e C l u o B l a c k 1 K i n 7 S a c e t a t e p r o t e i n + + - • F l s h c r 1 S C C F a s t G r e e n F C F p H 2 . 0 p r o t e i n • +• • F c d c r & 0 ' U r i o n 1 S 6 C + = p o s i t i v e , - = n e g a t i v e , P u = p u r p l e , P i = p i n k , * » a o d i f i c d , X = n o t t e s t e d . / R e s u l t s : P a r t 1 M o r p h o l o g y L i g h t m i c r o s c o p y : The d e v e l o p m e n t of a bud i n i t i a l from an a x i a l mother c e l l i s d e p i c t e d i n F i g s . 2-8. A q u i e s c e n t a x i a l c e l l ( F i g . l ) i s a l s o i n c l u d e d f o r c o m p a r i s o n . The f i r s t s i g n of bud i n i t i a t i o n , r e f e r r e d to i n the t e x t as s t a g e I , i s r e c o g n i z e d as a s m a l l p r o t r u s i o n of the c e l l w a l l i n the upper r i g h t q u a d r a n t of the a x i a l mother c e l l ( F i g . 2 ) . With time the bud p r o t r u s i o n c o n t i n u e s to expand and p a s s e s t h r o u g h d e v e l o p m e n t a l s t a g e s d e f i n e d as I I and I I I . D u r i n g t h e s e s t a g e s the p o s i t i o n of the n u c l e u s i n r e l a t i o n to the p r o t r u s i o n a r e a of the c e l l w a l l r e m a i n s unchanged (Compare F i g s . 3 and 4 w i t h F i g . 1 ) . Stage I I , however, i s r e c o g n i z e d as a s t a g e whereby the h e i g h t of the p r o t r u s i o n e q u a l s a p p r o x i m a t e l y 20% of the l e n g t h of the o u t e r l o n g i t u d i n a l c e l l w a l l , w h i l e i n s t a g e I I I the p r o t r u s i o n has d o u b l e d i t s h e i g h t . I n i t i a t i o n of n u c l e a r m i g r a t i o n towards the bud p r o t r u s i o n c h a r a c t e r i z e s s t a g e IV ( F i g . 5 ) , w h i l e i n s t a g e V the n u c l e u s has assumed a m e d i a l p o s i t i o n between the b a s a l p o r t i o n of the a x i a l mother c e l l and the w e l l d e v e l o p e d bud ( F i g . 6 ) . Bud p r o t r u s i o n d e v e l o p m e n t r e a c h e s a maximum by s t a g e VI ( F i g . 7 ) . D u r i n g t h i s time k a r y o k i n e s i s i s t h o u g h t to o c c u r , s i n c e the n e x t s t a g e , s t a g e V I I , i s c h a r a c t e r i z e d by the c o m p l e t i o n of a c r o s s w a l l , i . e . c y t o k i n e s i s ( F i g . 8 ) . The c y t o k i n e t i c p r o c e s s has r e s u l t e d i n the f o r m a t i o n of two d a u g h t e r c e l l s , a bud c e l l and a s u b t e n d i n g s i s t e r a x i a l c e l l . A t 10 t h i s t i m e , i t i s e v i d e n t t h a t the bud c e l l has t a k e n on a d e n s e r c y t o p l a s m i c o r g a n i z a t i o n r e s e m b l i n g t h a t of an a p i c a l m e r i s t e m a t i c c e l l , w h i l e the s u b t e n d i n g s i s t e r a x i a l c e l l has r e - e s t a b l i s h e d i t s v a c u o l a t e d , l e s s compact c y t o p l a s m i c o r g a n i z a t i o n t h a t i s c h a r a c t e r i s t i c of v e g e t a t i v e a x i a l c e l l s . Growth and s u b s e q u e n t d i v i s i o n of the bud c e l l would c o n s t i t u t e the l a t t e r phase of b r a n c h i n g . E l e c t r o n m i c r o s c o p y Q u i e s c e n t a x i a l c e l l : F i g . 9 shows the g e n e r a l u l t r a s t r u c t u r e of a q u i e s c e n t a x i a l c e l l . The n u c l e u s e x h i b i t s an i r r e g u l a r p r o f i l e and o c c u p i e s a c e n t r a l p o s i t i o n w i t h i n the c y t o p l a s m . A w e l l d e v e l o p e d n u c l e o l u s and e v e n l y d i s p e r s e d h e t e r o c h r o m a t i c m a t e r i a l a r e a l s o e v i d e n t . The most c o n s p i c u o u s f e a t u r e of t h e s e c e l l s , however, i s the h i g h d e g r e e of v a c u o l a t i o n w h i c h c a u s e s the c h l o r o p l a s t s and mitoc.hondr i a to assume a p e r i p h e r a l a r r a n g e m e n t . E n d o p l a s m i c r e t i c u l u m (ER) e l e m e n t s a r e o b s e r v e d b o t h p a r t i a l l y e n s h e a t h i n g the c h l o r o p l a s t s and d i s p e r s e d t h r o u g h o u t the c y t o p l a s m ( F i g . 9 ) . D i c t y o s o m e s a r e few i n number and d i s p l a y no p r e f e r e n t i a l l o c a t i o n w i t h i n the c y t o p l a s m . W i t h i n the v a c u o l e s remnants of membranous i n c l u s i o n s , i n d i c a t i v e of e x t e n s i v e a u t o p h a g i c a c t i v i t y , ( M a t i l e 1 974), a r e o b s e r v e d . I n a d d i t i o n , e l e c t r o n dense i n c l u s i o n s abound i n the p a r a m u r a l space w i t h the m a j o r i t y 11 of them l y i n g a d j a c e n t to the o u t e r l o n g i t u d i n a l w a l l . A c l o s e e x a m i n a t i o n of t h i s w a l l r e v e a l s a d i s t i n c t i v e l a y e r e d p a t t e r n . Four w a l l l a y e r s can be r e c o g n i z e d ( F i g . 9 , CW1 to CW4) of w h i c h the o u t e r m o s t i s c o v e r e d by a t h i n c u t i c l e ( F i g . 9 , C u ) . The w a l l l a y e r s CWl and CW3 a r e c h a r -a c t e r i z e d by the p r e s e n c e of an amorphous e l e c t r o n dense m a t e r i a l i n w h i c h a p o o r l y d e f i n e d m i c r o f i b r i l l a r component seems to be embedded. W a l l l a y e r s CW2 and CW4 show a l e s s e l e c t r o n dense b a c k g r o u n d and a more d i s t i n c t i v e f i b r i l l a r c o m p o s i t i o n c o n s i s t i n g of l o n g i t u d i n a l l y o r i e n t e d m i c r o f i b r i l s . W a l l l a y e r s CW3 and CW4 a r e c o n t i n u o u s w i t h the t r a n s v e r s e w a l l s and the i n n e r l o n g i t u d i n a l w a l l of the q u i e s c e n t a x i a l c e l l , w h i l e w a l l l a y e r s CWl and CW2 a r e r e s t r i c t e d to the o u t e r l o n g i t u d i n a l w a l l . Numerous plasmodesmata can a l s o be o b s e r v e d ; t h e s e a r e e x c l u s i v e l y c o n f i n e d to the t r a n s v e r s e w a l l s ( F i g . 9 ) . S tage I : I n t h i s s t a g e the n u c l e u s i s o c c u p i e s a m e d i a l p o s i t i o n w i t h i n the c e l l ( F i g . 1 0 ) . I t s p r o f i l e i s l e s s i r r e g u l a r than i n q u i e s c e n t a x i a l c e l l s . A c o n s p i c u o u s n u c l e o l u s and r a n d o m l y d i s p e r s e d h e t e r o c h r o m a t i n a r e a l s o e v i d e n t . I n a d d i t i o n , s e r i a l s e c t i o n i n g r e v e a l s a l a t e r a l ( w i t h r e s p e c t to the f u t u r e b r a n c h a x i s ) i n p o c k e t i n g of the n u c l e a r e n v e l o p e c o n t a i n i n g a p a i r of c e n t r i o l e s ( F i g . 1 0 ) . D i c t y o s o m e s o c c u p y a d i s t i n c t i v e p e r i n u c l e a r p o s i t i o n , f a v o r i n g the a c t i v e t r a n s f e r of n u c l e a r e n v e l o p e d e r i v e d v e s i c l e s to the f o r m a t i v e f a c e of the d i c t y o s o m e s ( F i g . 1 0 ) . 12 M i t o c h o n d r i a l p r o f i l e s have i n c r e a s e d i n number, w h i l e a n o t i c e a b l e d e c r e a s e i n the number of v a c u o l e s and a c o n c u r r e n t i n c r e a s e i n c y t o p l a s m i c mass and d e n s i t y t a k e s p l a c e . These e v e n t s a r e c h a r a c t e r i s t i c of de-d i f f e r e n t i a t i o n ( E s a u 1 960). O t h e r w i s e , the d i s t r i b u t i o n and m o r p h o l o g y of the r e m a i n i n g c e l l u l a r o r g a n e l l e s a r e s i m i l a r to t h o s e o b s e r v e d i n the q u i e s c e n t a x i a l c e l l . The f o u r w a l l l a y e r s r e m a i n d i s t i n c t i n the o u t e r l o n g i t u d i n a l w a l l ( F i g . 1 1 , CWl to CW4). T h e r e i s , however, a c o n s p i c u o u s l o c a l i z e d t h i c k e n i n g of the i n n e r m o s t l a y e r , CW4, i n the r e g i o n of the p r o t r u s i o n . W i t h i n t h i s r e g i o n , d i s t i n c t d i s c o n t i n u i t i e s a r e a l s o n o t e d between w a l l l a y e r s CWl and CW2 ( F i g . 1 1 ) . Stage I I : The p o s i t i o n of the n u c l e u s i n s t a g e I I ( F i g . 1 2 ) i s t h e same as i n s t a g e I . However, t h e r e i s a d e f i n i t e i n c r e a s e i n the s i z e of b o t h the n u c l e u s and the n u c l e o l u s . In a d d i t i o n , a second n u c l e o l a r body i s f r e q u e n t l y e n c o u n t e r e d . T h i s i s i n d e p e n d e n t from the l a r g e r n u c l e o l u s as d e m o n s t r a t e d by s e r i a l s e c t i o n i n g . E q u a l l y s t r i k i n g i s the i n a b i l i t y to d e t e c t h e t e r o c h r o m a t i c m a t e r i a l . A p a i r of c e n t r i o l e s r e m a i n s a s s o c i a t e d w i t h a l a t e r a l i n p o c k e t i n g of the n u c l e a r e n v e l o p e a t t h i s s t a g e . C h l o r o p l a s t and m i t o c h o n d r i a l p r o f i l e s have i n c r e a s e d i n number and seem to occupy the c y t o p l a s m of the bud p r o t r u s i o n . In the case of the c h l o r o p l a s t s , the l o n g a x i s o f t h e s e o r g a n e l l e s seems to be o r i e n t e d p a r a l l e l to the 13 p o r t i o n of the p r o t r u d i n g w a l l i n a c o n s i s t a n t f a s h i o n . An e x t e n s i v e r e o r g a n i z a t i o n of the endoraembrane system (ER, d i c t y o s o m e s and t h e i r a s s o c i a t e d v e s i c l e s ) i s a l s o o b s e r v e d , c o n s i s t i n g of the p r o l i f e r a t i o n of ER and d i c t y o s o m e s and t h e i r c o n c e n t r a t i o n i n the r e g i o n of the p r o t r u s i o n . A c h a r a c t e r i s t i c f e a t u r e of t h i s s t a g e i s the s t a c k e d a p p e a r a n c e of some of the ER c i s t e r n a e w i t h i n the p r o t r u s i o n ( F i g . 1 2 ) . C o n c u r r e n t w i t h t h e s e e v e n t s , a f u r t h e r d e c r e a s e i n the number of v a c u o l e s and an i n c r e a s e i n c y t o p l a s m i c mass i s d e t e c t e d . The p r e v i o u s l y a c q u i r e d c y t o p l a s m i c d e n s i t y i s m a i n t a i n e d . The f o u r w a l l l a y e r s c h a r a c t e r i s t i c of the o u t e r l o n g i t u d i n a l c e l l w a l l a r e now o n l y r e c o g n i z a b l e i n the b a s a l p o r t i o n of the p r o t r u s i o n ( F i g . 1 2 , CWl to CW4). C l o s e r to the apex of the p r o t r u s i o n o n l y two c e l l w a l l l a y e r s can be o b s e r v e d . T h i s r e d u c t i o n i n the number of w a l l l a y e r s r e s u l t s from the c o n v e r g e n c e of l a y e r s CWl,CW2 and CW3 i n t o a new, s i n g l e e l e c t r o n dense o u t e r w a l l l a y e r , CWO, which d i s p l a y s numerous h o l e - l i k e s t r u c t u r e s ( F i g . 1 2 ) . The i n n e r m o s t l a y e r , CWl, on the o t h e r hand, has i n c r e a s e d i n t h i c k n e s s w h i l e r e m a i n i n g c o n t i n u o u s w i t h l a y e r CW4 of the b a s a l p o r t i o n of the o u t e r l o n g i t u d i n a l c e l l w a l l . S tage I I I : T h i s s t a g e o c c u r s j u s t p r i o r t o , or a t the o n s e t o f , n u c l e a r m i g r a t i o n . I n F i g . 1 3 the n u c l e u s a p p e a r s to have become d i s p l a c e d from i t s o r i g i n a l p o s i t i o n near the i n n e r l o n g i t u d i n a l w a l l . O t h e r w i s e no v i s i b l e changes i n 14 n u c l e a r m o rphology were d e t e c t e d . C e n t r i o l e s were not e n c o u n t e r e d i n the s e c t i o n s I s t u d i e d but t h i s does n o t n e g a t e t h e r i r p r e s e n c e . T h e i r absence may be e x p a i n e d by the f a c t t h a t a s m a l l e r number of s e c t i o n s were a n a l y s e d . S u p p o r t i v e of t h i s i d e a i s the f a c t t h a t the c e n t r i o l e s were o b s e r v e d i n the next s t a g e of bud d e v e l o p m e n t . The a r r a n g e m e n t and m o r p h o l o g y of a l l o t h e r c e l l u l a r o r g a n e l l e s r e m a i n s v i r t u a l l y u n a l t e r e d , a l t h o u g h by now i t i s a p p a r e n t t h a t t h e r e a r e few, i f any, v a c u o l e s r e m a i n i n g w i t h i n the c y t o p l a s m . The c e l l w a l l i n the r e g i o n of the bud t i p s t i l l c o n s i s t s of o n l y two c e l l w a l l l a y e r s . However, t h e o u t e r e l e c t r o n dense l a y e r , CWO , seems l o o s e l y a t t a c h e d to the i n n e r w a l l l a y e r w h i c h a p p e a r s to be t h i n n e r t h a n i n t h e p r e v i o u s s t a g e ( F i g . 1 3 ) . Stage IV: T h i s s t a g e i s c h a r a c t e r i z e d by the o c c u r r e n c e of d i s t i n c t n u c l e a r m i g r a t i o n towards the bud i n i t i a l ( F i g . 1 5 ) . The n u c l e u s e x t e n d s beyond the c o n f i n e s of the b a s a l p o r t i o n of the a x i a l mother c e l l i n t o the e n l a r g e d p o r t i o n of the bud p r o t r u s i o n . T h i s i s accompanied by the r e a p p e a r a n c e of h e t e r o c h r o m a t i n . S e r i a l s e c t i o n i n g r e v e a l e d , as i n p r e v i o u s s t a g e s , a p a i r of c e n t r i o l e s c o n f i n e d to a l a t e r a l l y p o s i t i o n e d i n p o c k e t i n g of the n u c l e a r e n v e l o p e . No major changes i n o t h e r o r g a n e l l e s a r e n o t e d and the absence of c y t o p l a s m i c v a c u o l a t i o n r e m a i n s a dominant f e a t u r e of t h i s s t a g e . The p a t t e r n of the w a l l l a y e r i n g i s s i m i l a r to t h a t of 15 s t a g e I I I , but t h e r e a p p e a r s to be an i n c r e a s e i n the number of d i s c o n t i n u i t i e s w hich r e m a i n c o n f i n e d to the o u t e r c e l l w a l l l a y e r , CWO, i n the a r e a of the bud p r o t r u s i o n ( F i g . 1 6 ) . T h i s w a l l l a y e r has now become as t h i n as the o u t e r l a y e r , CWl, of the n o n - p r o t r u d i n g p o r t i o n of the o u t e r l o n g i t u d i n a l c e l l w a l l . M e a n w h i l e , the i n n e r m o s t c e l l w a l l l a y e r of the a p i c a l r e g i o n of the p r o t r u s i o n , CWl, i s now as t h i c k as the combined t h i c k n e s s of l a y e r s CW2 , CW3 , and CW4 of the b a s a l n o n - p r o t r u d i n g p o r t i o n of the o u t e r l o n g i t u d i n a l w a l l . A b e t t e r u n d e r s t a n d i n g of the change i n the p a t t e r n of the c e l l w a l l l a y e r i n g i s o b t a i n e d by c o m p a r i n g the b r i g h t f i e l d m i c r o g r a p h r e p r e s e n t i n g a bud a t d e v e l o p m e n t a l s t a g e IV ( F i g . 1 7 ) w i t h the m i c r o g r a p h of the same bud o b t a i n e d t h r o u g h f l u o r e s c e n c e m i c r o s c o p y ( F i g . 1 8 ) . The n o n - f 1 u o r e s c i n g t i p of the p r o t r u d i n g bud r e p r e s e n t s the a d d i t i o n of new, n o n - l a b e l l e d c e l l w a l l m a t e r i a l (Waaland and Waaland 1975). Note the c o n v e r g e n c e and d i s a p p e a r a n c e of the o r i g i n a l ( o l d ) w a l l f l u o r e s c e n c e as i t a p p r o a c h e s t h i s new w a l l m a t e r i a l . These o b s e r v a t i o n s s t r o n g l y s u g g e s t t h a t the c e l l w a l l l a y e r s CWO and CWl of the p r o t r u s i o n , c o n s i s t m a i n l y of newly s y n t h e s i z e d w a l l m a t e r i a l . The sequence of m i c r o f i b r i l l a r o r i e n t a t i o n s , from the base of the bud p r o t r u s i o n upwards i n r e l a t i o n to the l o n g i t u d i n a l a x i s of the bud, s h i f t s from a t r a n s v e r s e to a l o n g i t u d i n a l d i r e c t i o n . The t i p of the p r o t r u s i o n , however, e x h i b i t s a random or somewhat t r a n s v e r s e m i c r o f i b r i l o r i e n t a t i o n ( F i g . 1 6 ) . T h i s p a t t e r n of m i c r o f i b r i l 16 o r i e n t a t i o n i s c o n s i s t e n t l y o b s e r v e d i n a l l of the f o l l o w i n g s t a g e s of bud d e v e l o p m e n t . Stage V: Here the n u c l e u s l i e s mid-way between the a x i a l mother c e l l and the p r o t r u d i n g bud i n i t i a l ( F i g . 2 0 ) . The g e n e r a l a p p e a r a n c e of the n u c l e u s r e m a i n s unchanged. As i n e a r l i e r s t a g e s , a p a i r of c e n t r i o l e s a r e s t i l l l o c a t e d w i t h i n a l a t e r a l n u c l e a r i n p o c k e t i n g . The endomembrane system r e m a i n s e x t e n s i v e t h r o u g h o u t the c y t o p l a s m r e s u l t i n g i n a f a i r l y even d i s t r i b u t i o n of d i c t y o s o m e and ER p r o f i l e s . M i t o c h o n d r i a l p r o f i l e s a r e a l s o d i s t r i b u t e d t h r o u g h o u t the c y t o p l a s m and a p p e a r to be c l o s e l y a s s o c i a t e d w i t h the ER and d i c t y o s o m e s . C h l o r o p l a s t s r e m a i n p e r i p h e r a l l y p o s i t i o n e d w i t h t h e i r l o n g i t u d i n a l a x i s p a r a l l e l to the w a l l of the e x p a n d i n g p r o t r u s i o n . The most c o n s p i c u o u s f e a t u r e of t h i s s t a g e i s the r e s u m p t i o n of c y t o p l a s m i c v a c u o l a t i o n . The v a c u o l e s tend to o c c u p y a b a s a l p o s i t i o n w i t h i n the a x i a l mother c e l l . V a c u o l e s a p p e a r to be d e r i v e d from the s e q u e s t r a t i o n of c y t o p l a s m by smooth ER e l e m e n t s . T h i s i s f o l l o w e d by a d i l a t i o n and s e p a r a t i o n of the smooth ER membranes. Many v a c u o l e s o f t e n c o n t a i n a g r a n u l a r m a t r i x and membranous v e s i c l e s i n d i c a t i v e of a u t o p h a g i c v a c u o l e s ( M a t i l e 1 9 7 4 ) . They a l s o a p p e a r to f u s e w i t h one a n o t h e r c r e a t i n g l a r g e r v a c u o l e s . The p a t t e r n of via 1 1 l a y e r i n g i n s t a g e V r e m a i n s unchanged ( F i g . 1 9 ) from t h a t of s t a g e IV, a l t h o u g h t h e r e i s an o b v i o u s r u p t u r e i n the o u t e r w a l l l a y e r CWO of the bud 17 p r o t r u s i o n ( F i g . 2 0 ) . T h i s c o u l d be the r e s u l t of a c o n t i n -uous d e c r e a s e i n the t h i c k n e s s of t h i s o u t e r m o s t l a y e r w h i c h e v e n t u a l l y g i v e s way under the p r e s s u r e of the u n d e r l y i n g c y t o p l a s m . Hox^ever, i t s h o u l d be p o i n t e d out t h a t t h i s i s not a c o n s i s t e n t f e a t u r e of t h i s or s u b s e q u e n t s t a g e s . S tage VI : T h i s i s the s t a g e w h e r e i n n u c l e a r d i v i s i o n p r o b a b l y o c c u r s . The c o m p l e t e p r o c e s s of n u c l e a r d i v i s i o n was not r e s o l v e d . However, the p r e s e n c e of the c e n t r i o l e s a t o p p o s i t e p o l e s of the n u c l e u s ( F i g s . 21-24) s u g g e s t e d t h a t the c e n t r i o l a r r e p l i c a t i o n and m i g r a t i o n had o c c u r r e d . These e v e n t s a r e d i s t i n c t l y a s s o c i a t e d w i t h n u c l e a r d i v i s i o n . An e l e c t r o n dense m a t e r i a l l i e s between the base of the c e n t r i o l e s and the o u t e r membrane of the n u c l e a r e n v e l o p e ( F i g . 2 4 ) . T h i s m a t e r i a l a p p e a r s to a n c h o r the c e n t r i o l e to the n u c l e a r e n v e l o p e . E x t r a n u c l e a r m i c r o -t u b u l e s p o s i t i o n e d n e a r the c e n t r i o l e s and r a d i a t i n g towards the n u c l e a r e n v e l o p e can be o b s e r v e d a t t h i s time ( F i g . 2 4 ) . I t i s a l s o e v i d e n t t h a t the c h l o r o p l a s t s w i t h i n the b u d d i n g c e l l p o s s e s s a r e d u c e d number of t h y l a k o i d s compared to t h o s e of the a d j a c e n t q u i e s c e n t a x i a l c e l l s ( F i g . 2 5 ) . O t h e r w i s e , no o t h e r d i f f e r e n c e s can be d e t e c t e d i n r e l a t i o n to s t a g e V e x c e p t t h a t t h e r e a r e more v a c u o l e s w i t h i n the b a s a l p o r t i o n of the a x i a l mother c e l l . No o b v i o u s change can be d e t e c t e d i n the o r g a n i z a t i o n of the 18 c e l l w a l 1 . S t age V I I : T h i s s t a g e r e p r e s e n t s the f i n a l phase i n bud f o r m a t i o n . K a r y o k i n e s i s has a l r e a d y o c c u r r e d r e s u l t i n g i n the f o r m a t i o n of two d a u g h t e r n u c l e i , each c o n t a i n i n g a n u c l e o l u s t h a t i s c o n s i d e r a b l y s m a l l e r t h an the one o b s e r v e d i n the p r e v i o u s s t a g e . H e t e r o c h r o m a t i n i s a l s o v i s i b l e i n b o t h n u c l e i and s e r i a l s e c t i o n i n g r e v e a l s t h a t each n u c l e u s p o s s e s s e s a p a i r of c e n t r i o l e s l o c a t e d w i t h i n an i n p o c k e t i n g of the n u c l e a r e n v e l o p e ( F i g . 2 6 ) . The p r e s e n c e of a c r o s s w a l l between the two n u c l e i i n d i c a t e s t h a t c y t o k i n e s i s has a l s o o c c u r r e d . The d i s s i m i l a r i t y between the two d a u g h t e r c e l l s i s r e f l e c t e d i n t h e i r c y t o p l a s m i c o r g a n i z a t i o n . C e l l A c l o s e l y r e s e m b l e s a q u i e s c e n t a x i a l c e l l i n the f a c t t h a t i t i s h i g h l y v a c u o l a t e d and c o n s e q u e n t l y most of - i t s o r g a n e l l e s o c c u p y a p e r i p h e r a l p o s i t i o n ( F i g . 2 6 , A ) . C e l l 'B' l a c k s l a r g e v a c u o l e s and seems to p o s s e s s a l a r g e r amount of c y t o p l a s m i c o r g a n e l l e s ( F i g . 2 6 , B ) . O v e r a l l the s t r u c t u r a l o r g a n i z a t i o n of t h i s c e l l i s r e m i n i s c e n t of the m e r i s t e m a t i c c o n d i t i o n c h a r a c t e r i s t i c of the dominant a p i c a l c e l l of each f i l a m e n t . A c l o s e e x a m i n a t i o n of the c e l l w a l l s of the two d a u g h t e r c e l l s r e v e a l s t h a t o n l y the i n n e r m o s t w a l l l a y e r , CWI, of the bud c e l l i s c o n t i n u o u s w i t h c e l l w a l l l a y e r 4 (CW4) of the s i s t e r a x i a l c e l l ( F i g . 2 6 ) . L a y e r s CWl , CW2 and CW3 of the s i s t e r a x i a l c e l l e x t e n d o n l y s l i g h t l y 19 beyond the l e v e l of the c r o s s w a l l t h a t d e s i g n a t e s the bo u n d a r y between the two c e l l s . I t i s a p p a r e n t t h a t the newly formed c r o s s w a l l i s a l r e a d y t r a v e r s e d by numerous plasmodesmata and t h a t t h i s c r o s s w a l l r e p r e s e n t s the f i r s t t r a n s v e r s e w a l l of the f u t u r e b r a n c h a x i s ( F i g . 2 7 ) . 20 R e s u l t s : P a r t 2 C y t o c h e m i s t r y L i g h t m i c r o s c o p e C y t o c h e m i s t r y : In s t u d y i n g the d e v e l o p m e n t of a bud i n i t i a l from an a x i a l mother c e l l w i t h the l i g h t m i c r o s c o p e i t i s p o s s i b l e to d i s t i n g u i s h a l a y e r i n g p a t t e r n w i t h i n the o u t e r c e l l w a l l t h a t b e a r s a s t r i k i n g s i m i l a r i t y to t h a t o b s e r v e d w i t h the 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 e . E l e c t r o n m i c r o s c o p i c images s u g g e s t t h a t the l a y e r i n g p a t t e r n may r e f l e c t d i f f e r e n c e s i n c h e m i c a l c o m p o s i t i o n . I f t h i s i s the c a s e , t h e n t h i s h y p o t h e s i s c o u l d be t e s t e d u s i n g c y t o c h e m i c a l s t u d i e s . I n the c e l l w a l l of the a x i a l mother c e l l f o u r d i s t i n c t l a y e r s a r e p r e s e n t . From the o u t e r most to the i n n e r most l a y e r t h e y a r e d e s i g n a t e d CWl, CW2, CW3, and CW4. The l a y e r s CW3 and CW4 e x t e n d i n t o the c r o s s w a l l s of the a x i a l c e l l s w i t h CW3 assuming i n t h i s c a s e the p o s i t i o n of a ' m i d d l e l a m e l l a ' . In the w a l l of the bud i n i t i a l , o n l y two c e l l w a l l l a y e r s a r e e v i d e n t and t h e y a r e r e f e r r e d to as the o u t e r c e l l w a l l l a y e r , CWO, and the i n n e r c e l l x<rall l a y e r , CWl. The p o s i t i o n and o r i e n t a t i o n of a l l of the c e l l w a l l l a y e r s m e n t i o n e d a r e i l l u s t r a t e d i n F i g s . 2 8 - 3 1 . Based on a s e r i e s of c y t o c h e m i c a l t e s t s summarized i n T a b l e 1, i t a p p e a r s t h a t c e l l w a l l l a y e r s CWl, CW3 and CWO a r e composed m a i n l y of s u l f a t e d p o l y s a c c h a r i d e s and p r o t e i n , w i t h p e r h a p s a t r a c e of c e l l u l o s e i n CW3. Whereas CW2 , CW4 and CWl a p p e a r to c o n s i s t of b e t a l i n k e d a c i d i c p o l y s a c c h a r i d e s , w i t h some p r o t e i n i n CWl. I n brown a l g a e 21 s u l f a t e d p o l y s a c c h a r i d e s have been commonly termed ' f u c a n s ' and a c i d i c p o l y s a c c h a r i d e s ' a l g i n i c a c i d ' ( P e r c i v a l 1 9 7 9 ) . The d i s t r i b u t i o n of t h e s e two major p o l y s a c c h a r i d e s w i t h i n t h e c e l l w a l l of the d e v e l o p i n g bud i n i t i a l have been s c h e m a t i c a l l y p r e s e n t e d i n F i g s . 3 2 and 33. The r e s u l t s of the TBO and PAS s t a i n i n g p r o c e d u r e s (two of the most w i d e l y used s t a i n s i n brown a l g a l h i s t o l o g y ) a t f i r s t a p p e a r to c o n t r a d i c t t h i s i n t e r p r e t a t i o n . Red m e t a c h r o m a s i a p r o d u c e d by TBO i s s a i d to c h a r a c t e r i z e the p r e s e n c e of s u l f a t e d p o l y s a c c h a r i d e s and a p o s i t i v e r e a c t i o n w i t h PAS has been i n t e r p r e t e d as i n d i c a t i n g the p r e s e n c e of a l g i n i c a c i d ( M c C u l l y 1 9 7 0 ) . However, a c a r e f u l s t u d y of the l i t e r a t u r e r e v e a l s t h a t the i n t e r p r e t a t i o n of t h e s e s t a i n s must be made w i t h extreme c a u t i o n . Sample l i g h t m i c r o g r a p h s of TBO, PAS and some o t h e r s t a i n s a r e p r e s e n t e d i n F i g s . 3 4 - 3 7 . I n t e r p r e t a t i v e a s p e c t s of h i s t o l o g i c a l s t a i n i n g w i l l be d e a l t w i t h i n d e t a i l i n the d i s c u s s i o n . From e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s the o u t e r t h r e e c e l l w a l l l a y e r s CWl, CW2, and CW3 a p p e a r to c o n v e r g e upon the newly s y n t h e s i z e d o u t e r w a l l l a y e r CWO of the bud i n i t i a l . E v i d e n c e from the l i g h t m i c r o s c o p e r e v e a l s t h a t CWO i s n o t a c o n t i n u a t i o n of the o u t e r t h r e e c o n v e r g i n g c e l l w a l l l a y e r s of the b a s a l p o r t i o n of the bud mother c e l l . I n s t e a d i t seems to be s y n t h e s i z e d as a s u b t e n d i n g l a y e r t h a t assumes a p e r i p h e r a l p o s i t i o n i n the bud i n i t i a l c e l l w a l l a f t e r the c o n v e r g e n c e of the o u t e r t h r e e c e l l 22 w a l l l a y e r s of the a x i a l mother c e l l i s c o m p l e t e d . The i n n e r most c e l l w a l l l a y e r , CWI, of the bud i n i t i a l i s , on the o t h e r hand, an e x t e n s i o n of CW4 . However, CWI i s d e v e l o p e d i n the r e g i o n of the bud i n i t i a l to such an e x t e n t t h a t the o v e r a l l t h i c k n e s s of the two l a y e r e d c e l l w a l l of the bud p r o t r u s i o n r e m a i n s e q u i v a l e n t to t h a t of the f o u r l a y e r e d c e l l w a l l of the a x i a l mother c e l l . P o l a r i z e d l i g h t m i c r o s c o p y : P o l a r i z e d l i g h t r e v e a l e d the p r e s e n c e of b i r e f r i n g e n t l a y e r s w i t h i n the c e l l w a l l of b o t h the a x i a l mother c e l l and t h e bud i n i t i a l . T h i s b i r e f r i n g e n c e was c o n f i n e d to c e l l w a l l l a y e r s CW2 , CW4, and CWI ( s e e T a b l e 1) . C e l l w a l l e x t r a c t i o n s & C a l c o f l u o r f l u o r e s c e n c e : A t t e m p t s to e x t r a c t f u c a n s w i t h C a C ^ and a l g i n a t e s w i t h Na2CC>3 p r o d u c e d i n t e r e s t i n g r e s u l t s . O n l y e x t r a c t i o n s c a r r i e d out o v e r l o n g p e r i o d s of time a t h i g h t e m p e r a t u r e s p r o d u c e d s i g n i f i c a n t changes i n the s t a i n a b i l i t y of the c e l l w a l l . I t seems t h a t under t h e s e c o n d i t i o n s , C a C ^ removes b e t a -l i n k e d p o l y s a c c h a r i d e s ( a c c o r d i n g to the s t a i n i n g r e a c t i o n of the C a l c o f l u o r d y e ) , i . e . ' a l g i n a t e s ' , w h i l e Na^CO^ a p p e a r s to have no a f f e c t on t h e s e ' a l g i n a t e s ' ( s e e T a b l e 1) . 23 U l t r a s t r u e t u r a 1 l o c a l i z a t i o n o f enzyme a c t i v i t y  P e r o x i d a s e : My r e s u l t s s u g g e s t t h a t p e r o x i d a s e a c t i v i t y i s r e s t r i c t e d to the c e l l w a l l ( F i g s . 4 2 - 4 5 ) . The a p p e a r a n c e of e l e c t r o n dense r e a c t i o n p r o d u c t w i t h i n the m i t o c h o n d r i a ( F i g s . 42 and 45) i s most l i k e l y the r e s u l t of c y t o c h r o m e o x i d a s e a c t i v i t y ( A n g e r m u l l e r and F a h i m i 1981, O l a h and M u e l l e r 1 9 8 1 ) . C e l l w a l l p e r o x i d a s e a c t i v i t y i s a c o n s i s t e n t f e a t u r e of a l l a x i a l c e l l s and i s l o c a l i z e d i n c e l l w a l l l a y e r CWl, i n the m i d d l e l a m e l l a r e g i o n of the c r o s s w a l l s and i n s i d e t h e numerous plasmodesmata t h a t t r a v e r s e the c r o s s w a l l s ( F i g . 4 2 ) . However, the o n s e t of bud i n i t i a t i o n i s marked by the a p p e a r a n c e of p e r o x i d a s e a c t i v i t y i n a l l of the c e l l w a l l l a y e r s i n the r e g i o n of the bud p r o t r u s i o n ( F i g . 4 2 ) . The a c t i v i t y i s a l s o l o c a t e d beyond the r e g i o n of the bud p r o t r u s i o n i n c e l l w a l l l a y e r CW4 e x t e n d i n g i n t o the i n n e r most w a l l l a y e r of the e n t i r e a x i a l mother c e l l . Upon f u r t h e r e x p a n s i o n of the bud i n i t i a l , p e r o x i d a s e a c t i v i t y c a n s t i l l be d e t e c t e d w i t h i n the o u t e r m o s t c e l l w a l l l a y e r , CWO, and i t e x t e n d s p a r t i a l l y i n t o the o u t e r m o s t p o r t i o n of the s u b t e n d i n g c e l l w a l l l a y e r , CWl ( F i g . 4 3 ) . The r e m a i n i n g p o r t i o n of CWl g e n e r a l l y l a c k s p e r o x i d a s e a c t i v i t y but o c c a s s i o n a l y t h i n s u b - l a y e r s of r e a c t i o n p r o d u c t i n d i c a t i v e of p e r o x i d a s e a c t i v i t y can be d e t e c t e d ( F i g . 4 3 ) . The m i d d l e l a m e l l a of the c r o s s w a l l s e p a r a t i n g the bud c e l l from the s i s t e r a x i a l c e l l d i s p l a y s s i m i l a r p e r o x i d a s e a c t i v i t y to t h a t of the c r o s s w a l l s of the o t h e r a x i a l c e l l s ( F i g . 4 5 ) . F i g u r e 2 4 44 i s p r e s e n t e d as a c o n t r o l and d e m o n s t r a t e s the absence of p e r o x i d a s e a c t i v i t y when the m a t e r i a l was i n c u b a t e d i n the absence of s u b s t r a t e , i . e . E^C^. A d e n o s i n e t r i - p h o s p h a t a s e ( A T P a s e ) : ATPase a c t i v i t y a p p e a r s to be s t r i c t l y i n t r a c e l l u l a r . I t i s f r e q u e n t l y o b s e r v e d i n a s s o c i a t i o n w i t h the n u c l e a r e n v e l o p e , c h l o r o p l a s t e n v e l o p e and c h l o r o p l a s t e n d o p l a s m i c r e t i c u l u m , 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 s , and m i t o c h o n d r i a l c r i s t a e ( F i g s . 4 6 - 4 9 ) . The i n n e r s u r f a c e of the plasma membrane c o n s i s t e n t l y e x h i b i t s ATPase a c t i v i t y ( F i g . 4 8 ) . D u r i n g the p r o c e s s of bud i n i t i a t i o n and d e v e l o p m e n t some i n t r a c e l l u l a r r e - d i s t r i b u t i o n of ATPase a c t i v i t y seems to o c c u r . The n u c l e a r e n v e l o p e a p p e a r s to have more r e a c t i o n p r o d u c t d u r i n g the e a r l y s t a g e s of bud i n i t i a t i o n t h a n i n the l a t e r s t a g e s ( F i g . 4 6 ) and i t i s i n t e r e s t i n g to n o t e t h a t a t t h i s time t h e r e a r e many p e r i n u c l e a r d i c t y o s o m e s and e n d o p l a s m i c r e t i c u l u m p r o f i l e s w h i c h a l s o e x h i b i t the same i n t e n s e r e a c t i o n d e p o s i t s ( F i g s . 4 6 and 4 7 ) . No s i g n s of ATPase a c t i v i t y were o b s e r v e d w i t h i n the c e l l w a l l . C o n t r o l p r e p a r a t i o n s ( F i g s . 50 and 5 1 ) , i n w h i c h the s u b s t r a t e ATP was o m i t t e d or i n w h i c h the enzyme a c t i v i t y was d e s t r o y e d by h e a t , showed a d e f i n i t e d e c r e a s e i n r e a c t i o n p r o d u c t . A c i d p h o s p h a t a s e ( A c P a s e ) : AcPase a l s o seems to e x h i b i t an 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 . D e p o s i t i o n of r e a c t i o n p r o d u c t 25 i s c o n f i n e d to some c y t o p l a s m i c i n c l u s i o n s ( F i g s . 5 6 and 5 7 ) , the " c r i s t a e of m i t o c h o n d r i a ( F i g . 5 3 ) and p r i m a r i l y to the i n n e r s u r f a c e of the plasma membrane ( F i g s . 52 and 5 4 ) . T h i s d i s t r i b u t i o n p a t t e r n does n o t a p p e a r to change d u r i n g t h e c o u r s e of bud i n i t i a t i o n and d e v e l o p m e n t . No s i g n s of AcPase a c t i v i t y were d e t e c t e d w i t h i n the c e l l w a l l . I n the c o n t r o l p r e p a r a t i o n ( F i g . 5 5 ) sodium f l u o r i d e , an i n h i b i t o r of A c P a s e a c t i v i t y , was used and no AcPase a c t i v i t y was d e t e c t e d . 26 D i s c u s s i o n : P a r t 1 M o r p h o l o g y The g e n e r a l u 1 1 r a s t r u c t u r e of S p h a c e l a r i a f u r c i g e r a i s s i m i l a r to t h a t of S p h a c e l a r i a t r i b u l o i d e s ( G a l a t i s e t a l . 1977) and o t h e r brown a l g a e d e s c r i b e d i n the l i t e r a t u r e ( s e e R u s s e l l 1973 f o r a r e v i e w ) . T h e r e f o r e , I s h a l l r e s t r i c t my d i s c u s s i o n to t h o s e f e a t u r e s of S. f u r c i g e r a t h a t bear a more d i r e c t s i g n i f i c a n c e to the p r o c e s s of bud i n i t i a t i o n . Nuc l e u s : I t has been d e m o n s t r a t e d t h a t i n the moss Fun a r i a , s i d e b r a n c h f o r m a t i o n and o r i e n t a t i o n a r e i n f l u e n c e d by the p o s i t i o n o c c u p i e d by the n u c l e u s i n the a x i a l mother c e l l ( S c h m i e d e l and S c h n e p f 1979a, 1979b). My l i g h t m i c r o s c o p e o b s e r v a t i o n s , do not s u g g e s t a d i r e c t s p a t i a l r e l a t i o n s h i p between t h e p o s i t i o n of the n u c l e u s and the s i t e of the i n i t i a t i o n of the p r o t r u s i o n i n the a x i a l mother c e l l . The l o c a t i o n of the n u c l e u s r e m a i n s u n a l t e r e d u n t i l s t a g e IV a t w h i c h time i t o c c u p i e s a midway p o s i t i o n between the b a s a l and the p r o t r u d i n g p o r t i o n of the a x i a l mother c e l l of the bud i n i t i a l . T h i s type of b e h a v i o r has been o b s e r v e d d u r i n g t h e f o r m a t i o n of the p e r i a x i a l i n i t i a l i n the r e d a l g a Batrachospermum ( A g h a j a n i a n and Hommersand 1980) and i n the g l a n d i n i t i a l s o f the b l a d d e r w o r t U t r i c u l a r i a ( F i n e r a n 1 9 8 0 ) . I t i s g e n e r a l l y a c c e p t e d t h a t the o r i e n t a t i o n of the n u c l e u s and/or s p i n d l e i s d e t e r m i n e d a f t e r the n u c l e u s has 27 c o m p l e t e d i t s p r e - m i t o t i c m i g r a t i o n ( M i n e y u k i and F u r u y a 1 9 8 0 ) . My s t u d y s u p p o r t s t h i s view as the o r i g i n a l n u c l e a r o r i e n t a t i o n was m a i n t a i n e d d u r i n g t h e p r e - m i t o t i c m i g r a t i o n of the n u c l e u s i n t o the bud i n i t i a l . The f i n a l n u c l e a r o r i e n t a t i o n i n f l u e n c e s the o r i e n t a t i o n of the c e l l p l a t e ( M i n e y u k i and F u r u y a 1980) and i f t h i s i s the c a s e , t h e n the o r i e n t a t i o n of the c e l l p l a t e o b s e r v e d i n s t a g e V I I would i n d i c a t e t h a t the n u c l e a r o r i e n t a t i o n must have s h i f t e d by 90° j u s t p r i o r to k a r y o k i n e s i s . The b a s a l , r a t h e r t h a n l a t e r a l , p o s i t i o n of the c e n t r i o l e s i n the s u b t e n d i n g s i s t e r a x i a l c e l l ( C e l l 'A') s u g g e s t s t h a t such an e v e n t took p l a c e . The mechanism b e h i n d the m i g r a t i o n of the n u c l e u s i s n o t d e a l t w i t h i n our s t u d y , a l t h o u g h o t h e r a u t h o r s have s u g g e s t e d p r o t o p l a s m i c s t r e a m i n g ( B r a c k e r 1967, T r i n c i 1979) and/or the i n v o l v e m e n t of m i c r o t u b u l e s and m i c r o -f i l a m e n t s ( S c h n e p f and Heinzmann 1 980 , B a s s e l e t a l . 1981) as p r o b a b l e f a c t o r s . I t i s i n t e r e s t i n g to n o t e t h a t m i c r o -t u b u l e s were commonly o b s e r v e d to r u n between the c e n t r i o l e s and n u c l e a r e n v e l o p e i n S^ . f u r c i g e r a . Whether or n o t t h e y p l a y a r o l e i n n u c l e a r m i g r a t i o n i s unknown. However, a r e c e n t r e p o r t s u g g e s t s t h a t the a s s o c i a t i o n of the c e n t r i o l e s w i t h the n u c l e a r e n v e l o p e i s a l s o d e pendent upon the i n t e g r i t y of m i c r o t u b u l e s and m i c r o f i l a m e n t s (Maro and Bornens 1 9 8 0 ) . In S. f u r c i g e r a . the a s s o c i a t i o n of the c e n t r i o l e s w i t h the n u c l e a r e n v e l o p e r e s e m b l e s t h a t r e p o r t e d f o r o t h e r brown ( R u s s e l l 1973) and g r e e n a l g a e 28 ( A t k i n s o n e t a l . 1971, W i l s o n e t a l . 1 973 , C a c e r e s and R o b i n s o n 1 9 8 0 ) . Such an a s s o c i a t i o n may a s s i s t i n the p r o p e r p o s i t i o n i n g of the c e n t r i o l e s w i t h r e s p e c t to the n u c l e a r a x i s d u r i n g the r o t a t i o n of the n u c l e u s j u s t p r i o r to k a r y o k i n e s i s . T h i s i n t e r p r e t a t i o n becomes more a t t r a c t i v e w i t h the added o b s e r v a t i o n of an e l e c t r o n dense b r i d g i n g m a t e r i a l t h a t a p p e a r s to c o n n e c t the base of the c e n t r i o l e to the o u t e r membrane of the n u c l e a r e n v e l o p e . A s i m i l a r f i n d i n g was r e p o r t e d i n the fungus S a p r o l e g n i a , a f t e r the hyphae were g l y c e r i n a t e d d u r i n g the f i x a t i o n , a p r o c e d u r e w h i c h enhances the v i s i b i l i t y of the b r i d g i n g m a t e r i a l ( H e a t h 1 9 7 8 ) . The l a c k of r e p o r t s r e g a r d i n g t h i s e l e c t r o n dense b r i d g i n g m a t e r i a l s u g g e s t s t h a t i t i s a r a r i t y and/or a p o o r l y p r e s e r v e d s t r u c t u r e . A r e c e n t r e p o r t on the c e n t r i o l e - n u c l e u s a s s o c i a t i o n s u g g e s t e d t h a t the c e n t r i o l e was a n c h o r e d on the n o n - 1 i p o p r o t e i n p a r t of the n u c l e a r e n v e l o p e , however, the type of a t t a c h m e n t was n o t s p e c i f i e d (Maro and B o r e n s 1 9 8 0 ) . N u c l e a r d i v i s i o n t a k e s p l a c e most p r o b a b l y i n s t a g e V I . C o n s i s t e n t w i t h t h i s i n t e r p r e t a t i o n i s the o b s e r v e d d u p l i c a t i o n of the c e n t r i o l e s and t h e i r s u b s e q u e n t m i g r a t i o n to o p p o s i t e p o l e s of the n u c l e u s . A f t e r k a r y o k i n e s i s one s i s t e r n u c l e u s r e m a i n s w i t h i n the bud i n i t i a l w h i l e the o t h e r m i g r a t e s b a s i p e t a l l y . B a s i p e t a l m i g r a t i o n of the n u c l e u s has been r e p o r t e d i n e l o n g a t i n g f u n g a l hyphae ( T r i n c i 1979) and i n moss bud i n i t i a t i o n ( S c m i e d e l and S c h n e p f 1 9 7 9 a ) . F o l l o w i n g c y t o k i n e s i s each 29 d a u g h t e r c e l l p o s s e s s e s t h e r e f o r e i t s c o n t a i n i n g a n u c l e o l u s . The n u c l e o l i a l t h o u g h no s i g n i f i c a n t d i f f e r e n c e s a r e i n the n u c l e a r m o r p h o l o g y . C h l o r o p l a s t s and M i t o c h o n d r i a : The g e n e r a l t r e n d d u r i n g bud d e v e l o p m e n t i n f ur c i g e r a , i s the i n c r e a s e i n number of c h l o r o p l a s t s and m i t o c h o n d r i a d u r i n g the e a r l y s t a g e s of bud i n i t i a t i o n thus e n s u r i n g t h a t each d a u g h t e r c e l l r e c e i v e s a s u f f i c i e n t complement of t h e s e o r g a n e l l e s . C h l o r o p l a s t s and m i t o c h o n d r i a seem to m i g r a t e i n t o the bud p r o t r u s i o n p r i o r to the n u c l e a r m i g r a t i o n . How t h i s i s a c c o m p l i s h e d i s u n c l e a r and a g a i n p r o t o p l a s m i c s t r e a m i n g ( L a r s o n 1965, Uwate and L i n 1980) and/or m i c r o t u b u l e / m i c r o -f i l a m e n t i n v o l v e m e n t have been s u g g e s t e d ( A u f d e r h e i d e 1980, B r a d l e y and S a t i r 1980, T a y l o r and F u l l e r 1980, Uwate and L i n 1 9 8 0 ) . I f m i g r a t i o n o c c u r s t h r o u g h p r o t o p l a s m i c s t r e a m i n g , i t i s h a r d to i m a g i n e why the n u c l e u s does n o t s i m u l t a n e o u s l y m i g r a t e a l o n g w i t h the o t h e r o r g e n e l l e s . Even though d i f f e r e n t i a l t r a n s p o r t of v a r i o u s components i n the c y t o p l a s m i c s t r e a m i n g p r o c e s s has been o b s e r v e d i n g e r m i n a t i n g p o l l e n g r a i n s ( L a r s o n 1965), the o t h e r a l t e r n a t i v e seems more a t t r a c t i v e i n l i g h t of the r e c e n t work done on the mold Fu s ar ium accuminatum (Howard and A i s t 1 9 7 9 ) . Here the f r e e z e s u b s t i t u t i o n method r e v e a l e d the e x i s t e n c e of m i c r o t u b u l e s w i t h i n the h y p h a l apex t h a t a r e seldom p r e s e r v e d by r o u t i n e c h e m i c a l f i x a t i o n . I n a d d i t i o n , own n u c l e u s , each a r e r a t h e r s m a l l , o t h e r w i s e d e t e c t e d 30 m i t o c h o n d r i a were f r e q u e n t l y o b s e r v e d to be c l o s e l y a s s o c i a t e d w i t h m i c r o t u b u l e s (Howard and A i s t 1 9 7 9 ) . In S.  f u r c i g e r a , c y t o p l a s m i c m i c r o t u b u l e s , r a r e l y e n c o u n t e r e d , o n l y a p pear to r a d i a t e from t h e c e n t r i o l e s towards t h e n u c l e a r e n v e l o p e . Based on the r e s u l t s of F_j_ a c cumina turn i t i s unwise to r u l e out the p o s s i b i l i t y of m i c r o t u b u l e i n v o l v e m e n t i n the m i g r a t i o n of o r g a n e l l e s i n S_;_ f u r c i g e r a . Endomembrane s y s t e m : The endomembrane system combines II II it II c o n c e p t s of membrane f l o w and membrane d i f f e r e n t i a t i o n i n such a way t h a t the components of the system ( n u c l e a r e n v e l o p e , ER, G o l g i a p p a r a t u s , and v a r i o u s t r a n s i t i o n a l membrane e l e m e n t s ) a r e i n t i m a t e l y a s s o c i a t e d w i t h one a n o t h e r i n b o t h space and time ( M o r r e and M o l l e n h a u e r 1974, Morre e t a l . 1 97 9 ) . As the bud matures t h e r e i s i n c r e a s e d d e v e l o p m e n t of the endomembrane s y s t e m . Soon a f t e r the o n s e t of bud i n i t i a t i o n t h e r e i s an e x t e n s i v e p r o l i f e r a t i o n of ER such t h a t by s t a g e I I i t has a l r e a d y e x t e n d e d i t s e l f t h r o u g h o u t the c y t o p l a s m . Rough ER seems to p r e d o m i n a t e and i t i s w e l l known t h a t t h i s i s a c h a r a c t e r i s t i c f e a t u r e of c e l l s engaged i n the s y n t h e s i s of p r o t e i n f o r e x p o r t (De R o b e r t i s and De R o b e r t i s 1 9 8 0 ) . D i c t y o s o m e s a r e s c a r c e i n s t a g e I of bud d e v e l o p m e n t and t h e y e x h i b i t a p e r i n u c l e a r p o s i t i o n . V e s i c l e s a p p e a r to be p i n c h i n g o f f from the o u t e r n u c l e a r membrane and c o n t r i b u t i n g membrane m a t e r i a l to the f o r m i n g f a c e s of the a s s o c i a t e d d i c t y o s o m e s . S i m i l a r o b s e r v a t i o n s have been 31 r e p o r t e d i n o t h e r brown a l g a e ( C o l e 1969, R u s s e l l 1973, L o f t h o u s e and Capon 1975, G a l a t i s 1 9 7 7 ) . However, by s t a g e I I , t h e r e i s an i n c r e a s e i n the number of d i c t y o s o m e p r o f i l e s t h r o u g h o u t the c y t o p l a s m and t h i s p a r a l l e l s the d e v e l o p m e n t of the ER n e t w o r k . T h i s i s n o t s u p r i s i n g s i n c e numerous ER p r o f i l e s and a s s o c i a t e d t r a n s i t i o n v e s i c l e s were found a d j a c e n t to the d i c t y o s o m e s i n k e e p i n g w i t h the c o n c e p t t h a t d i c t y o s o m e s can a r i s e from the ER ( G r e e n 1969, M o r r e and M o l l e n h a u e r 1974 , M o r r e e t a l . 1979 , Uwate and L i n 1 980) . V a c u o l e s : The v a c u o l e s p r o v e d to be m o r p h o l o g i c a l l y the most dynamic o r g a n e l l e e n c o u n t e r e d d u r i n g bud i n i t i a t i o n . A t the e a r l i e s t s t a g e of bud i n i t i a t i o n the c y t o p l a s m a p p e a r s h i g h l y v a c u o l a t e d as i t does i n the q u i e s c e n t a x i a l c e l l s . W ith the i n c r e a s i n g e x p a n s i o n of the bud i n i t i a l the v a c u o l e s a ppear to s h r i n k and e v e n t u a l l y d i s a p p e a r by s t a g e I I I . I t was n o t e d t h a t , i n y e a s t c e l l s , the d u p l i c a t i o n of DNA o c c u r s when v a c u o l e s s h r i n k and f r a g m e n t t h e r e b y r e d u c i n g the o s m o t i c p r e s s u r e l e a d i n g to a n e t w a t e r l o s s w h i c h i n t u r n n e c e s s i t a t e s a r e d u c t i o n i n c e l l volume and an i n c r e a s e i n c y t o p l a s m i c d e n s i t y (Wiemken e t a l . 1 9 7 0 ) . I n t e r e s t i n g l y enough, d u r i n g the d e c r e a s e i n v a c u o l a t i o n e n c o u n t e r e d i n s t a g e I I and I I I of bud d e v e l o p m e n t i n j5. f u r c i g e r a , s e r i a l s e c t i o n i n g f a i l e d to r e v e a l any s i g n s of h e t e r o c h r o m a t i n w i t h i n the n u c l e u s ( s u p p o r t i v e of the i d e a t h a t DNA r e p l i c a t i o n i s o c c u r r i n g s i n c e h e t e r o c h r o m a t i n 32 d e c o n d e n s e s p r i o r to r e p l i c a t i o n ) and the c y t o p l a s m was v e r y d e n s e . Renewed v a c u o l a t i o n o c c u r s i n the b a s a l p o r t i o n of the a x i a l mother c e l l of the bud i n i t i a l a t s t a g e V and c o n t i n u e s u n t i l c y t o k i n e s i s . T h e r e f o r e , the s u b - t e n d i n g d a u g h t e r c e l l r e c e i v e s most of the v a c u o l e s and i t s c y t o p l a s m a p p e a r s l e s s e l e c t r o n dense than the bud i n i t i a l c e l l . A s i m i l a r s i t u a t i o n i s o b s e r v e d i n the b l a d d e r w o r t U t r i c u l a r i a d u r i n g the f o r m a t i o n of the g l a n d i n i t i a l ( F i n e r a n 1 9 8 0 ) . The c o n s t a n t l y c h a n g i n g v a c u o l a r p a t t e r n p r o b a b l y r e p r e s e n t s the m a n i p u l a t i o n of the m e c h a n i c a l d r i v i n g f o r c e b e h i n d the e x p a n s i o n of the bud i n i t i a l . T u r g o r p r e s s u r e , the r e s u l t of water b e i n g o s m o t i c a l l y a b s o r b e d i n t o the v a c u o l e (Wiebe 1978), has been i m p l i c a t e d as t h e d r i v i n g f o r c e b e h i n d p l a n t / f u n g a l e l o n g a t i o n ( B u l l e r 1958, Novotny and Forman 1975, P i c k e t t - H e a p s 1 9 7 7 ) . I t must be p o i n t e d out t h a t t u r g o r p r e s s u r e i s the same i n a l l d i r e c t i o n s and t h e d i r e c t i o n of growth t a k e n by the c e l l must r e s i d e i n the a n i s o t r o p i c ( u n e q u a l ) y i e l d i n g of the w a l l ( G r e e n 1962, Green 1980) C e l l W a l l : The r o l e of the c e l l w a l l i n bud e x p a n s i o n i s two f o l d ; i t must y i e l d to the d r i v i n g t u r g o r f o r c e and a t the same time 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 ( B a r t i n i c k i - G a r c i a and Lippman 1 972 , Bar t i n i c k i - G a r c i a 1973, Werz 1974, Novotny and Forman 1975, P i c k e t t - H e a p s 1977, Gooday 1979, Gooday and T r i n c i 1 9 8 0 ) . Such c o n d i t i o n s 33 of e l o n g a t i o n a p p e a r to be u n i v e r s a l among p l a n t s and f u n g i and i t a l s o seems to o p e r a t e w i t h i n f u r c i g e r a . A t the e a r l i e s t s i g n of bud i n i t i a t i o n the w a l l can be seen to p r o t r u d e ( y i e l d ) and t h e r e a p p e a r s to be a d e p o s i t i o n of new w a l l m a t e r i a l a d j a c e n t to the plasmalemma w h i c h h e l p s m a i n t a i n the s t r u c t u r a l i n t e g r i t y of the w a l l d u r i n g e x p a n s i o n . The mechanism b e h i n d the y i e l d i n g of the c e l l w a l l was n o t i n v e s t i g a t e d a t t h i s t i m e . However, l y s i s of e x i s t i n g c e l l w a l l components has been p o s t u l a t e d by o t h e r a u t h o r s ( B a r t i n i c k i - G a r c i a 1 973 , Werz 1 974 , B a l e t a l . 1 9 7 6 , T o t h 1 9 7 6 , F e v r e 1 9 7 7 , F e v r e 1 9 7 9 ) . The a p p e a r a n c e of d i s c o n t i n u i t i e s i n the p r o t r u d i n g c e l l w a l l t h r o u g h o u t bud i n i t i a t i o n . m i g h t be i n d i c a t i v e of a l y t i c e v e n t . The o r i g i n of the new c e l l w a l l m a t e r i a l a w a i t s f u r t h e r s t u d y , but o t h e r s t u d i e s on brown a l g a e have i m p l i c a t e d the i n v o l v e m e n t of the G o l g i a p p a r a t u s ( s e e R u s s e l l 1 9 7 3 f o r a r e v i e w ) . M o r p h o l o g i c a l l y , the c e l l w a l l of the e x p a n d i n g bud i n i t i a l i s of p a r t i c u l a r i n t e r e s t . In the e a r l y s t a g e s of bud i n i t i a t i o n , f o u r w e l l d e f i n e d w a l l l a y e r s e x i s t w i t h i n the o u t e r l o n g i t u d i n a l w a l l . However, as the bud p r o t r u s i o n d e v e l o p s i t becomes e v i d e n t t h a t the o u t e r 3 w a l l l a y e r s c o n v e r g e upon a new o u t e r w a l l l a y e r , CWO , and t h a t the i n n e r m o s t s u b - t e n d i n g l a y e r a c c e p t s newly s y n t h e s i z e d w a l l m a t e r i a l and t h i c k e n s i n such a way t h a t the o v e r a l l t h i c k n e s s of the w a l l of the p r o t r u s i o n r e m a i n s e q u i v a l e n t to t h a t of the the c e l l w a l l of the a d j a c e n t a x i a l c e l l s . 34 With r e s p e c t to the l o n g i t u d i n a l a x i s of the bud, the m i c r o f i b r i l l a r o r i e n t a t i o n of the w a l l l a y e r s CWl,CW2, and CW3 d u r i n g bud d e v e l o p m e n t g r a d u a l l y s h i f t from a t r a n s v e r s e to a l o n g i t u d i n a l d i r e c t i o n . T h i s i s i n agreement w i t h the. " m u l t i - n e t growth h y p o t h e s i s " c o n c e r n i n g c e l l w a l l e x p a n s i o n (Houwink and R o e l o f s e n 1 9 5 4 ) . One of the g e n e r a l l y a c c e p t e d a s s u m p t i o n s , of t h i s h y p o t h e s i s i s t h a t m i c r o f i b r i l s become p a s s i v e l y a l i g n e d i n the d i r e c t i o n of growth by s l i p p i n g p a s t one a n o t h e r (Houwink and R o e l o f s e n 1 9 5 4 , Green 1 9 6 9 , R o l a n d and V i a n 1 9 7 9 ) . As bud e x p a n s i o n c o n t i n u e s , the new i n n e r c e l l w a l l l a y e r , CWl, c o n s i s t s of o n l y l o n g i t u d i n a l l y o r i e n t e d m i c r o f i b r i l s . The o n l y p o s s i b l e e x p l a n a t i o n u s i n g the m u l t i - n e t growth h y p o t h e s i s i s t h a t new c e l l w a l l m a t e r i a l i s d e p o s i t e d i n random o r i e n t a t i o n a t the t i p and n o t a l o n g the t u b u l a r p o r t i o n of the p r o t r u s i o n . Then, as the bud i n i t i a l grows t h e r e i s a p a s s i v e r e - a l i g n m e n t i n the d i r e c t i o n of the l o n g i t u d i n a l a x i s of the bud. The e l e c t r o n m i c r o g r a p h s f a v o u r t h i s e x p l a n a t i o n and f u r t h e r s u p p o r t comes from the C a l c o f l u o r s t u d y which d e m o n s t r a t e s t h a t d e p o s i t i o n of new c e l l w a l l m a t e r i a l o c c u r s a t the t i p of the p r o t r u s i o n o n l y . T h i s c o n c e p t of c e l l w a l l e x p a n s i o n i s i n k e e p i n g w i t h the view t h a t t i p growth i s c h a r a c t e r i s t i c of many f i l a m e n t o u s a l g a e and f u n g i ( P i c k e t t - H e a p s 1 9 7 7 ) . 3 5 D i s c u s s i o n : P a r t 2 C y t o c h e m i s t r y L i g h t m i c r o s c o p e C y t o c h e m i s t r y : M o r p h o l o g i c a l and c y t o c h e m i c a l s t u d i e s of the c e l l w a l l and i t s c h e m i c a l b e h a v i o r d u r i n g bud i n i t i a t i o n c a r r i e d out w i t h the l i g h t m i c r o s c o p e have r e v e a l e d a d i s t i n c t i v e l a y e r e d p a t t e r n w i t h i n the o u t e r c e l l w a l l of b o t h the a x i a l mother c e l l and the p r o t r u d i n g bud i n i t i a l t h a t i s comparable to the l a y e r e d p a t t e r n o b s e r v e d w i t h the e l e c t r o n m i c r o s c o p e . C o r r e c t i n t e r p r e t a t i o n of the r e s u l t s h i n g e s on an a wareness of the l i m i t a t i o n s of each c y t o c h e m i c a l method employed and a c a r e f u l c o r r e l a t i o n of our c y t o c h e m i c a l r e s u l t s w i t h the e x i s t i n g b i o c h e m i c a l and m o r p h o l o g i c a l d a t a on brown a l g a l c e l l w a l l s . Two main p o l y s a c c h a r i d e s o c c u r i n the c e l l w a l l s of brown a l g a l t i s s u e s ; a) p o l y u r o n i d e a l g i n i c a c i d composed of c o n t i g u o u s b l o c k s of a l p h a 1 , 4 - l i n k e d L - g u l u r o n i c a c i d and b e t a 1 , 4 - l i n k e d D-manuronic a c i d b) f u c a n s ( f u c o i d i n , f u c o i d a n , a s c o p h y l l a n , s a r g a s s a n , and g l u c u r o n o x y l o f u c a n ) c o m p r i s i n g a f a m i l y of p o l y d i s p e r s e h e t e r o m o l e c u 1 e s c o n t a i n i n g i n a d d i t i o n to f u c o s e , v a r y i n g p r o p o r t i o n s of g a l a c t o s e , mannose, x y l o s e and g l u c u r o n i c a c i d ( P e r c i v a l 1 9 7 9 ) . Fucans can c o n t a i n from 3-20% u r o n i c a c i d ( P e r c i v a l 1 9 7 9 ) . D i f f e r e n t brown a l g a e c o n t a i n c h e m i c a l l y d i f f e r e n t a l g i n a t e s (Haug e t a l . 1967) and the age of the t i s s u e i s known to a f f e c t the r a t i o of m a n u r o n i c a c i d to g u l u r o n i c a c i d w i t h i n the a l g i n i c a c i d p o lymer ( P e r c i v a l 1 9 7 9 ) . A l l 36 brown a l g a e c o n t a i n a wide range of s u l f a t e d p o l y s a c c h a r i d e s ( E v a n s and C a l l o w 1972) and the d e g r e e to w h i c h an a l g a i s exposed to the a i r has a d i r e c t b e a r i n g on the amount of s u l f a t e d p o l y s a c c h a r i d e m a t e r i a l p r e s e n t , s i n c e i t i s t h o u g h t t h a t i t s h y g r o s c o p i c n a t u r e s e r v e s to r e d u c e d e s i c c a t i o n d u r i n g e x p o s u r e to a i r ( L e s t a n g and Q u i l l e t 1 9 7 4 ) . Thus, i t i s a p p a r e n t t h a t brown a l g a l c e l l w a l l s a r e not o n l y b i o c h e m i c a l l y d i v e r s i f i e d but a l s o a r e s u b j e c t e d to e n v i r o n m e n t a l a l t e r a t i o n s . Add t h i s to the i m p r e c i s e n a t u r e of h i s t o l o g i c a l s t a i n i n g and i t i s no wonder t h a t a v a r i e t y of i n t e r p r e t a t i o n s can be a p p l i e d to any one s e t of h i s t o l o g i c a l r e s u l t s . The r e s u l t s of the two c l a s s i c a l s t a i n s , TBO and PAS, used i n brown a l g a l h i s t o l o g y a r e p o i n t s i n q u e s t i o n . I f the r e s u l t s of t h e s e two s t a i n s a r e examined a t the e x c l u s i o n of o t h e r h i s t o l o g i c a l s t a i n s one m i g h t i n t e r p r e t the r e s u l t s of the s t a i n s to i n d i c a t e t h a t CWl, CW3, and CWO c o n s i s t of a c i d i c p o l y s a c c h a r i d e s ( p u r p l e m e t a c h r o m a s i a w i t h TBO ( M c C u l l y 1965, 1968, T o t h 1976) and a p o s i t i v e r e a c t i o n w i t h PAS ( M c C u l l y 1 965 , 1 968) whereas CW2 , CW4, and CWl c o n s i s t of s u l f a t e d p o l y s a c c h a r i d e s ( p i n k m e t a c h r o m a s i a w i t h TBO ( M c C u l l y 1965, 1968, T o t h 1976) and a r e v e r y p o o r l y , i f a t a l l , PAS p o s i t i v e ( M c C u l l y 1965, 1 9 6 8 ) . However, the a p p l i c a t i o n of o t h e r h i s t o l o g i c a l t e c h n i q u e s s u g g e s t j u s t the o p p o s i t e ( s e e T a b l e 1 ) . T h i s prompted a more t h o r o u g h i n v e s t i g a t i o n i n t o the l i t e r a t u r e r e g a r d i n g the n a t u r e of the TBO and PAS s t a i n i n g r e a c t i o n s 37 and t h e i r s i g n i f i c a n c e . TBO i s a t h i a z i n dye e x h i b i t i n g m e t a c h r o m a s i a w i t h h i g h m o l e c u l a r w e i g h t compounds h a v i n g f r e e c a r b o x y l , s u l f a t e and p h o s p h a t e groups ( M c C u l l y 1966, Cook 1977). M e t a c h r o m a s i a e x i s t s i n t h r e e f o r m s , a l p h a ( o r t h o c h r o m a t i c b l u e ) , b e t a ( p u r p l e ) , and gamma ( r e d ) (Cook 1977). M e t a c h r o m a s i a i s t h o u g h t to o c c u r t h r o u g h the p o l y m e r i z a t i o n of monomeric u n i t s of the dye when the n e g a t i v e c h a r g e s of the c h r o m o t r o p e ( e . g . a c i d i c and s u l f a t e d p o l y s a c c h a r i d e s ) a t t r a c t s u f f i c i e n t p o s i t i v e l y -c h a r g e d p o l a r g r o u p s on the dye to cause a g g r e g a t e s to form and p o l y m e r i z e (Cook 1977). T h i s r e s u l t s i n a c o l o u r s h i f t from b l u e upwards towards r e d (Cook 1977). M e t a c h r o m a s i a i s i n c o n s i s t e n t and i s i n f l u e n c e d by many f a c t o r s ( L i s o n 1936, Cook 1977). The r e s u l t s o b t a i n e d w i t h the v a r i o u s TBO methods p r e s e n t e d i n t h i s paper ( s e e T a b l e 1) s u g g e s t t h a t s u l f a t e d p o l y s a c c h a r i d e s a r e s t a i n i n g p u r p l e and p i n k a t pH 0.5 (as d e t e r m i n e d by the use of low pH a t which p o i n t c a r b o x y l g r o u p s a r e not supposed to be i o n i z e d ( M c C u l l y 1970, Novotny and Forman 1975) and c a r b o x y l g r o u p s p i n k to r e d a t pH 6.8. I t i s o f t e n r e p o r t e d i n the l i t e r a t u r e t h a t r e d / p i n k m e t a c h r o m a s i a i s c h a r a c t e r i s t i c of s u l f a t e d groups ( L i s o n 1936, M c C u l l y 1 9 6 5 ) . However, i t i s w e l l known t h a t s u l f a t e d p o l y s a c c h a r i d e s a r e found i n a n i m a l s and non-t e r r e s t r i a l p l a n t s but a r e a b s e n t from t e r r e s t r i a l p l a n t s ( M c C a n d l e s s 1979, P e r c i v a l 1 9 7 9 ) . Y e t r e d m e t a c h r o m a s i a u s i n g TBO was d e m o n s t r a t e d i n s i e v e t ubes and companion 38 c e l l s of h i g h e r p l a n t s ( O ' B r i e n e t a l . 1964), as was p i n k m e t a c h r o m a s i a i n P i n g u i c u l a , a genus of c a r n i v o r o u s p l a n t s ( H e s l o p - H a r r i s o n and H e s l o p - H a r r i s o n 1 9 8 1 ) . I t becomes a p p a r e n t , t h e r e f o r e , t h a t c a u t i o n s h o u l d be e x e r c i s e d when as s u m i n g the p r e s e n c e of s u l f a t e d p o l y s a c c h a r i d e s based on TBO r e d / p i n k m e t a c h r o m a s i a . I n f a c t , the m e t a c h r o m a t i c c o l o u r o b s e r v e d a p p e a r s to be a f u n c t i o n of the m o l e c u l a r w e i g h t and/or n e g a t i v e c h a r g e of the c h r o m o t r o p e and n o t h i n g more. Thus I f e e l t h a t my r e s u l t s w i t h TBO m e t a c h r o m a s i a a r e , i n s t e a d , i n p e r f e c t agreement w i t h t h o s e u s i n g the o t h e r h i s t o l o g i c a l t e c h n i q u e s shown i n T a b l e 1 w h i c h s u g g e s t t h a t c e l l w a l l l a y e r s CWI, CW3, and CWO a r e composed p r i m a r i l y of s u l f a t e d p o l y s a c c h a r i d e s w h i l e CW2 , CW4, and CWI a r e p r i m a r i l y a c i d i c p o l y s a c c h a r i d e s . One f i n a l p o i n t c o n c e r n i n g TBO s t a i n i n g i s the q u e s t i o n of the c o n c e n t r a t i o n of the dye and the d u r a t i o n of the s t a i n i n g p r o c e d u r e . T h e r e a r e q u e s t i o n s to be asked s u c h as, a t what p o i n t does the s t a i n become n o n - s p e c i f i c , i . e . when i s the s p e c i m e n o v e r - s t a i n e d ? A 1:10,000 to 1:50,000 d i l u t i o n was o r i g i n a l l y recommended f o r TBO ( L i s o n 1 9 3 6 ) , but no time i n t e r v a l f o r s t a i n i n g g i v e n . C u r r e n t l y p h y c o l o g i s t s a r e commonly u s i n g a 1:2,000 d i l u t i o n f o r one m i n u t e . Our s t u d i e s i n d i c a t e t h a t t h i s e l e v a t e d c o n c e n t r a t i o n of 1:2,000 f o r one m i n u t e p r o d u c e s an o v e r -s t a i n e d s p e c i m e n . T h i s i s r e c o g n i z e d by the l o s s of f i n e d e t a i l and s t a i n i n g s p e c i f i c i t y . How t h e n s h o u l d one c a r r y out TBO s t a i n i n g ? VJe recommend t h a t a range of dye 39 c o n c e n t r a t i o n s and d u r a t i o n s of s t a i n i n g be p e r f o r m e d and c o m p a r i s o n s be made w i t h o t h e r r e s u l t s o b t a i n e d w i t h a wide v a r i e t y of o t h e r h i s t o l o g i c a l t e c h n i q u e s . In t h i s way the c o r r e c t c o n c e n t r a t i o n of the dye and the s t a i n i n g time can be b e s t a s s e s s e d . I n our case a c o n c e n t r a t i o n of 1:10,000 and a s t a i n i n g time of t h i r t y s e c o n d s seemed to g i v e the b e s t r e s u l t s . The p r i n c i p l e b e h i n d the PAS r e a c t i o n i s t h a t p e r i o d i c a c i d w i l l b r i n g about o x i d a t i v e c l e a v a g e of the c a r b o n to c a r b o n bond i n 1 , 2 - g l y c o l s or t h e i r amino or a l k y l a m i n o d e r i v a t i v e s to form d i a l d e h y d e s . These a l d e h y d e s w i l l r e a c t w i t h f u c h s i n - s u l f u r o u s a c i d , w hich combines w i t h the b a s i c p a r a r o s a n i 1 i n e to form a magenta c o l o u r e d compound (Cook 1977 ) . I t has been r e p o r t e d t h a t a l g i n i c a c i d i s PAS p o s i t i v e ( M c C u l l y 1 9 7 0 ) . However, a d d i t i o n a l r e p o r t s e x i s t w h i c h s t a t e t h a t the PAS t e c h n i q u e i s a s e n s i t i v e and s p e c i f i c t e s t f o r a group of compounds r e f e r r e d to as c a r b o h y d r a t e - p r o t e i n complexes w h i c h always c o n t a i n g a l a c t o s e and f u c o s e , as w e l l as hexosamine and p r o b a b l y s i a l i c a c i d , o f t e n mannose, and r a r e l y g l u c o s e ( L e b l o n d e t a l . 1 9 5 7 ) . The d e g r e e of r e a c t i v i t y depends on the amount of c a r b o h y d r a t e - p r o t e i n complex and the number of m o n o s a c c h a r i d e u n i t s combined so as to l e a v e 1 , 2 - g l y c o l g r o u p s f r e e to r e a c t . I n t e r e s t i n g l y enough, f u c a n s a r e known to c o n t a i n p r o t e i n , g a l a c t o s e and f u c o s e ( P e r c i v a l 1979) and thus s h o u l d be PAS p o s i t i v e . One s t u d y r e v e a l e d t h a t p e r i o d a t e i s p o o r l y r e d u c e d i n the p r e s e n c e of a l g i n i c 40 a c i d (Druramond e t a l . 196 2 ) , w h i l e a n o t h e r s t u d y d e m o n s t r a t e d t h a t p e r i o d a t e c o u l d r e a c t w i t h e v e r y t h i r d and f o u r t h f u c o s e u n i t i n the f u c o i d i n e x t r a c t e d from Fucus  ves i c u l o s u s ( C o n c h i e and P e r c i v a l 1 9 5 0 ) . I t i s a p p a r e n t t h a t the o n l y s a f e a s s u m p t i o n t h a t can be made w i t h r e s p e c t to a p o s i t i v e PAS r e a c t i o n i s the p r e s e n c e of 1,2- g l y c o l g r o u p s . I n t h e o r y and p r a c t i c e , b o t h a c i d i c and s u l f a t e d p o l y s a c c h a r i d e s of brown a l g a e can and do meet t h i s c r i t e r i u m ( C o n c h i e and P e r c i v a l 1 950 , L e b l o n d e t a l . 1 957 , F e d e r and O ' B r i e n 1 9 6 8 ) . T h e r e f o r e , the s i g n i f i c a n c e of a PAS p o s i t i v e r e a c t i o n s h o u l d n o t be i n t e r p r e t e d as a n y t h i n g more t h a n the s i m p l e d e m o n s t r a t i o n of 1 , 2 - g l y c o l g r o u p s . F i n a l l y , the p r e s e n c e of p r o t e i n w i t h i n the " f u c a n " l a y e r s , as r e v e a l e d by h i s t o l o g i c a l s t a i n i n g , i s not u n u s u a l ( L a r s e n e t a l . 1966, Evans and C a l l o w 1972, M e d c a l f 1977 ) and i t may s e r v e as a s t r u c t u r a l l i n k a g e among the c a r b o h y d r a t e s ( M e d c a l f 1 9 7 7 ) . However, a d d i t i o n a l p r o t e i n s u b - l a y e r s were o b s e r v e d i n the i n n e r a l g i n a t e w a l l l a y e r of the bud i n i t i a l . These p r o t e i n s u b - l a y e r s may p l a y a s t r u c t u r a l r o l e but t h e i r abundance and r e s t r i c t i o n t o the bud i n i t i a l c e l l w a l l s u g g e s t s o t h e r w i s e . I t i s more l i k e l y t h a t t h e s e p r o t e i n s u b - l a y e r s a r e e n z y m a t i c i n n a t u r e . The c y t o c h e m i c a l d e m o n s t r a t i o n of i n t e n s e p e r o x i d a s e a c t i v i t y i n the c e l l w a l l of the bud i n i t i a l l e n d s s u p p o r t to t h i s h y p o t h e s i s . P o l a r i z e d l i g h t m i c r o s c o p y : T h i s t e c h n i q u e f a s c i l i t a t e s 41 the l o c a l i z a t i o n of p o l y s a c c h a r i d e s t h a t p o s s e s s b i r e f r i n g e n t p r o p e r t i e s ( M c C u l l y 1 9 7 0 ) . A l g i n i c a c i d and c e l l u l o s e a r e known to be b i r e f r i n g e n t ( A n d e r s e n 1 9 5 6 ) . B i r e f r i n g e n c e i n our s t u d y was c o n f i n e d to c e l l w a l l l a y e r s CWI, CW4, and CWI. The r e s u l t s of the o t h e r h i s t o l o g i c a l t e c h n i q u e s employed s u g g e s t t h a t t h e s e t h r e e c e l l w a l l l a y e r s a r e composed of b e t a - l i n k e d a c i d i c p o l y s a c c h a r i d e s but n o t c e l l u l o s e . Thus i t would a p p e a r t h a t b i r e f r i n g e n c e c o n f i r m s the c h e m i c a l n a t u r e of t h e s e t h r e e c e l l w a l l l a y e r s and s u g g e s t s t h a t ' a l g i n i c a c i d ' i s the major c h e m i c a l component. I n d e e d , A n d e r s e n (1956) r e p o r t s t h a t a l g i n i c a c i d b i r e f r i n g e n c e was found i n the 'composed m i d d l e l a m e l l a ' and t h i s i s i n agreement w i t h our r e s u l t s . C e l l w a l l e x t r a c t i o n s & C a l c o f l u o r f l u o r e s c e n c e : The r e s u l t s of the c e l l wal 1 e x t r a c t i o n s s u g g e s t t h a t a 1% C a C ^ s o l u t i o n a t 60°C f o r 18 hou r s e x t r a c t s b e t a - l i n k e d p o l y s a c c h a r i d e s whereas a 1% N a 2 C 0 3 s o l u t i o n under the same c o n d i t i o n s has no a f f e c t . T h i s i s c o n t r a d i c t o r y to the e x p e c t e d r e s u l t s s i n c e C a C l 2 i s supposed to i n h i b i t the e x t r a c t i o n of a l g i n a t e s and a l g i n i c a c i d i s known to be a l k a l i n e s o l u b l e . I t i s p o s s i b l e t h a t C a l c o f l u o r does n o t s t a i n a l g i n i c a c i d but r a t h e r f u c a n s . T h i s i s , however, u n l i k e l y s i n c e the s p e c i f i c i t y of C a l c o f l u o r has been e x t e n s i v e l y i n v e s t i g a t e d and i t was d e m o n s t r a t e d to r e a c t w i t h b e t a 1,4- and b e t a 1 , 3 - l i n k e d p o l y s a c c h a r i d e s and n o t a l p h a - l i n k e d p o l y s a c c h a r i d e s as o c c u r i n f u c a n s ( H a i g l e r e t 42 a l . 1980, Wood 19 8 0 ) . Thus i t seems t h a t the e x t r a c t i o n d a t a a r e i n c o n f l i c t w i t h the r e s t of the r e s u l t s , but t h i s i s n o t r e a l l y s u p r i s i n g s i n c e i t i s known t h a t t h e s e e x t r a c t i o n s a r e not p r e c i s e and t h a t i t i s n o t uncommon to f i n d t h a t 7-8% of the e x t r a c t i o n i s n o n - s p e c i f i c ( M c C u l l y 1970, Novotny and Forman 1 9 7 5 ) . In a d d i t i o n , t h e s e e x t r a c t i o n t e c h n i q u e s have been a d a p t e d from t h o s e used on f r e s h , l i v e m a t e r i a l and the a f f e c t s of f i x a t i o n and embedment i n p l a s t i c s upon the e x t r a c t i o n t e c h n i q u e s have n o t been t h o r o u g h l y i n v e s t i g a t e d . 43 U 1 1 r a s t r u e t u r a 1 l o c a l i z a t i o n of enzyme a c t i v i t y  P e r o x i d a s e : I n t r a c e l l u l a r p e r o x i d a s e a c t i v i t y was n o t d e t e c t e d i n t h i s s t u d y . T h i s may r e s u l t from the f a c t t h a t i s o z y m e s of p e r o x i d a s e have d i f f e r e n t s u b s t r a t e s p e c i f i c i t i e s , and d i f f e r e n t pH and t e m p e r a t u r e o p t i m a ( K l i s u r s k a and Dencheva 1980) and t h e r e f o r e c y t o c h e m i c a l t e c h n i q u e s may not be s e n s i t i v e enough to d e t e c t a l l of them. P e r o x i d a s e a c t i v i t y was o b s e r v e d i n the c e l l w a l l and p lasmode sma t a of the a x i a l mother c e l l of S. f u r c i g e r a and i t a p p e a r e d to be p a r t i c u l a r l y i n t e n s e i n the c e l l w a l l of the bud p r o t r u s i o n . T h i s i s n o t s u p r i s i n g i n l i g h t of the e x i s t i n g r e p o r t s on p e r o x i d a s e l o c a l i z a t i o n w i t h i n the c e l l w a l l of p l a n t s from v a r i o u s t a x o n o m i c groups ( H a l l and S e x t o n 1972, Yung and N o r t h c o t e 1975, F r y 1979, Henry 1979, Benayoun e t a l . 1 9 8 1 ) . A t the o n s e t of bud i n i t i a t i o n , the e x i s t i n g o u t e r c e l l w a l l of the a x i a l mother c e l l must s o f t e n and become more e x t e n s i b l e . D e p o s i t i o n of new c e l l w a l l m a t e r i a l a l l o w s f o r the c o n t i n u e d e x p a n s i o n of the bud i n i t i a l . The i n t e n s e p e r o x i d a s e a c t i v i t y e n c o u n t e r e d a t the o n s e t of bud i n i t i a t i o n i n the r e g i o n of the c e l l w a l l p r o t r u s i o n i n d i c a t e s t h a t p e r o x i d a s e may be i n v o l v e d i n c e l l w a l l l o o s e n i n g / l y s i s as was s u g g e s t e d f o r the p e r o x i d a s e a c t i v i t y i n the a b s c i s s i o n zone t i s s u e of t o b a c c o (Henry 1 9 7 9 ) . A d e c r e a s e i n c e l l w a l l p e r o x i d a s e a c t i v i t y o c c u r s as the bud p r o t r u s i o n c o n t i n u e s to expand r e s u l t i n g i n the p r o g r e s s i v e r e s t r i c t i o n of p e r o x i d a s e 44 a c t i v i t y to a t h i n o u t e r m o s t w a l l l a y e r , CWO, of the bud i n i t i a l c e l l w a l l . T h i s l e n d s s u p p o r t to the p r e v i o u s s u g g e s t i o n t h a t p e r o x i d a s e a c t i v i t y i s p r i m a r i l y i n v o l v e d i n c e l l w a l l l o o s e n i n g i n r e s p o n s e to c e l l e x p a n s i o n and i t a l s o i n d i r e c t l y s u p p o r t s the m u l t i - n e t growth h y p o t h e s i s . T h i s h y p o t h e s i s s t a t e s t h a t the newly d e p o s i t e d m i c r o f i b r i l s become p a s s i v e l y a l i g n e d i n the d i r e c t i o n of growth (Houwink and R o e l o f s o n 1 9 5 4 ) . C o n v e r s e l y , the newly d e p o s i t e d m i c r o f i b r i l s a r e not s u b j e c t e d to a c t i v e c e l l w a l l l o o s e n i n g a g e n t s , e.g.: p e r o x i d a s e . T h e r e f o r e , i t i s n o t s u p r i s i n g to o b s e r v e a l a c k of p e r o x i d a s e a c t i v i t y i n the newly d e p o s i t e d c e l l w a l l l a y e r CWl of the bud i n i t i a l . The f a c t t h a t p e r o x i d a s e a c t i v i t y i s always a s s o c i a t e d w i t h the o u t e r m o s t w a l l l a y e r s , CWl and CWO, may a l s o r e f l e c t an a n t i - m i c r o b i a l a c t i v i t y as p o s t u l a t e d f o r the g r e e n a l g a e ( M e n z e l 1980) and the b l i g h t r e s i s t a n t c o t t o n ( V e n e r e 1 9 8 0 ) . A T P a s e : "ATPase a c t i v i t y i s a s s o c i a t e d w i t h a number of d i f f e r e n t p h y s i o l o g i c a l p r o c e s s e s i n b o t h p l a n t and a n i m a l c e l l s " ( H a l l 1 9 7 1 ) . These i n c l u d e i o n t r a n s p o r t ( H a l l 1971, G i l d e r and Cronshaw 1973, Bentwood and Cronshaw 1 9 7 6 ) , n u c l e i c a c i d m e t a b o l i s m ( H a l l 1 971), o x i d a t i v e p h o s p h o r y l a t i o n ( H a l l 1971) and c e l l w a l l s y n t h e s i s (Coupe and D'Auzac 1 9 8 0 ) . ATPase a c t i v i t y has been l o c a l i z e d i n the plasma membrane, n u c l e u s , m i t o c h o n d r i a , c h o l o r o p l a s t s , l y s o s o m e s , d i c t y o s o m e s , e n d o p l a s m i c r e t i c u l u m , d e v e l o p i n g 45 v a c u o l e s , p l a smod e sma t a and the c e l l w a l l ( H a l l 1971, G i l d e r and Cronshaw 1973, Wheeler e t a l . 1979, Coupe and D'Auzac 1980). The p r o c e s s of bud i n i t i a t i o n and d e v e l o p m e n t i n _S. f ur c i g e r a i n v o l v e s r e - o r g a n i z a t i o n a l changes of the n u c l e u s , endomembrane system and c e l l w a l l . These s t r u c t u r e s a r e t h e r e f o r e l i k e l y p l a c e s f o r ATPase a c t i v i t y . ' Our r e s u l t s i n d i c a t e t h a t ATPase a c t i v i t y f u n c t i o n s i n t r a c e 1 l u l a r l y and i t s d i s t r i b u t i o n p a t t e r n a p p e a r s u n a l t e r e d d u r i n g bud i n i t i a t i o n and d e v e l o p m e n t . However, ATPase a c t i v i t y i s found c o n s i s t e n t l y a d j a c e n t to the plasma membrane r e g a r d l e s s of the d e v e l o p m e n t a l s t a g e of the bud i n i t i a l . T h i s i s not s u p r i s i n g i n l i g h t of i t s known r o l e i n i o n t r a n s p o r t ( H a l l 1971, G i l d e r and Cronshaw 1973, Bentwood and Cronshaw 1 9 7 6 ) . Ion t r a n s p o r t i s an i m p o r t a n t b i o l o g i c a l phenomenon which s e r v e s to m a i n t a i n the pH and o s m o t i c p r e s s u r e i n the c e l l and w h i c h a c t i v a t e s a number of enzymes and r e g u l a t e s the exchange and a b s o r p t i o n of some s u b s t a n c e s (Ban 1 9 8 0 ) . F u r t h e r m o r e , the plasma membrane i s a l s o known to f u n c t i o n as an a s s e m b l y s i t e f o r w a l l m i c r o f i b r i l s u s i n g m u l t i - e n z y m e complexes ( R o l a n d 1973 , van der Woude e t a l . 1974 , R o l a n d and V i a n 1979, Young 1980) and a r e c e n t r e p o r t s u g g e s t s t h a t ATPase may be i n v o l v e d i n c e l l w a l l growth and s y n t h e s i s (Coupe and D'Auzac 1980). Thus t h i s may e x p l a i n the r a t h e r h i g h ATPase a c t i v i t y found a l o n g the plasma membrane d u r i n g the p r o c e s s of bud i n i t i a t i o n and d e v e l o p m e n t . 46 A c P a s e : The r o l e of p l a n t A c P a s e s r e m a i n s u n c l e a r (Bentwood and Cronshaw 1 9 7 6 ) . I t i s known t h a t AcPase h y d r o l y s e s a v a r i e t y of o r t h o p h o s p h a t e e s t e r s (Akiyama and S u z u k i 1980) and t h a t a d e f i c i e n c y i n i n o r g a n i c p h o s p h a t e l e a d s to i n c r e a s e d AcPase a c t i v i t y (Blum 1965, Sommer and Blum 1 96 5 , C a l l e j a e t a l . 1 9 8 0 ) . T h e r e a r e a l s o r e p o r t s of c e l l w a l l d e g r a d a t i o n and a s s o c i a t e d AcPase a c t i v i t y ( H a l p e r i n 1969, G i o r d a n i 1 9 8 0 ) . R e c e n t l y , AcPase was i s o l a t e d from sycamore c e l l w a l l s and i t was p o s t u l a t e d t h a t t h i s enzyme m i g h t p l a y a r o l e i n the l o c a l h y d r o l y s i s of c e l l w a l l p o l y s a c c h a r i d e s w h i c h would a s s i s t i n c e l l w a l l e x t e n s i o n ( C r a s n i e r 1 9 8 0 ) . AcPase a c t i v i t y was not d e t e c t e d i n the c e l l w a l l s of S_. f u r c i g e r a . However, f a i l u r e to d e t e c t a r e a c t i o n p r o d u c t i n the c e l l w a l l s h o u l d n o t be t a k e n to i m p l y the absence of AcPase a c t i v i t y . G l u t a r a l d e h y d e f i x a t i o n i s known to b l o c k AcPase a c t i v i t y i n the c e l l w a l l s of t o b a c c o c e l l s (De Jong e t a l . 1967) and r e c e n t l y i t was shown t h a t d i f f e r e n t s u b s t r a t e s can d i s t i n g u i s h between d i f f e r e n t s u b - c e l l u l a r l o c a t i o n s of AcPase a c t i v i t y ( O p a r k a e t a l . 1 9 8 1 ) . O n l y one s u b s t r a t e was employed i n t h i s s t u d y and t h e r e f o r e the r e s u l t s must be i n t e r p r e t e d w i t h c a u t i o n . I n S_. f u r c i g e r a , AcPase a c t i v i t y a p p e a r e d to be p r i m a r i l y a s s o c i a t e d w i t h the plasma membrane. M i n o r r e a c t i o n d e p o s i t s were d e t e c t e d i n the m i t o c h o n d r i a and a few v e s i c u l a r b o d i e s . S i m i l a r f i n d i n g s have been r e p o r t e d f o r o t h e r o r g a n i s m s ( O l i v e i r a 1975, Bentwood and Cronshaw 47 1 976 , Murray 1 980 , T r o n c h i n e t a l . 1 9 8 0 , ) . A f u n c t i o n f o r t h i s A cPase a c t i v i t y can not be g i v e n based on the r e s u l t s p r e s e n t e d h e r e , but o t h e r a u t h o r s have s u g g e s t e d t h a t A c P a s e may be i n v o l v e d i n the t r a n s p o r t of e x t r a c e l l u l a r m e t a b o l i t e s i n t o the c e l l ( C r a s n i e r 1 9 8 0 ) . T h e r e f o r e , i t seems r e a s o n a b l e to s u g g e s t t h a t the a s s o c i a t i o n of AcPase a c i t i v i t y w i t h the plasma membrane i n j>. f u r c i g e r a may a s s i s t i n the h y d r o l y s i s and t r a n s p o r t of e x t r a c e l l u l a r m e t a b o l i t e s a c r o s s the plasma membrane. Whatever i t s f u n c t i o n , i t does seem t h a t the AcPase a c t i v i t y l o c a l i z e d i n t h i s s t u d y i s n e c e s s a r y f o r b o t h b u d d i n g and n o n - b u d d i n g c e l l s as i t shows s i m i l a r d i s t r i b u t i o n p a t t e r n s i n b o t h b u d d i n g and n o n - b u d d i n g c e l l s . 48 Summary Bud i n i t i a t i o n and d e v e l o p m e n t i n S p h a c e l a r i a  f u r c i g e r a i s a complex p r o c e s s t h a t i n v o l v e s the de-d i f f e r e n t i a t i o n of a q u i e s c e n t a x i a l c e l l . T a b l e 2 summarizes the major c e l l u l a r a l t e r a t i o n s e n c o u n t e r e d d u r i n g t h i s p r o c e s s . F o r p u r p o s e s of s i m p l i f i c a t i o n i t i s s u b - d i v i d e d i n t o t h r e e p h a s e s . Phase I : A l o c a l i z e d t h i c k e n i n g and s u b s e q u e n t p r o t r u s i o n of the o u t e r c e l l w a l l of the a x i a l mother c e l l i n the v i c i n i t y of the n u c l e u s c h a r a c t e r i z e s the e a r l i e s t d e t e c t a b l e s t a g e of bud i n i t i a t i o n . The c y t o p l a s m i c f a c t o r s t h a t a r e r e s p o n s i b l e f o r i n i t i a t i n g bud d e v e l o p m e n t a r e not r e v e a l e d i n t h i s s t u d y . However, they a r e p r o b a b l y i n v o l v e d i n a) s y n t h e s i s and d e p o s i t i o n of new c e l l wal 1 m a t e r i a l and b) e n z y m a t i c 1 o o s e n i n g / 1 y s i s of the e x i s t i n g c e l l w a l l a l l o w i n g f o r the e x p a n s i o n , i . e . p r o t r u s i o n , of the bud i n i t i a l . R e s u l t s of the C a l c o f l u o r s t u d y prove t h a t new c e l l w a l l m a t e r i a l i s i n d e e d b e i n g d e p o s i t e d and c y t o c h e m i s t r y r e v e a l s the p o s s i b l e i n v o l v e m e n t of p e r o x i d a s e a c t i v i t y . P e r o x i d a s e a c t i v i t y has been l i n k e d to c e l l w a l l breakdown (Henry 1 9 7 9 ) . T u r g o r p r e s s u r e , the r e s u l t of water b e i n g a b s o r b e d i n t o the v a c u o l e ( s ) (Wiebe 1980) i s p r o b a b l y the i n i t i a l d r i v i n g f o r c e b e h i n d the e x p a n s i o n of the bud i n i t i a l . The d i r e c t i o n of growth t a k e n by the c e l l , however, i s d ependent on the a n i s o t r o p i c 49 ( u n e q u a l ) y i e l d i n g of the c e l l w a l l as t u r g o r p r e s s u r e i s the same i n a l l d i r e c t i o n s ( G r e e n 1962, 1 9 8 0 ) . A f t e r the i n i t i a l w a l l p r o t r u s i o n i s e s t a b l i s h e d , c y t o p l a s m i c m o d i f i c a t i o n s t h a t f o l l o w e n t a i l a l o s s of c y t o p l a s m i c v a c u o l e s and an i n c r e a s e i n c y t o p l a s m i c mass and d e n s i t y . T h e r e i s a marked i n c r e a s e i n the number of c h l o r o p l a s t s and m i t o c h o n d r i a and a n o t i c e a b l e p r o l i f e r a t i o n of the endomembrane s y s t e m . These c y t o p l a s m i c e v e n t s c o n t r i b u t e to the o v e r a l l s y n t h e t i c a c t i v i t y of the a x i a l mother c e l l and a l l o w f o r c o n t i n u e d e x p a n s i o n of the bud i n i t i a l . O r g a n e l l e m i g r a t i o n i n t o the bud p r o t r u s i o n b e g i n s , w i t h the e x c e p t i o n of the n u c l e u s w h i c h r e t a i n s i t s b a s a l p o s i t i o n w i t h i n the a x i a l mother c e l l . However, the n u c l e u s i s n o t t o t a l l y i n a c t i v e as i t does i n c r e a s e i n s i z e i n p r e p a r a t i o n f o r m i t o s i s . The r e d u c t i o n i n s i z e and number of v a c u o l e s must cause a d e c r e a s e i n t u r g o r p r e s s u r e and the i n c r e a s e i n c y t o p l a s m i c volume p r o b a b l y t a k e s o v e r as the main d r i v i n g f o r c e w h i c h p e r p e t u a t e s the e x p a n s i o n of the bud i n i t i a l . C o n t i n u e d i n c o r p o r a t i o n of new c e l l w a l l m a t e r i a l i n t o the o u t e r l o n g i t u d i n a l c e l l w a l l m a i n t a i n s the s t r u c t u r a l i n t e g r i t y of the c e l l w a l l d u r i n g the e x p a n s i o n of the bud p r o t r u s i o n . T h i s new c e l l w a l l m a t e r i a l i s d e p o s i t e d i n a l a y e r e d p a t t e r n w h i c h d i f f e r s s i g n i f i c a n t l y from the l a y e r e d p a t t e r n o b s e r v e d i n the o r i g i n a l c e l l w a l l of the a x i a l mother c e l l . The c e l l w a l l of the a x i a l mother c e l l c o n s i s t s of f o u r c e l l w a l l l a y e r s , each one made up 50 p r e d o m i n a t l y of " f u c a n s " or " a l g i n a t e s " . I t b e g i n s w i t h an o u t e r f u c a n l a y e r , f o l l o w e d by an a l g i n a t e l a y e r , t h e n a n o t h e r f u c a n l a y e r and t e r m i n a t e s w i t h an i n n e r a l g i n a t e l a y e r ( F i g . 3 2 ) . W i t h i n the bud i n i t i a l o n l y two c e l l w a l l l a y e r s a r e p r e s e n t , a t h i n o u t e r f u c a n l a y e r and a t h i c k i n n e r a l g i n a t e l a y e r ( F i g . 3 3 ) . I t has been r e p o r t e d tha-t a l g i n i c a c i d i s c o n f i n e d to the f i b r i l l a r a r e a s of the w a l l and f u c o i d a n to the amorphous m a t r i x ( M c C u l l y 1966, Novotny and Forman 1 9 7 5 ) . The i n i t i a l e l e c t r o n m i c r o s c o p i c s t u d y c o n f i r m s t h i s s u g g e s t i o n as the f u c a n l a y e r s a p p e a r amorphous and the a l g i n a t e l a y e r s a p pear f i b r i l l a r and h i g h l y o r g a n i z e d . Phase I I ; Once the a x i a l mother c e l l has e s t a b l i s h e d a " v a c u o l e - f r e e " c y t o p l a s m , n u c l e a r m i g r a t i o n towards the p l a c e to be o c c u p i e d by the f u t u r e c e l l p l a t e commences. The c y t o p l a s m a t t h i s p o i n t has a v e r y dense a p p e a r a n c e as i t i s r i c h l y i n v e s t e d w i t h r i b o s o m e s which a r e l i k e l y engaged i n p r o t e i n s y n t h e s i s . D u r i n g t h i s p hase, d e p o s i t i o n of new c e l l w a l l m a t e r i a l a t the t i p of the bud p r o t r u s i o n c o n t i n u e s . P e r o x i d a s e a c t i v i t y becomes i n c r e a s i n g l y r e s t r i c t e d to the o u t e r m o s t p o s i t i o n of the w a l l of the bud p r o t r u s i o n . T h i s i m p l i e s t h a t c e l l w a l l l o o s e n i n g / l y s i s of the newly d e p o s i t e d c e l l w a l l m a t e r i a l i s n o t as i m p o r t a n t as i t was f o r the e x i s t i n g a x i a l mother c e l l w a l l . T h i s may be a c c o u n t e d f o r i f the new c e l l w a l l m i c r o f i b r i l s become p a s s i v e l y a l i g n e d i n the d i r e c t i o n of growth w i t h o u t the 51 a i d of c e l l w a l l l o o s e n i n g a g e n t s as p r e d i c t e d by Houwink and R o e l o f s e n ( 1 9 5 4 ) . Phase I I I : A f t e r the n u c l e u s has r e a c h e d the s i t e of the f u t u r e c e l l p l a t e t h e r e i s a r e a p p e a r a n c e of c y t o p l a s m i c v a c u o l e s w i t h i n the b a s a l p o r t i o n of the a x i a l mother c e l l . C e n t r i o l e d u p l i c a t i o n and m i g r a t i o n to o p p o s i t e p o l e s of the n u c l e u s o c c u r and k a r y o k i n e s i s soon f o l l o w s w i t h the f o r m a t i o n of two d a u g h t e r n u c l e i . The d e p o s i t i o n of a c r o s s w a l l between the tx*o n u c l e i r e s u l t s i n an u n e q u a l c e l l d i v i s i o n . The bud c e l l c y t o p l a s m i s n o n - v a c u o l a t e d and r i c h i n o r g a n e l l e s and w i l l c o n t i n u e to grow and d i v i d e , u l t i m a t e l y f o r m i n g a m u l t i c e l l u l a r b r a n c h . On the o t h e r hand, the s i s t e r a x i a l c e l l i s h i g h l y v a c u o l a t e d and poor i n o r g a n e l l e s and t h e s e a r e c y t o l o g i c a l f e a t u r e s c h a r a c t e r i s t i c of the q u i e s c e n t c e l l i t has become. P e r o x i d a s e a c t i v i t y i s now c o n f i n e d to the o u t e r m o s t f u c a n l a y e r of b o t h the bud c e l l and the s i s t e r a x i a l c e l l . I t l i k e l y s e r v e s to p r o t e c t the c e l l s by p e r f o r m i n g an a n t i -m i c r o b i a l f u n c t i o n ( M e n z e l 1980, Venere 1980) as i t may no l o n g e r be r e q u i r e d f o r c e l l w a l l l o o s e n i n g / l y s i s . T h i s s t u d y has p r o v i d e d the ground work f o r u n d e r s t a n d i n g bud i n i t i a t i o n and d e v e l o p m e n t . I t has a l s o opened up the p o s s i b i l i t y of f u t u r e s t u d i e s . S t e r e o l o g i c a 1 and m o r p h o m e t r i c s t u d i e s of the c e l l o r g a n e l l e s , e s p e c i a l l y the v a c u o l e s , s h o u l d p r o v i d e a more d e t a i l e d a p p r o a c h to the m o r p h o l o g i c a l a n a l y s i s of bud i n i t i a t i o n and 52 d e v e l o p m e n t . The i n f l u e n c e of the p o s i t i o n of the n u c l e u s , i f any, i n d e t e r m i n i n g the d e v e l o p m e n t of the bud i n i t i a l c o u l d be s t u d i e d u s i n g drugs 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 w h i c h can b l o c k n u c l e a r m i g r a t i o n ( S c h n e p f and Heinzmann 1980) and thus h e l p i n e l u c i d a t i n g the r o l e i f any p l a y e d by m i c r o t u b u l e s and m i c r o f i l a m e n t s i n t h i s p r o c e s s . A d d i t i o n a l c y t o c h e m i c a l t e c h n i q u e s c o u l d be used to d e m o n s t r a t e the o t h e r s u b - c e l l u l a r l o c a t i o n s of the v a r i o u s i s o z y m e s of p e r o x i d a s e , ATPase and A c P a s e . T h i s would p r o v i d e a more c o m p l e t e p i c t u r e of the b e h a v i o r of t h e s e enzymes d u r i n g bud i n i t i a t i o n and d e v e l o p m e n t and p e r h a p s b e t t e r r e v e a l t h e i r r o l e s , i f any, i n c e l l w a l l d e g r a d a t i o n and d e p o s i t i o n . The s i t e of new c e l l w a l l s y n t h e s i s and d e p o s i t i o n c o u l d be e l u c i d a t e d u s i n g the p h o s p h o t u n g s t i c a c i d ( B a r s o t t i and M a r i n o z z i 1980) and T h i e r y ( T h i e r y and Rambourg 1974) t e c h n i q u e s whereby g l y c o p r o t e i n s and c a r b o h y d r a t e s can be u l t r a s t r u c t u r a l l y l o c a l i z e d . F i n a l l y , e x p e r i m e n t a l m a n i p u l a t i o n s of such v a r i a b l e s as t e m p e r a t u r e , l i g h t , and n u t r i e n t s c o u l d be p e r f o r m e d to g a i n a b e t t e r u n d e r s t a n d i n g of the e n v i r o n m e n t a l ' f a c t o r s a f f e c t i n g b r a n c h i n i t i a t i o n and d e v e l o p m e n t . 53 T a b l e 2 O r g a n e l l e s C y t o p l a s m C e l l W a l l Phase I nuc1eus ne a r bud wa 1 1 pr o-t r u s i o n d e c r e a s e i n # of v a c u o l e s l o c a l i z e d t h i c k e n i n g i n r e g i o n of bud pro-t r u s i o n . i n c r e a s e i n # of m i t o -c h o n d r i a and c h l o r o p l a s t s p r o l i f e r a t i o n of ER i n c r e a s e i n n u c l e a r s i z e i n c r e a s e i n c y t o p l a s m i c mass and d e n s i t y i n t e n s e p e r o x i d a s e a c t i v i t y t h r o u g h o u t bud w a l l p r o t r u s i o n r e d u c t i o n i n # of w a l l l a y e r s from 4 to 2 a t t i p of bud p r o t r u s i o n Phase I I n u c l e a r m i g r a - " v a c u o l e - f r e e " c o n t i n u e d d e p o s i t i o n of t i o n towards new w a l l m a t e r i a l i . e . f u t u r e c e l l f u c a n s and a l g i n a t e s p l a t e d e n s e • c y t o p l a s m p e r o x i d a s e a c t i v i t y con-f i n e d to o u t e r h a l f of bud wal1 Phase I I I n u c l e u s a t f u t u r e c e l l p l a t e r eap pe ar ance v a c u o l e s i n i n b a s a l p o r -t i o n of a x i a l mo t h e r c e l l of c o n t i n u e d d e p o s i t i o n of of new w a l l m a t e r i a l and m a i n t a i n e n c e of 2 l a y e r e d a p p e a r a n c e of bud wa 1 1 c e n t r i o l e dup-l i c a t i o n and m i g r a t i o n to o p p o s i t e p o l e s o f n u c l e u s dense c y t o p l a s m r e s t r i c t e d to bud p r o t r u s i o n pe rox i d a s e o u t e r wal 1 p r o t r u s i o n a c t i v i t y i n l a y e r of bud o n l y k a r y o k i n e s i s " v a c u o l e - f r e e " and dense c y t o p l a s m i n bud c e l l f o r m a t i o n of r e s u l t i n g i n and a s i s t e r c r o s s wal1 a bud c e l l a x i a l c e l l h i g h l y v a c u o -l a t e d s i s t e r a x i a l c e l l 54 KEY FOR FIGURES C, c h l o r o p l a s t , CrW, c r o s s w a l l , C t , c e n t r i o l e , Cu, c u t i c l e , CVJ1 , c e l l w a l l l a y e r 1, CW2 , c e l l w a l l l a y e r 2, CW3 , c e l l w a l l l a y e r 3, CW4, c e l l w a l l l a y e r 4, CWl, i n n e r c e l l w a l l l a y e r , CWO, o u t e r c e l l w a l l l a y e r , D, d i c t y o s o m e , ER, e n d o p l a s m i c r e t i c u l u m , N, n u c l e u s , Nu, n u c l e o l u s , M, m i t o c h o n d r i o n , ML, m i d d l e l a m e l l a , Mt, m i c r o t u b u l e , V, v a c u o l e , P, plasmodesmata . 55 F i g s . 1 to 8 N o marski d i f f e r e n t i a l c o n t r a s t o b s e r v a t i o n s of bud S. . f u r c i g e r a . i n t e r f e r ence i n i t i a t i o n i n F i g . l Q u i e s c e n t a x i a l c e l l . The n u c l e u s o c c u p i e s a m e d i a l p o s i t i o n w i t h i n the c y t o p l a s m ( a r r o w h e a d ) . F i g s . 2 and 3 E a r l y s t a g e s i n bud i n i t i a t i o n ( s t a g e s I and I I ) . Arrow d e n o t e s s i t e of i n i t i a l c e l l w a l l p r o t r u s i o n c h a r a c t e r i z i n g s t a g e I ( F i g . 2 ) . F i g s . 4 to 7 Sequence of s t a g e s i n bud i n i t i a t i o n ( s t a g e s I I I to V I ) . Observe the p r o g r e s s i v e change i n the p o s i t i o n of of the n u c l e u s ( a r r o w h e a d ) i n r e l a t i o n to the d e v e l o p i n g bud. F i g . 8 C o m p l e t i o n of c e l l d i v i s i o n ( s t a g e V I I ) r e s u l t s i n the f o r m a t i o n of two d a u g h t e r c e l l s , a bud c e l l 'B' and a s u b t e n d i n g s i s t e r a x i a l c e l l 'A'. Arrowheads i n d i c a t e the p o s i t i o n of the n u c l e u s w i t h i n each c e l l . Arrows i n d i c a t e the p o s i t i o n of the t r a n s v e r s e w a l l between the two c e l l s . 56 Q u i e s c e n t a x i a l c e l l , n ormal complement d i s t r i b u t e d i n a c y t o p l a s m . Four w a l l can be r e c o g n i z e d l o n g i t u d i n a l w a l l . T h i s c e l l d i s p l a y s a of o r g a n e l l e s h i g h l y v a c u o l a t e d l a y e r s (CWI to CW4) i n the o u t e r A x i a l mother c e l l ( s t a g e I ) . A s l i g h t p r o -t r u s i o n of the o u t e r l o n g i t u d i n a l w a l l o c c u r s i n the v i c i n i t y of the n u c l e u s and marks the b e g i n n i n g of bud i n i t i a t i o n . The i n p o c k e t i n g of the n u c l e a r e n v e l o p e (*) was o b s e r v e d to c o n t a i n a p a i r of c e n t r i o l e s . Note t h a t the c y t o p l a s m has become l e s s v a c u o l a t e d . I n c r e a s e d m a g n i f i c a t i o n of i n s e t i n F i g . 1 0 . Note the f o u r l a y e r c o m p o s i t i o n of the c e l l w a l l and the i n c r e a s e i n t h i c k n e s s of c e l l w a l l l a y e r 4 (CW4) i n the r e g i o n of the p r o t r u s i o n . Arrowheads d e n o t e the e x i s t e n c e of d i s c o n t i n u i t i e s w i t h i n c e l l w a l l l a y e r 1 ( C W l ) . 57 A x i a l mother c e l l ( s t a g e I I ) . C e l l w a l l l a y e r s 1, 2 and 3 (CWI, CW2 and CW3) o b s e r v e d i n the b a s a l p o r t i o n of the o u t e r l o n g i t u d i n a l w a l l c o n v e r g e i n t o a s i n g l e o u t e r c e l l w a l l l a y e r (CWO) i n the r e g i o n of the p r o t r u s i o n . Arrowheads d e n o t e d i s c o n t i n u i t i e s w i t h i n the o u t e r c e l l w a l l l a y e r (CWO). A x i a l mother c e l l ( s t a g e I I I ) . The most c h a r a c t e r i s t i c f e a t u r e of t h i s s t a g e i s the a l m o s t t o t a l absence of v a c u o l e s . I n c r e a s e d m a g n i f i c a t i o n of i n s e t i n F i g . 1 3 d e p i c t i n g the l o o s e a t t a c h m e n t (*) t h a t seems to e x i s t between the o u t e r (CWO) and i n n e r (CWI) c e l l w a l l l a y e r s a t the t i p of the p r o t r u s i o n . 58 F i g . 1 5 A x i a l mother c e l l ( s t a g e I V ) . The n u c l e u s o c c u p i e s a p o s i t i o n w i t h i n the bud c y t o p l a s m w h i c h i s i n d i c a t i v e of the o c c u r r e n c e of n u c l e a r m i g r a t i o n i n t o the bud p r o t r u s i o n . Note a l s o the c o n t i n u e d absence of v a c u o l e s from the c y t o p l a s m . F i g . 1 6 O r g a n i z a t i o n a l d e t a i l of the c e l l w a l l a t the t i p of the p r o t r u s i o n ( i n s e t i n F i g . 1 5 ) . The o u t e r (CWO) and i n n e r (CWl) w a l l l a y e r s d i f f e r i n t h i c k n e s s . Note the random a r r a n g e m e n t of the m i c r o f i b r i l l a r m a t e r i a l of CWl. Arrowheads p o i n t to d i s c o n t i n u i t i e s w i t h i n CWO. F i g . 1 7 B r i g h t f i e l d m i c r o g r a p h of a bud i n i t i a l . The o u t e r c e l l w a l l l a y e r s c o n v e r g e ( a r r o w s ) i n t o a t h i n n e r o u t e r w a l l l a y e r . The l a t t e r c o v e r s a t h i c k i n n e r w a l l l a y e r ( a r r o w h ead s ) . F i g . 1 8 C a l c o f l u o r White f l u o r e s c e n c e m i c r o s c o p y of the same . c e l l d e p i c t e d i n F i g . 1 7 . The d o t t e d l i n e d e f i n e s the boundary between the o l d f l u o r e s c i n g c e l l w a l l and the newly s y n t h e s i z e d n o n - f 1 u o r e s c i n g c e l l w a l l . 59 51 A F i g . 1 9 M a g n i f i e d view of i n s e t i n F i g . 2 0 . Note the u n i n t e r r u p t e d m e r g i n g of the o l d c e l l w a l l l a y e r CW4 w i t h t h a t of the newly d e p o s i t e d c e l l w a l l l a y e r CWl. F i g . 2 0 The n u c l e u s comes to r e s t midway between the b a s a l p o r t i o n of the a x i a l mother c e l l ( s t a g e V) and t h e p r o t r u s i o n . Note the r u p t u r e ( a r r o w h e a d s ) of the c e l l w a l l l a y e r CWO a t the t i p of the p r o t r u s i o n and the r e a p p e a r a n c e of v a c u o l e s i n t h e b a s a l p o r t i o n of the a x i a l mother c e l l . 60 F i g s . 2 1 and 22 Two s e r i a l s e c t i o n s of the n u c l e u s o b s e r v e d i n F i g . 2 5 . Note t h a t the c e n t r i o l e s ( a r r o w h e a d s ) a p p e a r on o p p o s i t e s i d e s of the n u c l e u s , a f e a t u r e a s s o c i a t e d w i t h k a r y o k i n e s i s . F i g s . 2 3 and 24 H i g h m a g n i f i c a t i o n o b s e r v a t i o n of 1 the c e n t r i o l e s found a t the p o s i t i o n s i n d i c a t e d i n F i g s . 21 and 22. Note the o c c u r r e n c e and o r i e n t a t i o n of the e x t r a -n u c l e a r m i c r o t u b u l e s and the o r g a n i z a t i o n of the e l e c t r o n dense b r i d g i n g m a t e r i a l ( a r r o w h e a d s ) i n r e l a t i o n to the n u c l e a r e n v e l o p e . F i g . 2 5 A x i a l mother c e l l ( s t a g e V I ) . Note the i n -c r e a s e i n the number of c y t o p l a s m i c v a c u o l e s w i t h i n the b a s a l p o r t i o n of the a x i a l mother c e l l . 61 &l A Stage V I I i s c h a r a c t e r i z e d by the c o m p l e t i o n of c e l l d i v i s i o n . A c r o s s w a l l i s d e p o s i t e d between the bud c e l l 'B' and the s u b t e n d i n g s i s t e r a x i a l c e l l 'A'. Note the p o s i t i o n of the c e n t r i o l e s (*) as r e v e a l e d by s e r i a l s e c t i o n i n g . Numerous v a c u o l e s abound i n c e l l 'A', w h i l e c e l l 'B' e x h i b i t s a d e n s e r , v i r t u a l l y non-v a c u o l a t e d c y t o p l a s m . A p o r t i o n of the c r o s s w a l l (CrW) s e p a r a t i n g c e l l 'A' from c e l l 'B' i n F i g . 2 6 i s shown a t h i g h e r m a g n i f i c a t i o n to r e v e a l the p r e s e n c e of numerous plasmodesmata ( a r r o w s ) . 62 <oZ A F i g s . 2 8 to 31 A s c h e m a t i c d i a g r a m d e p i c t i n g the change i n a p p e a r a n c e of t h r e e p r o m i n e n t m o r p h o l o g i c a l f e a t u r e s ( n u c l e u s , v a c u o l e s and the c e l l w a l l ) o b s e r v e d d u r i n g the p r o c e s s of bud i n i t i a t i o n and d e v e l o p m e n t . The d o t t e d l i n e d e l i n e a t e s the boundary between the o l d c e l l w a l l l a y e r s and the newly d e p o s i t e d c e l l w a l l l a y e r s . F o r d e t a i l s see c o n c l u s i o n . 63 F i g s . 3 2 & 33 Two s c h e m a t i c d r a w i n g s d e p i c t i n g the d i s t r i b u t i o n of the two major c e l l w a l l p o l y s a c c h a r i d e s i d e n t i f i e d by l i g h t m i c r o s c o p y c y t o c h e m i s t r y . Compare the d i s t r i b u t i o n p a t t e r n of an e a r l y bud i n i t i a l ( F i g . 3 2 ) w i t h t h a t of a more advanced bud i n i t i a l ( F i g . 3 3 ) . 64 64- A F i g . 3 4 to 41 L i g h t m i c r o s c o p e c y t o c h e m i s t r y . F i g . 3 4 F i g . 3 5 F i g . 3 6 F i g . 3 7 F i g . 3 8 F i g . 3 9 F i g . 4 0 F i g . 41 Compare w i t h F i g . 35. 0.5. Note the absence c e l l w a l l l a y e r CWl. S a f r a n i n 0 / c a r b o x y l group O n l y c e l l w a l l l a y e r s CWl and TBO a t pH 6.8. TBO a t pH of s t a i n i n g i n PAS r e a c t i o n , r e a c t s s t r o n g l y F a s t G r e e n d a r k g r e e n wa11 l a y e r s of p r o t e i n . A n i l i n e Blue p r o t e i n sub-c e l l w a l l of C e l l w a l l and CWl does me thy l a t i o n. CW3 s t a i n . l a y e r CWO n o t . F C F / S a f r a n i n 0. Note the c o l o u r p r o d u c e d w i t h i n c e l l CWl and CWO a r e i d i c a t i v e B l a c k / S a f r a n i n 0. Note the - l a y e r s ( a r r o w s ) w i t h i n the the bud i n i t i a l . R uthenium Red. Note t h a t c e l l w a l l l a y e r CWO r e a c t s s t r o n g l y whereas CWl does n o t . I KI/H 2 s o 4 . O n l y a s l i g h t r e a c t i o n i s p r e s e n t i n c e l l w a l l l a y e r s CW3 and CWl. 65 A F i g s . 4 2 t o 4 5 D e m o n s t r a t i o n of p e r o - i H - « ; p •, r , t - J - U " i U o s e a c t i v i t y d u r i n g bud i n i t i a t i o n and d e v e l o p m e n t . F i g . 4 2 T h e c e l l w a l l a n d p l a smod e sma t a e x h i b i t p e r o x i d a s e a c t i v i t y . N o t e t h e e x t e n t o f t h e r e a c t i o n p r o d u c t w i t h i n t h e c e l l w a l l o f t h e b u d p r o t r u s i o n . F i g . 4 3 A m o r e a d v a n c e d b u d i n i t a l t h a n t h a t p r e s e n t e d i n F i g . 4 2 . N o t e t h e l a c k o f p e r o x i d a s e a c t i v i t y i n t h e i n n e r h a l f o f c e l l w a l l l a y e r CWI. F i g . 4 4 A c o n t r o l p r e p a r a t i o n i n c u b a t e d i n t h e a b s e n c e o f s u b s t r a t e . N o t e t h e l a c k o f p e r o x i d a s e a c t i v i t y . F i g . 4 5 P e r o x i d a s e a c t i v i t y w i t h i n t h e m i d d l e l a m e l l a o f t h e c r o s s w a l l w h i c h s e p e r a t e s t h e b u d c e l l f r o m i t s s i s t e r a x i a l c e l l a f t e r c y t o k i n e s i s . 66 ( , U F i g s . 4 6 to 51 D e m o n s t r a t i o n of ATPase a c t i v i t y . F i g . 4 6 ATPase a c t i v i t y i s a s s o c i a t e d w i t h the n u c l e u s , d i c t y o s o m e s and c h l o r o p l a s t s . F i g . 4 7 ATPase a c t i v i t y w i t h i n a d i c t y o s o m e . F i g . 4 8 ATPase a c t i v i t y a s s o c i a t e d w i t h the i n n e r s u r f a c e of the plasma membrane. F i g . 4 9 ATPase a c t i v i t y e x h i b i t e d by the m i t o c h o n -d r i a l c r i s t a e . F i g . 5 0 C o n t r o l p r e p a r a t i o n i n c u b a t e d i n the absence of s u b s t r a t e . Note a g e n e r a l r e d u c t i o n i n ATPase a c t i v i t y . F i g . 5 1 A n o t h e r c o n t r o l p r e p a r a t i o n i n which e n z y m a t i c a c t i v i t y was d e s t r o y e d by h e a t . Note the l o s s of ATPase a c t i v i t y . 67 A F i g s . 5 2 to 57 D e m o n s t r a t i o n of AcPase a c t i v i t y . F i g . 5 2 The i n n e r s u r f a c e of the plasma membrane e x h i b i t s AcPase a c t i v i t y . F i g . 5 3 AcPase a c t i v i t y was d e t e c t e d w i t h i n the c r i s t a e of the m i t o c h o n d r i a . F i g . 5 4 I n t e n s e AcPase a c t i v i t y i s d i s p l a y e d a l o n g the e n t i r e i n n e r s u r f a c e of the plasma membrane of a bud c e l l . F i g . 5 5 C o n t r o l p r e p a r a t i o n i n w h i c h the i n c u b a t i i o n was c a r r i e d out i n the p r e s e n c e of the i n h i b i t o r sodium f l u o r i d e . Note the l o s s of AcPase a c t i v i t y . F i g . 5 6 & 57 AcPase a c t i v i t y i s e x h i b i t e d by v a r i o u s c y t o p l a s m i c v e s i c l e s ( * ) . 6 68 6fc A L i t e r a t u r e C i t e d A g h a j a n i a n , J.G. and Hommersand, M.H., 1 980: Growth and d i f f e r e n t i a t i o n of a x i a l and l a t e r a l f i l a m e n t s in-Batrachospermum s i r o d o t i i (Rhod o phy t a ) . J . P h y c o l 1 5 - 2 8 . 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