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Mechanisms of diclofop methyl uptake in oat protoplasts (Avena sativa ’Cascade’) Tritter, Susan 1983

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MECHANISMS OF DICLOFOP METHYL UPTAKE IN OAT PROTOPLASTS ( Avena s a t i v a 'Cascade by SUSAN TRITTER B.SC.CAGR.), UNIVERSITY OF GUELPH. 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f P l a n t S c i e n c e We a c c e p t t h i s t h e s i s as co n f o r m i n g t o the r e q u i r e d s t a n d a r d THE U n i v e r s i t y o f B r i t i s h Columbia A p r i l , 1983. ^(c)Susan T r i t t e r jg83 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f f^ftUl So£kJCLjF~ The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT I s o l a t e d o a t p r o t o p l a s t s ( A v e n a s a t i v a L . ' C a s c a d e ' ) were u s e d t o compare t h e u p t a k e o f t h e h e r b i c i d e d i c l o f o p m e t h y l ( m e t h y l 2 - ( 4 - ( 2 , 4 - d i e h l o r o p h e n o x y ) p h e n o x y ) p r o p a n o a t e ) and t h e a c i d f o r m d i c l o f o p ( 2 - ( 4 - ( 2 , 4 - d i c h l o r o p h e n o x y ) p h e n o x y ) p r o p i o n i c a c i d and t o s t u d y h e r b i c i d e u p t a k e as a b a s i s f o r t h e 2 , 4 - D - d i c l o f o p m e t h y l a n t a g o n i s m . D i c l o f o p m e t h y l was r a p i d l y t a k e n up by p r o t o p l a s t s ( < 5 s e c o n d s ) t o a c o n s t a n t l e v e l w h i c h d i d n o t ch a n g e s i g n i f i c a n t l y w i t h i n c u b a t i o n t i m e s up t o 1.0 h o u r . F o r t y p e r c e n t o f added 1 4 C - d i c l o f o p m e t h y l was t a k e n up w i t h i n 5.0 s e c o n d s w h i l e d i c l o f o p u p t a k e was l i m i t e d o v e r t h e same t i m e p e r i o d . D i c l o f o p m e t h y l u p t a k e was l i n e a r w i t h h e r b i c i d e c o n c e n t r a t i o n i n t h e u p t a k e s o l u t i o n up t o 50uM. T h e r e was no s i g n i f i c a n t d i f f e r e n c e b e t ween u p t a k e by b u r s t o r i n t a c t p r o t o p l a s t s p r e p a r a t i o n s . L a r g e a c c u m u l a t i o n s o f l 4 C - d i c l o f o p m e t h y l were f o u n d i n t h e membrane f r a c t i o n s o f t h e p r o t o p l a s t s . D i c l o f o p m e t h y l u p t a k e a p p e a r e d t o be a p a s s i v e , n o n - m e d i a t e d p r o c e s s i n w h i c h t h e h e r b i c i d e was p a r t i t i o n e d i n t o t h e l i p i d p h a s e o f t h e p r o t o p l a s t membrane. D i c l o f o p m e t h y l was c o n v e r t e d t o t h e a c i d f o r m i n t h e t r e a t m e n t s o l u t i o n by h y d r o l a s e enzymes a p p a r e n t l y a s s o c i a t e d w i t h t h e e x t e r n a l s u r f a c e o f t h e p r o t o p l a s t p l a s m a membrane. 2,4-D d i d n o t a f f e c t d i c l o f o p m e t h y l o r d i c l o f o p u p t a k e a t c o n c e n t r a t i o n s f r o m 10uM t o 2.0mM n o r was t h e c o n v e r s i o n o f d i c l o f o p m e t h y l t o d i c l o f o p a f f e c t e d by by 2,4-D e x c e p t a t h i g h n o n - p h y s i o l o g i c a l c o n c e n t r a t i o n s . D i c l o f o p m e t h y l and d i c l o f o p d i d n o t a d v e r s e l y a f f e c t membrane p e r m e a b i l i t y o f t h e p r o t o p l a s t s a t t h e i i i h e r b i c i d e c o n c e n t r a t i o n s used i n t h e s e e x p e r i m e n t s . P r o t o p l a s t p r e p a r a t i o n s proved to be a u s e f u l and s i m p l e system t o s t u d y the uptake and metabolism o f h e r b i c i d e s i n p l a n t s . i v i v TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 5 D i c l o f o p m e t h y l 5 P h y s i o l o i c a l e f f e c t s 5 B a s i s o f d i c l o f o p m e t h y l s e l e c t i v i t y 7 T r a n s l o c a t i o n 10 Mode o f a c t i o n 11 A n t a g o n i s m 12 H e r b i c i d e U p t a k e and T r a n s l o c a t i o n 16 Membrane t r a n s p o r t 17 E f f e c t s o f H e r b i c i d e s on U p t a k e and T r a n s l o c a t i o n . 22 MATERIALS AND METHODS 24 P l a n t M a t e r i a l 24 I s o l a t i o n o f P r o t o p l a s t s 24 Membrane P e r m e a b i l t y 26 N e u t r a l r e d a s s a y 26 A t o m i c a b s o r p t i o n a s s a y 27 D i c l o f o p M e t h y l and D i c l o f o p U p t a k e 28 D i s t r i b u t i o n o f R a d i o l a b e l 30 D e n s i t y g r a d i e n t f r a c t i o n a t i o n s 30 S o l u b i l i t y f r a c t i o n a t i o n 32 pH P r o f i l e o f H y d r o l a s e A c t i v i t y 35 V RESULTS AND DISCUSSION 36 P r o t o p l a s t V i a b i l i t y 36 Membrane P e r m e a b i l t y 38 D i c l o f o p M e t h y l and D i c l o f o p U p t a k e 49 D i s t r i b u t i o n o f D i c l o f o p M e t h y l and M e t a b o l i t e s i n Oat P r o t o p l a s t s . . . . . 57 2,4-D E f f e c t s on D i c l o f o p M e t h y l U p t a k e and M e t a b o l i t e s 66 GENERAL DISCUSSION 73 CONCLUSION 78 BIBLIOGRAPHY 79 APPENDIX 86 v i L I S T OF T A B L E S p a g e T a b l e 1 a . T h e e f f e c t o f e t h a n o l o n l e a k a g e o f n e u t r a l r e d d y e f r o m o a t p r o t o p l a s t s 40 T a b l e 1 b . T h e e f f e c t o f e t h a n o l o n t h e l e a k a g e o f K+ f r o m o a t p r o t o p l a s t s 41 T a b l e 2 a . T h e e f f e c t o f d i c l o f o p m e t h y l o n l e a k a g e o f n e u t r a l r e d d y e f r o m o a t p r o t o p l a s t s 43 T a b l e 2 b . T h e e f f e c t o f d i c l o f o p m e t h y l o n l e a k a g e o f K+ f r o m o a t p r o t o p l a s t s 44 T a b l e 3 a . T h e e f f e c t o f d i c l o f o p o n t h e l e a k a g e o f n e u t r a l r e d d y e f r o m o a t p r o t o p l a s t s 45 T a b l e 3 b . T h e e f f e c t o f d i c l o f o p o n t h e l e a k a g e o f K+ f r o m o a t p r o t o p l a s t s 46 T a b l e 4. T i m e p r o f i l e o f DM u p t a k e 50 T a b l e 5 . T i m e p r o f i l e o f d i c l o f o p u p t a k e 51 T a b l e 6. U p t a k e o f DM a n d d i c l o f o p b y i n t a c t a n d b u r s t p r o t o p l a s t s 54 T a b l e 7 a . D i s t r i b u t i o n o f r a d i o l a b e l i n t h e p r o t o p l a s t s u s p e n s i o n 58 T a b l e 7 b . P e r c e n t c o m p o s i t i o n o f r a d i o l a b e l i n t h e p r o t o p l a s t s a n d t h e e x t e r n a l s o l u t i o n 58 T a b l e 8. P r o t e i n a n d p h o s p h o r u s c o n t e n t o f t h e w a t e r s o l u b l e a n d i n s o l u b l e p r o t o p l a s t f r a c t i o n s . . . 60 T a b l e 9. D i s t r i b u t i o n o f r a d i o l a b e l w i t h i n t h e p r o t o p l a s t s a n d t h e e x t e r n a l s o l u t i o n a s a p e r c e n t o f t o t a l r e c o v e r a b l e r a d i o l a b e l . . . . 6 2 T a b l e 1 0 . D e t e r m i n a t i o n o f h y d r o l a s e a c t i v i t y i n p r o t o p l a s t i n c u b a t i o n s o l u t i o n s 63 T a b l e 1 1 . T h e e f f e c t o f 2,4-D o n DM u p t a k e 67 T a b l e 1 2 . T h e e f f e c t o f 2,4-D o n d i c l o f o p u p t a k e 68 T a b l e 1 3 . T h e e f f e c t o f 2,4-D o n t h e c o n v e r s i o n o f DM t o d i c l o f o p . 69 v i i L I S T OF F I G U R E S p a g e . F i g u r e 1 . S t r u c t u r e o f d i c l o f o p m e t h y l (DM) a n d d i c l o f o p 1 F i g u r e 2 . P a t h w a y o f d i c l o f o p m e t h y l m e t a b o l i s m i n p l a n t s 8 F i g u r e 3 - D e n s i t y g r a d i e n t f r a c t i o n a t i o n t u b e s . . . . 31 F i g u r e 4. F l o w c h a r t o f f r a c t i o n a t i o n o f p r o t o p l a s t s i n t o e x t e r n a l s o l u t i o n , w a t e r - s o l u b l e a n d w a t e r - i n s o l u b l e c o m p o n e n t s 33 F i g u r e 5 . C o n t a i n m e n t o f n e u t r a l r e d d y e i n v i a b l e p r o t o p l a s t s 37 F i g u r e 6. E x c l u s i o n o f E v a n ' s b l u e d y e f r o m v i a b l e p r o t o p l a s t s 38 F i g u r e 7 . C o n c e n t r a t i o n p r o f i l e o f d i c l o f o p m e t h y l u p t a k e 53 F i g u r e 8 . pH p r o f i l e o f h y d r o l a s e a c t i v i t y 7 1 v i i i ACKNOWLEDGEMENTS I w o u l d l i k e t o t h a n k D r . F.B. H o l l , A s s o c i a t e P r o f e s s o r , D e p a r t m e n t o f P l a n t S c i e n c e , U n i v e r s i t y o f B r i t i s h C o l u m b i a f o r h i s e n c o u r a g e m e n t and v a l u a b l e a d v i c e t h r o u g h o u t t h e r e s e a r c h and w r i t i n g o f t h i s t h e s i s . A s p e c i a l t h a n k s t o D r . B.G. Todd, M a n i t o b a D e p a r t m e n t o f A g r i c u l t u r e , f o r h i s g u i d a n c e and s u g g e s t i o n s i n t h e i n i t i a l p l a n n i n g o f t h e t h e s i s p r o p o s a l . I g r a t e f u l l y a c k n o w l e d g e t h e c o n t r i b u t i o n s o f D r . V.C. R u n e c k l e s and D r . P . A . J o l l i f f e , members o f my t h e s i s c o m m i t t e e , and D r . G.W. E a t o n and P a t Bowen f o r t h e i r t i m e and p a t i e n c e i n d i s c u s s i o n o f s t a t i s t i c a l a n a l y s i s . I a l s o w i s h t o t h a n k H o e c h s t C h e m i c a l I n c . f o r d o n a t i o n o f t h e r a d i o l a b e l l e d m a t e r i a l s and h e r b i c i d e s , and A g r i c u l t u r e Canada and t h e D e p a r t m e n t o f P l a n t S c i e n c e f o r f i n a n c i a l s u p p o r t d u r i n g t h e c o u r s e o f t h i s t h e s i s r e s e a r c h . -1-I N T R O D U C T I O N D i c l o f o p m e t h y l ( m e t h y l 2 - ( 4 - ( 2 , 4 - d i c h l o r o p h e n o x y ) p h e n o x y ) p r o p a n o a t e ) i s a s e l e c t i v e p o s t - e m e r g e n c e h e r b i c i d e w h i c h c o n t r o l s a v a r i e t y o f g r a s s y w e e d s i n c e r e a l a n d o i l s e e d c r o p s . D i c l o f o p m e t h y l (DM) i s a member o f t h e p h e n o x y - p h e n o x y p r o p i o n i c a c i d h e r b i c i d e s e r i e s f i r s t d i s c o v e r e d b y H o e c h s t I n c . i n 1971 a n d i s t h e m o s t e f f e c t i v e w i l d o a t h e r b i c i d e i n t h i s s e r i e s ( N e s t l e r e t a l . 1 9 7 8 ) . S i n c e 1971 d i c l o f o p m e t h y l h a s b e e n d e v e l o p e d i n t o a f o r m u l a t e d p r o d u c t now w i d e l y u s e d o n t h e C a n a d i a n p r a i r i e s . T h e h e r b i c i d e h a s t w o p h y t o t o x i c f o r m s , t h e m e t h y l e s t e r , d i c l o f o p m e t h y l , a n d t h e f r e e a c i d , d i c l o f o p ( F i g u r e 1 ) . Figure 1. Structure of diclofop methyl (DM) and diclofop. DICLOFOP METHYL (DM) methyl-2(-4(2,4-dichlorophenoxy)phenoxy)propanoate DICLOFOP 2-(k-(2,4-dichlorophenoxy)phenoxy)propionic acid - 2 -The h e r b i c i d e i s f o r m u l a t e d a s t h e m e t h y l e s t e r . However upon a p p l i c a t i o n t o p l a n t s ( G o r b a c h e t a l . 1977; S h i m a b u k u r o e t a l . 1979; Todd, 1979) o r s o i l (Smith, 1 9 7 7 ) i t i s r a p i d l y h y d r o l y s e d t o t h e a c i d f o r m . I n s p i t e o f t h e r e s e a r c h e f f o r t on t h e r e s p o n s e o f p l a n t s t o d i c l o f o p m e t h y l and d i c l o f o p , t h e mec h a n i s m o f p h y t o t o x i c a c t i o n r e m a i n s unknown. 2,4-D, MCPA and d i c a m b a a r e u s e f u l b r o a d l e a f h e r b i c i d e s w i d e l y u s e d t o c o n t r o l weeds i n c e r e a l s . T h e o r e t i c a l l y , t h e c o m b i n a t i o n o f DM and a b r o a d l e a f h e r b i c i d e s h o u l d p r o v i d e e x c e l l e n t b r o a d s p e c t r u m c o n t r o l o f b o t h g r a s s y and b r o a d l e a f w e e d s . I n p r a c t i c e , t h e c o m b i n a t i o n o f DM w i t h 2,4-D (To d d 1 9 7 9 ) , w i t h MCPA ( Q u e r e s h i and Vanden B o r n , 1979) o r w i t h d i c a m b a ( O ' S u l l i v a n and Vanden B o r n 1975) r e s u l t s i n a r e d u c t i o n o f t h e p h y t o t o x i c a c t i o n o f DM and l o s s o f c o n t r o l o f g r a s s y w e e d s . However, DM had no e f f e c t on t h e h e r b i c i d a l a c t i v i t y o f t h e b r o a d l e a f h e r b i c i d e s ( O ' S u l l i v a n and Vanden B o r n , 1975; Todd, 1 9 7 9 ) . The s i t e and n a t u r e o f t h e i n t e r a c t i o n between t h e s e h e r b i c i d e s a r e unknown. I n v e s t i g a t i o n o f t h e mechanism and movement a c r o s s t h e plasmalemma o f p l a n t c e l l s i s impeded by t h e p r e s e n c e o f t h e c e l l w a l l , i n t e r c e l l u l a r s p a c e s and t h e l o g i s t i c s o f h a n d l i n g c o m p l e x t i s s u e s . The use o f i s o l a t e d p r o t o p l a s t s p r o v i d e s a s i m p l i f i e d and r e l a t i v e l y homogeneous s y s t e m t o s t u d y u p t a k e m e c h a n i s m s . The u s e o f i s o l a t e d p r o t o p l a s t s e l i m i n a t e p r o b l e m s o f r a d i o l a b e l l e d m a t e r i a l t a k e n up i n t o i n t e r c e l l u l a r s p a c e s o r a d s o r b e d by t h e c u t i c l e and c e l l w a l l c o n s t i t u e n t s and r e d u c e s e r r o r s f r o m d i f f u s i o n r e s i s t a n c e . I n r e c e n t s t u d i e s , p r o t o p l a s t s h a v e been u s e d t o s t u d y u p t a k e and membrane -3-t r a n s p o r t o f i o n s ( M e t t l e r and L e o n a r d , 1979; L i n , 1980) amino a c i d s ( R u b i n s t e i n and T a t t a r , 1980; Guy e t a l . 1978) and s u g a r s (Guy e t a l . 1978, 1 9 8 0 ) . I t has been demons t r a t ed t h a t i s o l a t i o n and p u r i f i c a t i o n p r o c e d u r e s d i d no t i n h i b i t t he a b i l i t y o f c e l l s to t ake up amino a c i d s . Q u a l i t a t i v e and q u a n t i t a t i v e c o m p a r i s o n s between p r o t o p l a s t s and l e a f segments i n d i c a t e d t h a t p h y s i o l o g i c a l f u n c t i o n s a re a p p a r e n t l y not a l t e r e d by i s o l a t i o n p r o c e d u r e s ( R u b i n s t e i n and T a t t a r , 1978) . P u b l i s h e d r e p o r t s i n d i c a t e p rob lems a s s o c i a t e d w i t h p r o t o p l a s t up take measurements i n c l u d e ma in tenance o f o s m o t i c s t a b i l i t y , and s e p a r a t i o n o f p r o t o p l a s t s f rom the non-absorbed t r a c e r i n the i n c u b a t i o n medium. H e r b i c i d e up take by p l a n t s i s most o f t e n measured by exposu re o f p l a n t t i s s u e t o r a d i o l a b e l l e d compounds. The amount o f h e r b i c i d e p e n e t r a t i n g the t i s s u e or removed f rom the ambient s o l u t i o n has been used as a measure o f p l a n t u p t a k e . There has been no d i s t i n c t i o n between h e r b i c i d e up take i n t o the c u t i c l e , f r e e spaces and c e l l w a l l s f rom h e r b i c i d e wh i ch p e n e t r a t e s the c e l l membrane and e n t e r s the c e l l s . The e f f e c t s o f DM on the growth o f wheat c e l l s u s p e n s i o n c u l t u r e s was s t u d i e d by D a v i s and B rezeanu ( 1 9 7 9 ) . N o n - p h y t o t o x i c c o n c e n t r a t i o n s o f DM (4uM) and r e l a t i v e l y s h o r t i n c u b a t i o n t i m e s (12 hours ) d i d not a f f e c t g rowth o f the c u l t u r e and r e s u l t e d i n o n l y minor u l t r a s t r u c t u r a l damage t o the c e l l s . The m e t a b o l i s m o f DM i n the wheat c e l l s u s p e n s i o n c u l t u r e s appeared s i m i l a r to t h a t i n i n t a c t p l a n t s (Dusky e t a l . 1980) . The o b j e c t i v e s o f the s t u d y r e p o r t e d i n t h i s t h e s i s i n c l u d e t h e d e v e l o p m e n t o f a p r o t o p l a s t s y s t e m t o i n v e s t i g a t e t h e u p t a k e o f DM and d i c l o f o p i n o a t s and t o d e t e r m i n e t h e e f f e c t o f 2,4-D on u p t a k e as a t a r g e t f o r t h e DM - 2,4-D a n t a g o n i s m . The r e s u l t s o f t h e p r o t o p l a s t s s t u d y a r e e v a l u a t e d i n t e r m s o f t h e i r r e l e v a n c e t o t h e m e c hanisms w h i c h may o p e r a t e i n i n t a c t p l a n t s . -5-LITERATURE REVIEW DICLOFOP METHYL P h y s i o l o g i c a l e f f e c t s Many adve r se p l a n t r e s p o n s e s have been obse r ved f o l l o w i n g d i c l o f o p m e t h y l (DM) a p p l i c a t i o n . Chrow ley and P r e n d e v i l l e (1979) obse r ved an i n c r e a s e i n l e a f c e l l membrane p e r m e a b i l i t y (measured by c o n d u c t i v i t y o f the e x t e r n a l s o l u t i o n ) 12 hours a f t e r f o l i a r a p p l i c a t i o n o f DM to w i l d oa t l e a f segments and b e f o r e v i s i b l e i n j u r y s i g n s were e v i d e n t . C h l o r o s i s and n e c r o s i s o f t r e a t e d p l a n t s were not obse r ved u n t i l 96 h o u r s a f t e r DM a p p l i c a t i o n . In t o l e r a n t wheat s p e c i e s , no i n c r e a s e i n membrane p e r m e a b i l i t y was e v i d e n t 96 hou r s a f t e r t r e a t m e n t . Dec reased l e v e l s o f c h l o r o p h y l l a and b , and a c o i n c i d e n t appearance o f c h l o r o s i s were obse r ved i n w i l d oa t s h o o t s f o l l o w i n g DM t r e a t m e n t s (Chow and L a B e r g e , 1 9 7 8 ) . P h o t o s y n t h e s i s and t r a n s l o c a t i o n o f the p h o t o s y n t h a t e s were r educed by the DM t r e a t m e n t , by 24 and 63% r e s p e c t i v e l y . A c o r r e s p o n d i n g i n c r e a s e i n g l u c o s e , s u c r o s e and f r u c t o s e i n s h o o t s was obse r ved (Chow, 1976; Chow and L a B e r g e , 1 9 7 8 ) , w h i l e ATP l e v e l s were r educed by 44% compared to u n t r e a t e d c o n t r o l p l a n t s . Hoppe (1981) s t u d i e d the e f f e c t s o f DM on the l i p i d components o f w i l d oa t and maize p l a n t s . D i c l o f o p m e t h y l i n h i b i t e d l i p i d b i o s y n t h e s i s , r educed p h o s p h o l i p i d c o n t e n t , i n d u c e d a c c u m u l a t i o n s o f c a r b o h y d r a t e s i n r o o t t i p s and i n t e r f e r e d w i t h membrane s t r u c t u r e . In v i ew o f t h e s e e f f e c t s -6-i t was suggested t h a t DM may i n t e r f e r e w i t h f a t t y a c i d b i o s y n t h e s i s . H i s t o l o g i c a l s t u d i e s o f a d v e n t i t i o u s r o o t t i p s o f wheat and w i l d o a t t r e a t e d w i t h DM showed a reduced m i t o t i c i n d e x , i n d i c a t i n g i n h i b i t i o n o f c e l l d i v i s i o n (Owino, 1 9 7 7 ) . D i c l o f o p m e t h y l a r r e s t e d the c e l l s a t a stage i n the c e l l c y c l e p r e c e e d i n g m i t o s i s . The m i t o t i c index i n w i l d o a t s was more s e v e r e l y reduced a t lower DM c o n c e n t r a t i o n s and a f t e r s h o r t e r exposure p e r i o d s than i n wheat. Growth o f the e l o n g a t i o n r e g i o n o f a d v e n t i t i o u s r o o t s o f w i l d o a t s was s i g n i f i c a n t l y i n h i b i t e d a f t e r a 24 hour exposure as was i n i t i a t i o n o f new a d v e n t i t i o u s r o o t s (Owino, 1 9 7 7 ) . I t was co n c l u d e d t h a t the r o o t meristem t i s s u e i s a s e n s i t i v e t a r g e t s i t e f o r DM a c t i v i t y . A r e d u c t i o n i n r o o t growth and development a f t e r DM tr e a t m e n t has been observed by many a u t h o r s . Hoppe ( 1 9 8 0 ) r e p o r t e d DM reduced r a d i c l e growth, induced n e c r o s i s i n the m e r i s t e m a t i c a r e a s and e l o n g a t i o n zones o f r o o t t i p s and a r r e s t e d c e l l d i v i s i o n . Root development o f w i l d o a t s was i n h i b i t e d when DM was a p p l i e d e i t h e r p r e p l a n t i n c o r p o r a t e d (Wu and Santelmann 1976) or post emergent ( F r e i s e n , 1 9 7 6 ) . Shimabukuro e t a l . ( 1 9 7 8 ) proposed the r e d u c t i o n i n r o o t and shoot e l o n g a t i o n r e s u l t e d from i n t e r f e r e n c e o f DM w i t h a u x i n - m e d i a t e d growth. Both d i c l o f o p and DM i n h i b i t e d a u x i n induced growth i n r o o t and c o l e o p t i l e segments. The a u t h o r s c o n c l u d e d d i c l o f o p m e t h y l was an a u x i n a n t a g o n i s t . - 7 -B a s i s o f d i c l o f o p m e t h y l s e l e c t i v i t y D i c l o f o p m e t h y l i s w i d e l y u s e d as a s e l e c t i v e h e r b i c i d e t o c o n t r o l g r a s s y weed s p e c i e s i n wheat and b a r l e y ( A n d e r s o n 1976). The b a s i s o f s e l e c t i v i t y i s a p p a r e n t l y r e l a t e d t o d i f f e r e n t i a l r a t e s o f DM m e t a b o l i s m t o n o n - p h y t o t o x i c compounds ( F i g u r e 2) ( G o r b a c h e t a l . 1977; S h i m a b u k u r o e t a l . 1979; Todd, 1979). O t h e r s e l e c t i v e m e c h a n i s m s , i n c l u d i n g d i f f e r e n t i a l s p r a y r e t e n t i o n ( T o d d , 1977), h e r b i c i d e p e n e t r a t i o n ( S h i m a b u k u r o e t a l . 1979; D o n a l d and S h i m a b u k u r o 1981; T o d d , 1977), r o o t u p t a k e ( T o d d , 1977) and t r a n s l o c a t i o n ( B r e z e a n u e t a l . 1976; S h i m a b u k u r o e t a l . 1979; Todd, 1977) h a v e been s t u d i e d , b u t have n o t p r o v e n t o be p r i m a r y f a c t o r s i n s e l e c t i v i t y between s u s c e p t i b l e and t o l e r a n t s p e c i e s . N e v e r t h e l e s s , i n some c a s e s , e g . g r e e n f o x t a i l , e n h a n c e d s p r a y r e t e n t i o n and h e r b i c i d e p e n e t r a t i o n may have a s e c o n d a r y r o l e i n d i c l o f o p m e t h y l s e l e c t i v i t y ( T o d d , 1977) . G o r b a c h e t a l . (1977) s t u d i e d t h e d e g r a d a t i o n p r o d u c t s o f d i c l o f o p m e t h y l m e t a b o l i s m i n w h e a t . The main m e t a b o l i t e s i n c l u d e d p h y t o t o x i c d i c l o f o p ( 2 - ( 4 - ( 2 , 4 - d i c h l o r o p h e n o x y ) p h e n o x y ) p r o p a n o i c a c i d ) , non t o x i c h y d r o x y d i c l o f o p ( 2 - ( 4 -( 2 , 4 - d i c h l o r o - 5 - h y d r o x y - p h e n o x y ) p h e n o x y ) p r o p i o n i c a c i d ) and t h e i s o m e r s o f h y d r o x y d i c l o f o p w i t h h y d r o x y l g r o u p s a t t h e 3' and 6' p o s i t i o n s . Todd (1979) l a t e r i d e n t i f i e d an a d d i t i o n a l p h y t o t o x i c m e t a b o l i t e , 4 - ( 2 , 4 - d i c h l o r o p h e n o x y ) p h e n o l , r e f e r r e d t o a s p h e n o x y p h e n o l . T hese d i f f e r e n t m e t a b o l i c p r o d u c t s o f DM may be f o u n d f r e e i n t h e p l a n t o r p r e s e n t as c o n j u g a t e s ( S h i m a b u k u r o e t a l . 1979; Todd, 1979; G o r e c k a e t a l . 1981). The d i f f e r e n t r a t e s o f m e t a b o l i s m t o t h e s e compounds i n v a r i o u s - 8 -Figure 2, Pathway of DM metabolism i n plants, combined from Todd ( 1 9 7 9 ) and Shimabukuro et a l . ( 1 9 7 9 ) C l n Diclofop methyl (phytotoxic) C-COOCH. Cl CH. C l ^ r ^ 0 ^ ^ \ o - ( J - C O O H Diclofop (phytotoxic) H wheat and barley a l l species CH-°\j>°VJ>T00R Ester conjugate Hydroxy d i c l o f o p (non phytotoxic) Phenoxy phenol (non-phytotoxic) 0 - C-COOH i H 0-R Phenoxy conjugate (non-phytotoxic) -9-p l a n t s a c c o u n t f o r t h e s e l e c t i v i t y between s p e c i e s . I n b o t h s u s c e p t i b l e and t o l e r a n t s p e c i e s DM i s r a p i d l y c o n v e r t e d t o t h e a c i d f o r m by h y d r o l a s e enzymes ( G o r b a c h e t a l . 1977; S h i m a b u k u r o e t a l . 1979; T o d d , 1979; Todd and S t o b b e , 1980). B o t h DM and d i c l o f o p h ave h e r b i c i d a l a c t i v i t y . Todd (1979) c a l c u l a t e d t h e h a l f - l i f e o f d i c l o f o p m e t h y l , b e f o r e c o n v e r s i o n t o d i c l o f o p , t o be 1.0 h o u r s i n w i l d o a t and 1.8 h o u r s i n w h e a t . I n t o l e r a n t s p e c i e s t h e a c i d d i c l o f o p i s i r r e v e r s i b l y d e t o x i f i e d by a r y l h y d r o x y l a t i o n t o f o r m n o n - t o x i c h y d r o x y d i c l o f o p ( G o r b a c h e t a l . 1977; S h i m a b u k u r o e t a l . 1979; T o d d , 1979.) However, i n s u s c e p t i b l e s p e c i e s , d e t o x i f i c a t i o n by h y d r o x y l a t i o n o c c u r s a t a v e r y s l o w r a t e . The m a j o r m e t a b o l i c p a t h w a y i n s u s c e p t i b l e p l a n t s o c c u r s v i a f o r m a t i o n o f g l y c o s y l c o n j u g a t e s o f d i c l o f o p . T h i s c o n j u g a t i o n i s r e v e r s i b l e and i s t h o u g h t t o r e p r e s e n t a p o o l o f t o x i c a n t w h i c h c a n e x e r t f u r t h e r h e r b i c i d a l a c t i v i t y upon h y d r o l y s i s o f t h e g l y c o s y l c o n j u g a t e ( S h i m a b u k u r o e t a l . 1979; G o r e k a e t a l . 1981; Todd, 1979.) - 1 0 -T r a n s l o c a t i o n T r a n s l o c a t i o n o f DM and i t s m e t a b o l i t e s i s l i m i t e d i n p l a n t s ( B r e z e a n u e t a l . 1 9 7 6 ; S h i m a b u k u r o e t a l . 1979; T odd, 1979; Todd and S t o b b e , 1980; B o l d t and Putmann, 1 9 8 1 ) . Todd and S t o b b e ( 1 9 8 0 ) r e p o r t e d o n l y 1.6% o f r e c o v e r e d r a d i o l a b e l was t r a n s l o c a t e d f r o m t h e t r e a t m e n t zone i n 72 h o u r s . H owever, O l s o n and N a l e w a j a ( 1 9 8 2 ) r e p o r t e d h i g h e r t r a n s l o c a t i o n r a t e s ; t e n p e r c e n t o f t h e a b s o r b e d r a d i o l a b e l had moved f r o m t h e t r e a t e d a r e a 96 h o u r s a f t e r t r e a t m e n t . A l t h o u g h DM movement i n p l a n t s i s l i m i t e d , t r a n s l o c a t i o n i n b o t h t h e b a s i p e t a l and a c r o p e t a l d i r e c t i o n s h a s been o b s e r v e d ( T o d d , 1 9 7 9 ) . B r e z e a n u e t a l . ( 1 9 7 6 ) f o u n d 96 % o f t h e r e c o v e r e d r a d i o l a b e l i n t h e t r e a t m e n t z o n e ; o f t h e t r a n s l o c a t e d m a t e r i a l 0.7% o f a b s o r b e d r a d i o l a b e l a p p e a r e d i n t h e t r e a t e d l e a f a b o v e t h e t r e a t m e n t z o n e , 2.2% b e l o w t r e a t m e n t z o n e , 0 .5% i n r o o t s and 0.3% i n t h e s h o o t s . T h e s e r e s u l t s and t h o s e o f o t h e r a u t h o r s ( S h i m a b u k u r o e t a l . 1979; T odd, 1979) show a t e n d e n c y f o r DM t o move p r e f e r e n t i a l l y i n t h e b a s i p e t a l d i r e c t i o n . R a d i o l a b e l l e d h e r b i c i d e t r a n s l o c a t e d o u t o f t h e t r e a t m e n t zone a c c u m u l a t e d i n a r e a s o f h i g h m e t a b o l i c a c t i v i t y ( T odd 1 9 7 9 ) . T h e r e was no d e t e c t a b l e d i f f e r e n c e i n t h e t r a n s l o c a t i o n p a t t e r n s o f s u s c e p t i b l e and t o l e r a n t s p e c i e s ( B r e z e a n u e t a l . 1976; S h i m a b u k u r o e t a l . 1979; T odd, 1 9 7 9 ; ) . The d i s t r i b u t i o n p a t t e r n o f m e t a b o l i c p r o d u c t s i n s u s c e p t i b l e s p e c i e s i n d i c a t e d movement o f t h e e s t e r was n e g l i g i b l e c ompared t o t h e a c i d ( T o d d , 1 9 7 9 ) . The a c i d d i c l o f o p showed b o t h a c r o p e t a l and b a s i p e t a l movement, w i t h r e l a t i v e l y l a r g e amounts i n a r e a s a b o v e and b e l o w t h e t r e a t m e n t z o n e , and i n r o o t s and s h o o t s - 1 1 -( Todd , 1 9 7 9 ; ) . In w i l d oa t r o o t s 24 h o u r s a f t e r a p p l i c a t i o n , 8 2 . 1 % o f r a d i o l a b e l was i n the a c i d form compared t o 5.0% as DM. In the t r e a t m e n t z o n e , absorbed r a d i o l a b e l was 46 . 2 % DM and 23-8 % as d i c l o f o p . S i m i l a r t r e n d s i n movement and d i s t r i b u t i o n were obse r ved by Sh imabukuro e t a l . ( 1 9 7 9 ) . Mode o f A c t i o n Sh imabukuro e t a l . (1979) p roposed t h a t the two p h y t o t o x i c f o r m s , d i c l o f o p and d i c l o f o p m e t h y l , had d i f f e r e n t modes o f a c t i o n i n s u s c e p t i b l e p l a n t s . They sugges t ed t h a t g rowth i n h i b i t i o n was caused by DM, as d i c l o f o p m e t h y l was a s t r o n g e r a u x i n a n t a g o n i s t t han d i c l o f o p whereas u l t r a s t r u c t u r a l c e l l u l a r damage was t hough t to be caused by the a c i d d i c l o f o p . However, Todd and Stobbe (1980) argued the o p p o s i t e , s u g g e s t i n g DM caused the u l t r a s t r u c t u r a l damage and c h l o r o s i s , w h i l e d i c l o f o p i n h i b i t e d shoot and r o o t e l o n g a t i o n i n the m e r i s t e m a t i c a r e a s . D i c l o f o p p roved to be more m o b i l e i n p l a n t s than DM, and c o n c e n t r a t i o n s o f the a c i d i n m e r i s t e m a t i c r e g i o n s exceeded t h a t o f the e s t e r f o r m . A d d i t i o n a l s u p p o r t f o r the second p r o p o s a l , i s p r o v i d e d by : 1. Sh imabukuro e t a l . ( 1 9 7 8 ) r e p o r t e d d i c l o f o p was a b e t t e r i n h i b i t o r o f r o o t g rowth than DM. 2 . In s u s c e p t i b l e p l a n t s s e l e c t i v e p l acement o f the h e r b i c i d e on l e a f b l a d e s r e s u l t e d i n c h l o r o s i s o f the l e a f t i s s u e . P l acement on the shea th near the shoot me r i s t em r e s u l t e d i n both c h l o r o s i s and growth i n h i b i t i o n (Hoe rau f and Sh imabukuro , 1979) i n d i c a t i n g the p r e s e n c e o f the h e r b i c i d e near the me r i s t em was n e c e s s a r y f o r g rowth i n h i b i t i o n . - 1 2 -D i c l o f o p i s the major m e t a b o l i t e found i n the m e r i s t e m a t i c r e g i o n s o f f o l i a r - t r e a t e d w i l d oa t p l a n t s (Todd , 1 9 7 9 ) . 3 . Dona ld and Sh imabukuro (1981) i n j e c t e d w i l d oa t stems w i t h d i c l o f o p and d i c l o f o p m e t h y l . Growth o f the t h i r d l e a f was i n h i b i t e d e q u a l l y by both forms o f the h e r b i c i d e . I f DM was r e s p o n s i b l e f o r i n h i b i t i o n o f new shoot g r o w t h , i t would be e x p e c t e d t h a t t r e a t m e n t w i t h d i c l o f o p a l o n e would not show a s i m i l a r r e s p o n s e . An t agon i sm I n c o m p a t a b i l i t y o f 2,4-D and DM i n t ank m i x t u r e s was s u g g e s t e d as the b a s i s o f the a n t a g o n i s t i c i n t e r a c t i o n between t h e s e h e r b i c i d e s ( Q u e r e s h i and Vanden B o r n , 1 9 7 9 ) . A breakdown i n the e m u l s i o n o f DM and MCPA amine was o b s e r v e d , but not i n the MCPA e s t e r f o r m u l a t i o n s . In a d d i t i o n , MCPA amine was more a n t a g o n i s t i c t o d i c l o f o p m e t h y l a c t i v i t y than the e s t e r f o r m u l a t i o n . S i n c e no c o m p l e x i n g or d e g r a d a t i o n o f DM was d e t e c t e d when mixed w i t h 2,4-D e s t e r or amine f o r m u l a t i o n s ( O ' S u l l i v a n and Vanden B o r n , 1979; Todd and S t o b b e , 1980) i t was c o n c l u d e d t h a t the a c t i v e i n g r e d i e n t r a t h e r than the f o r m u l a t i o n or s u r f a c t a n t s was r e s p o n s i b l e f o r the a n t a g o n i s m . Q u e r e s h i and Vanden Born (1979) r e p o r t e d a r e d u c t i o n i n up t ake o f DM i n t o p l a n t t i s s u e when MCPA was combined w i t h d i c l o f o p m e t h y l . D i c l o f o p m e t h y l a l o n e r e s u l t e d i n 24% o f a p p l i e d h e r b i c i d e p e n e t r a t i n g the p l a n t t i s s u e . In the p r e s e n c e o f MCPA e s t e r and MCPA amine p e n e t r a t i o n was r educed to 15 .0 and 7.7% r e s p e c t i v e l y 24 hou r s a f t e r a p p l i c a t i o n s . However Todd and Stobbe (1980) and B o l d t and Putmann (1981) -13-r e p o r t e d no e f f e c t o f 2,4-D on DM u p t a k e and p e n e t r a t i o n i n t o w i l d o a t l e a v e s . S i x t y p e r c e n t o f t h e a p p l i e d DM was t a k e n up w i t h i n 24 h o u r s o f a p p l i c a t i o n w h e t h e r a l o n e o r i n c o m b i n a t i o n w i t h 2,4-D. S e v e r a l r e p o r t s h ave shown t h a t t h e a d d i t i o n o f 2,4-D (Todd and S t o b b e , 1980) o r MCPA ( Q u e r e s h i and Vanden B o r n , 1979) t o t a n k m i x t u r e s c a n r e d u c e t h e r a t e o f h y d r o l y s i s o f DM t o d i c l o f o p . T w e n t y f o u r h o u r s a f t e r DM a p p l i c a t i o n , 26% o f r e c o v e r e d r a d i o l a b e l i n t h e t r e a t m e n t zone was p r e s e n t a s d i c l o f o p m e t h y l . When DM was c o m b i n e d w i t h 2,4-D, 69.6% o f r e c o v e r e d r a d i o l a b e l was t h e e s t e r f o r m . T h e s e d a t a i n d i c a t e s an i n h i b i t i o n o f h y d r o l y s i s o f d i c l o f o p m e t h y l by 2,4-D (Todd and S t o b b e , 1 9 8 0 ) . S i m i l a r r e s u l t s were o b t a i n e d by Q u e r e s h i and Vanden B o r n ( 1 9 7 9 ) where c r u d e e x t r a c t s o f w i l d o a t l e a v e s c o n v e r t e d d i c l o f o p m e t h y l t o d i c l o f o p i n d i c a t i n g t h e p r e s e n c e o f h y d r o l y t i c a c t i v i t y i n c e l l - f r e e p r e p a r a t i o n s . The a d d i t i o n o f MCPA t o t h e c r u d e s u p e r n a t a n t i n h i b i t e d h y d r o l y s i s by 3 0 % . C o n f l i c t i n g r e s u l t s were r e p o r t e d by H i l l e t a l . ( 1 9 8 0 ) i n w h i c h 2,4-D had no e f f e c t on t h e r a t e o f h y d r o l y s i s o f DM i n  v i t r o u s i n g a p a r t i a l l y p u r i f i e d enzyme p r e p a r a t i o n . When DM and 2,4-D a r e a p p l i e d t o f o l i a g e as a t a n k m i x , g r o w t h o f w i l d o a t c o n t i n u e s , i n d i c a t i n g an a n t a g o n i s t i c h e r b i c i d e i n t e r a c t i o n . However, Todd and S t o b b e ( 1 9 8 0 ) d e m o n s t r a t e d t h a t when 2,4-D and DM were c o m b i n e d and a p p l i e d t o r o o t s , o r when 2,4-D was a p p l i e d t o r o o t s and DM was a p p l i e d t o t h e f o l i a g e , no a n t a g o n i s t i c i n t e r a c t i o n was o b s e r v e d . S h o o t and r o o t g r o w t h were i n h i b i t e d by d i c l o f o p m e t h y l . The a n t a g o n i s t i c i n t e r a c t i o n was n o t a t t h e s i t e o f a c t i o n i n t h e r o o t and s h o o t m e r i s t e m s , as a c o m b i n e d a p p l i c a t i o n t o t h e r o o t s w o u l d have shown an a n t a g o n i s t i c r e s p o n s e . I t was c o n c l u d e d t h a t when 2,4-D and DM were a p p l i e d t o f o l i a g e , 2,4-D must a c t t o r e d u c e t h e amount o f DM r e a c h i n g t h e s i t e o f a c t i o n ( T odd and S t o b b e , 1980) . The t r a n s l o c a t i o n p a t t e r n o f DM was a l s o a l t e r e d by t h e a d d i t i o n o f 2,4-D amine (Todd and S t o b b e , 1980). A l t h o u g h t h e q u a n t i t y o f r a d i o l a b e l l e d h e r b i c i d e m o v i n g away f r o m t h e t r e a t m e n t zone was u n a f f e c t e d , t h e d i r e c t i o n o f movement was a l t e r e d by 2,4-D. A c r o p e t a l movement o f r a d i o l a b e l i n t h e t r e a t e d l e a f was e n h a n c e d by a d d i t i o n o f 2,4-D. However, t h e q u a n t i t y o f d i c l o f o p m e t h y l r e a c h i n g t h e r o o t s and s h o o t was s i g n i f i c a n t l y l e s s i n 2 , 4 - D - t r e a t e d p l a n t s . I t was s u g g e s t e d t h a t t h e r e d u c t i o n i n h y d r o l y s i s o f d i c l o f o p m e t h y l t o d i c l o f o p a f t e r 2,4-D t r e a t m e n t r e s u l t e d i n h i g h e r l e v e l s o f d i c l o f o p m e t h y l i n t r e a t e d a r e a s , c a u s i n g i n c r e a s e d c h l o r o s i s and d e s t r u c t i o n o f t h e l e a f t i s s u e w h i c h a n y p r e c l u d e d b a s i p e t a l , s y m p l a s t i c t r a n s l o c a t i o n ( T odd and S t o b b e , 1980). O l s o n and N a l e w a j a (1982) c o n d u c t e d s i m i l a r e x p e r i m e n t s and a l s o o b s e r v e d a r e d u c t i o n i n b a s i p e t a l t r a n s l o c a t i o n o f r a d i o l a b e l l e d d i c l o f o p m e t h y l a f t e r MCPA t r e a t m e n t s . However, upward movement o f t h e r a d i o l a b e l was u n a l t e r e d by MCPA and t h e amount r e m a i n i n g i n t h e t r e a t m e n t z o n e i n c r e a s e d . B a s i p e t a l movement was i n h i b i t e d i f MCPA was p l a c e d 1.0 cm abo v e 1.0 cm b e l o w o r on t h e same zo n e a s d i c l o f o p m e t h y l . A s i m i l a r e f f e c t was o b s e r v e d f o r r a d i o l a b e l l e d g l u c o s e and s u c r o s e f o l l o w i n g MCPA a p p l i c a t i o n i n d i c a t i n g a g e n e r a l n o n - s p e c i f i c r e d u c t i o n i n b a s i p e t a l t r a n s l o c a t i o n ( O l s o n and N a l e w a j a , 1982). The -15-i n h i b i t i o n o f b a s i p e t a l t r a n s l o c a t i o n o f d i c l o f o p m e t h y l , g l u c o s e o r s u c r o s e c o u l d be m i m i c e d by d a r k t r e a t m e n t s . MCPA a p p l i e d t o p i c a l l y t o w i l d o a t l e a v e s n e a r t h e s i t e o f d i c l o f o p m e t h y l a p p l i c a t i o n i n h i b i t e d t h e downward movement a s much a s a t r e a t m e n t s p r a y e d on t h e e n t i r e p l a n t . The a u t h o r s c o n c l u d e d t h a t t h e i n h i b i t i o n o f downward t r a n s l o c a t i o n by MPCA was d e p e n d e n t on MCPA n e a r t h e s i t e o f d i c l o f o p m e t h y l a p p l i c a t i o n . -16-HERBICIDE UPTAKE AND TRANSLOCATION The a c t i v i t y o f a s y s t e m i c h e r b i c i d e i s dependent upon i t s a b i l i t y to r e a c h the s i t e o f a c t i o n i n the p l a n t . Thus , f a c t o r s wh ich l i m i t movement t h r o u g h the p l a n t may l i m i t t he e f f i c i e n c y o f the h e r b i c i d e . The a c t i o n o f a f o l i a r - a p p l i e d h e r b i c i d e depends upon the e f f i c i e n c y o f c u t i c u l a r p e n e t r a t i o n , the a b i l i t y to p e n e t r a t e the plasmalemma and e n t e r the s y m p l a s t , t r a n s p o r t e f f i c i e n c y w i t h i n the a p o p l a s t or s y m p l a s t , and r a t e o f m e t a b o l i s m and i m m o b i l i z a t i o n a t m e t a b o l i c a l l y n o n - a c t i v e s i t e s ( K i r k w o o d , 1 9 7 6 ) . The f o l i a g e o f a p l a n t i s cove r ed w i t h a l i p o i d a l c u t i c l e wh i ch a c t s as a b a r r i e r between the p l a n t and the e x t e r n a l e n v i r o n m e n t . The c u t i c l e merges w t i h the u n d e r l y i n g c e l l u l o s e o f the parenchyma c e l l w a l l s and i s c o n t i n u o u s w i t h the m i d d l e l a m e l l a . ( K i r k w o o d , 1 9 7 6 , 1978; P r i c e , 1976; 1 9 7 8 ) . Movement a c r o s s the c u t i c u l a r membrane i s a p h y s i c a l p r o c e s s d i r e c t l y i n f l u e n c e d by the m o l e c u l a r s i z e , pH, degree o f d i s s o c i a t i o n , p o l a r i t y , f o r m u l a t i o n and l i p i d s o l u b i l t y o f the h e r b i c i d e . Any f a c t o r wh i ch i n c r e a s e s l i p i d s o l u b i l i t y o f the h e r b i c i d e w i l l enhance c u t i c u l a r p e n e t r a t i o n . For h e r b i c i d a l a c t i v i t y , movement f rom the l i p i d c u t i c l e i n t o the aqueous phase o f the u n d e r l y i n g c e l l w a l l s and i n t e r c e l l u l a r spaces o f the a p o p l a s t i s e s s e n t i a l ( K i r k w o o d , 1 9 7 6 ) . B i n d i n g o f h e r b i c i d e s w i t h i n the c u t i c u l a r wax can i m m o b i l i z e the compound and r e n d e r i t n o n - t o x i c . D e s o r p t i o n o f l i p o p h i l i c compounds f rom the c u t i c l e i s c o n s i d e r e d a r a t e - l i m i t i n g s t e p i n h e r b i c i d e up take ( K i r k w o o d , 1 9 7 6 ) . K i r kwood (1972) found MCPB t o be immob i l e -17-and i n a c t i v e i n b r o a d beans a f t e r a b s o r p t i o n i n t h e c u t i c u l a r membranes. S i m i l a r l y Todd (1979) and H a l l (1981) c o n c l u d e d t h a t l a r g e p o r t i o n s o f r a d i o l a b e l l e d DM r e m a i n i n g i n t h e t r e a t m e n t z o n e s o f a p l a n t l e a f a r e i m m o b i l i z e d w i t h i n t h e c u t i c l e l a y e r . D i c l o f o p m e t h y l i s n e v e r c o m p l e t e l y h y d r o l y z e d i n t r e a t e d p l a n t s ( T o d d , 1979) i n d i c a t i n g t h a t a s i g n i f i c a n t p r o p o r t i o n o f h e r b i c i d e may r e m a i n d i s s o l v e d i n t h e c u t i c l e i n t h e e s t e r f o r m . H e r b i c i d e e s t e r f o r m u l a t i o n s w h i c h a r e s t r o n g l y l i p o p h i l i c e a s i l y p e n e t r a t e t h e c u t i c l e and may s u b s e q u e n t l y h y d r o l y z e and p a r t i t i o n i n t o t h e a q u e o u s p h a s e . D i c l o f o p m e t h y l may f u n c t i o n i n t h i s manner i f t h e n e c c e s s a r y h y d r o l a s e enzymes a r e p r e s e n t a t t h e c u t i c l e - c e l l w a l l i n t e r f a c e . H e r b i c i d e s w h i c h can d i f f u s e a c r o s s t h e c u t i c l e t o t h e a q u e o u s a p o p l a s t may t h e n move t h r o u g h t h e f r e e s p a c e and c e l l w a l l t o t h e v a s c u l a r t i s s u e ( P r i c e , 1978). Movement i n t h e a p o p l a s t i s s t r o n g l y a c r o p e t a l f o l l o w i n g w a t e r movement t o t h e l e a f t i p s and m a r g i n s . Compounds w i t h i n t h e a p o p l a s t move by d i f f u s i o n and mass f l o w u n l e s s t h e y a r e bound t o p l a n t c o m p o n e n t s o r c r o s s t h e plasmalemma f r o m t h e a p o p l a s t i n t o t h e s y m p l a s t ( P r i c e , 1976). T h e o r e t i c a l l y , no h e r b i c i d e c a n be c o m p l e t e l y a p o p l a s t i c s i n c e t o be p h y t o t o x i c t h e h e r b i c i d e must c r o s s t h e plasmalemma t o r e a c h a s i t e o f a c t i o n i n t h e s y m p l a s t ( P e t e r s o n and E d g i n g t o n , 1976). Compounds w i t h i n t h e s y m p l a s t may move f r o m c e l l t o c e l l v i a t h e p l a s m o d e s m a t a . I t h a s been s u g g e s t e d t h a t t h e p l a s m o d e s m a t a may c o n n e c t s p e c i a l i z e d p a r e n c h y m a c e l l s t o t h e s e i v e t u b e c e l l s i n t h e p h l o e m ( G e i g e r , 1971). However, -18-r e s e a r c h by G e i g e r e t a l . ( 1 9 7 1 ) and G i a q u i n t a ( 1 9 7 6 ) s u g g e s t s s u g a r s a r e l o a d e d i n t o p h l o e m f r o m t h e a p o p l a s t . P h l o e m l o a d i n g o f h e r b i c i d e s may f o l l o w a s i m i l a r r o u t e . I t h a s been assumed t h a t l o n g d i s t a n c e h e r b i c i d e t r a n s p o r t i n t h e p h l o e m i s s i m i l a r t o t h a t f o r n o r m a l p l a n t a s s i m i l a t e s . P h l o e m m o b i l i t y i s e n h a n c e d by f u n c t i o n a l g r o u p s t h a t r e a d i l y c o n j u g a t e w i t h s i m p l e s u g a r s and amino a c i d s . The r e m a i n d e r o f t h e m o l e c u l e m a i n t a i n s a l i p o p h i l i c / h y d r o p h i l i c b a l a n c e t h a t d o e s n o t s t r o n g l y f a v o r w a t e r o r l i p i d s ( C r i s p , 1 9 7 2 ) . The m e t a b o l i c p r o d u c t s o f d i c l o f o p m e t h y l , d i c l o f o p , h y d r o x y d i c l o f o p and p h e n o x y p h e n o l a r e p r e s e n t i n p l a n t s as s u g a r c o n j u g a t e s ( S h i m a b u k u r o e t a l . 1 9 7 9 ; Todd, 1 9 7 9 , G o r e c k a e t a l . 1 9 8 1 ) . Membrane t r a n s p o r t The i n t e r a c t i o n o f s m a l l m o l e c u l e s w i t h t h e plasmalemma d e t e r m i n e s how e a s i l y t h e m o l e c u l e moves i n t o and o u t o f c e l l s . P a s s a g e t h r o u g h t h e plasmalemma may be p a s s i v e a l o n g a c o n c e n t r a t i o n g r a d i e n t , o r a c t i v e , r e q u i r i n g e n e r g y t o a c c u m u l a t e a compound a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . T r a n s p o r t , w h e t h e r a c t i v e o r p a s s i v e , may a l s o be m e d i a t e d by a c a r r i e r as i n d i c a t e d by s a t u r a t i o n k i n e t i c s , s u b s t r a t e s p e c i f i c i t y , and i n h i b i t i o n by s p e c i f i c c o m p e t i t i v e i n h i b i t o r s ( L e h n i n g e r 1 9 7 6 ) . E a r l y s t u d i e s on membrane t r a n s p o r t r e l a t e d membrane p e r m e a b i l i t y d i r e c t l y t o t h e l i p o p h i l i c i t y o f t h e compound and i n v e r s e l y t o m o l e c u l a r s i z e and s h a p e . U p t a k e i n t o N i t e l l a c e l l s was e a s i e s t f o r l i p o p h i l i c m o l e c u l e s ( C o l l a n d e r , 1 9 5 4 ) . -19-The r e l a t i v e l i p o p h i l i c i t y o f the compound was d e t e r m i n e d by c a l c u l a t i o n o f the l o g o f the p a r t i t i o n c o e f f i c i e n t (P) i n a two phase aqueous and l i p i d sys tem . An o c t a n o l and water sys tem i s o f t e n used to d e t e r m i n e the p a r t i t i o n c o e f f i c e n t and l i p o p h i l i c i t y o f h e r b i c i d e s and f u n g i c i d e s ( B r i g g s e t a l . 1 9 7 6 ) . The a u t h o r s s u g g e s t e d membrane p e r m e a b i l t y was h i g h f o r h e r b i c i d e s w i t h l o g P i n the range o f 0-2 and was lower f o r more p o l a r m o l e c u l e s . T r a n s l o c a t i o n o f a h e r b i c i d e r e a ched a maximum a t l i p o p h i l i c i t y o f l o g P = 2 and d e c l i n e d a t h i g h e r v a l u e s , s u g g e s t i n g an optimum l i p o p h i l i c i t y f o r t r a n s l o c a t i o n ( B r i g g s e t a l . 1 9 7 6 ) . P e n n i s t o n (1969) p roposed t h a t h y d r o p h i l i c compounds have poo re r b i o l o g i c a l a c t i v i t y because o f t h e i r i n a b i l i t y t o p a r t i t i o n i n t o l i p i d membranes and e n t e r the s y m p l a s t . Ve r y l i p o p h i l i c compounds p a r t i t i o n s t r o n g l y i n t o l i p i d membranes but a re l e s s a b l e to move i n t o the aqueous m o b i l e phase ( P e n n i s t o n , 1 9 6 9 ) . H a l l (1981) c a l c u l a t e d the l o g o f the p a r t i t i o n c o e f f i c e n t s f o r DM, d i c l o f o p and 2,4-D to be 3 . 1 1 , 2 . 3 4 , and 0 .29 r e s p e c t i v e l y a t pH 5 - 3 . More r e c e n t r e s e a r c h has shown t h a t p o l a r compounds can c r o s s membranes more e f f e c t i v e l y than would be expec t ed on the s o l e b a s i s o f l i p i d p a r t i t i o n i n g . P r o t e i n s embedded i n the c e l l membranes may p r o v i d e a l t e r n a t e pathways f o r more p o l a r compounds ( P r i c e , 1976, 1 9 7 8 ) . Most p l a n t c e l l s e x p e r i e n c e a n e g a t i v e p o t e n t i a l d i f f e r e n c e a c r o s s the p lasma membrane c r e a t e d by e l e c t r i c a l and c h e m i c a l g r a d i e n t s w h i c h c o n t r o l d i f f u s i o n o f m o l e c u l e s i n t o and ou t o f the c e l l . However, many compounds may a c cumu la t e i n the c e l l t o c o n c e n t r a t i o n s h i g h e r than would be expec t ed i f - 2 0 -e l e c t r o c h e m i c a l p o t e n t i a l was the o n l y f a c t o r i n v o l v e d i n u p t a k e . Weak a c i d s p e n e t r a t e membranes i n the u n d i s s o c i a t e d form and d i s s o c i a t e i n the more a l k a l i n e c y t o p l a s m . I f t he membrane i s permeab le to the u n d i s s o c i a t e d form and l e s s pe rmeab le or impermeable t o the d i s s o c i a t e d f o r m , a c i d a c c u m u l a t e s i n the c y t o p l asm ( P r i c e , 1978) . Weak bases may a l s o c r o s s the membrane i n the u n d i s s o c i a t e d f o r m , rema in u n d i s s o c i a t e d i n the c y t o p l a s m , but may become c a t i o n i c i n the v a c u o l e ( P r i c e , 1976, 1 9 7 8 ) . Compounds may a c cumu la t e i n c e l l s t h r o u g h a c t i v e t r a n s p o r t ; i o n s and m o l e c u l e s c r o s s membranes a t r a t e s and q u a n t i t i e s t h a t exceed the l i m i t s o f s i m p l e d i f f u s i o n and the e l e c t r o c h e m i c a l e q u i l i b r i u m . A c t i v e t r a n s p o r t r e q u i r e s m e t a b o l i c energy to c r e a t e a hydrogen i o n g r a d i e n t a l o n g wh ich o t h e r compounds d i f f u s e ( P o o l e , 1 9 7 8 ) . A c t i v e t r a n s p o r t o f o r g a n i c compounds has been w i d e l y s t u d i e d i n a n i m a l s and m i c r o o r g a n i s m s and to a l e s s e r e x t e n t i n p l a n t s . However , l i t t l e e v i d e n c e o f a c t i v e t r a n s p o r t o f h e r b i c i d e s i s a v a i l a b l e . Rubery (1978) d e s c r i b e d two mechanisms f o r 2,4-D e n t r y i n t o s u s p e n s i o n - c u l t u r e d c e l l s . The u n d i s s o c i a t e d form c r o s s e d the plasmalemma by d i f f u s i o n i n an u n s a t u r a t e d , non-media ted p r o c e s s and accumu la t ed i n the c e l l when the i n c u b a t i o n medium was more a c i d i c than the c y t o p l a s m . 2,4-D was a l s o t aken up by a s a t u r a b l e , c a r r i e r - m e d i a t e d p r o c e s s s p e c i f i c f o r i n d o l e a c e t i c a c i d and 2 , 4 - D . I t was sugges t ed t h a t up take o f a u x i n and 2,4-D may o c c u r by c o - t r a n s p o r t w i t h hydrogen i o n s . Smej tek and P a u l i s - I l l a n g e g a s k a r e (1979) obse r ved p a s s i v e d i f f u s i o n o f n o n - d i s s o c i a t e d 2,4-D t h r o u g h an a r t i f i c a l l i p i d -21-b i l a y e r but no t the d i s s o c i a t e d f o r m . The components r e q u i r e d f o r med i a t ed t r a n s p o r t o f the l a t t e r form a re not p r e s e n t i n a r t i f i i c a l b i l a y e r s y s t e m s . M a r t i n and E d g i n g t o n (1981) demons t r a t ed 2 ,4-D a c c u m u l a t i o n i n p o t a t o t u b e r d i s c s to a c o n c e n t r a t i o n 15 t i m e s g r e a t e r than the ambient s o l u t i o n . A f t e r a f r e e z e - t h a w c y c l e t o k i l l the t i s s u e , the 2 ,4-D c o n c e n t r a t i o n e q u i l i b r a t e d between the p o t a t o t i s s u e and the ambient s o l u t i o n . Uptake s t u d i e s o f o t h e r h e r b i c i d e s , a t r a z i n e ( P e t e r s o n and E d g i n g t o n 1976) and g l y p h o s a t e ( M a r t i n and E d g i n g t o n , 1 9 8 1 ) , d i d not show a c c u m u l a t i o n a g a i n s t c o n c e n t r a t i o n g r a d i e n t s w h i l e H a l l ( 1 9 8 1 ) , w o r k i n g w i t h p o t a t o t u b e r d i s k s , r e p o r t e d d i c l o f o p m e t h y l and d i c l o f o p a c c u m u l a t i o n a g a i n s t a c o n c e n t r a t i o n g r a d i e n t , wh i ch was r e l e a s e d a f t e r a f r e e z e - t h a w c y c l e . He c o n c l u d e d these h e r b i c i d e s were t aken up by an a c t i v e p r o c e s s . -22 -EFFECTS OF HERBICIDES ON UPTAKE AND TRANSLOCATION U p t a k e and t r a n s l o c a t i o n i n p l a n t s may be i n h i b i t e d o r s t i m u l a t e d by t h e a c t i o n o f h e r b i c i d e s . Smejek and P a u l i s - I l l a n g e s k a r e ( 1 9 7 9 ) s t u d i e d t h e e f f e c t s o f 2,4-D on i o n t r a n s p o r t i n an a r t i f i c i a l l i p i d b i l a y e r s y s t e m . The u n d i s s o c i a t e d f o r m o f 2,4-D s t i m u l a t e d t h e t r a n s p o r t o f p o s i t i v e i o n s and i n h i b i t e d t h e t r a n s p o r t o f n e g a t i v e i o n s . I t was p r o p o s e d t h a t 2,4-D m o l e c u l e s were a b s o r b e d w i t h i n t h e membrane i n t e r f a c i a l r e g i o n , s u c h t h a t t h e o r i e n t a t i o n o f t h e 2,4-D d i p o l e c a u s e d a d e c r e a s e i n t h e e l e c t r i c a l p o t e n t i a l o f t h e membrane and d e c r e a s e d t h e r a t e o f u p t a k e n e g a t i v e l y c h a r g e d i o n s . However, t h e s e a f f e c t s were s m a l l a t a b i o l o g i c a l pH when t h e d i s s o c i a t e d 2,4-D i o n was p r e s e n t . S i m i l a r l y , K e n nedy ( 1 9 7 9 ) r e p o r t e d 2,4-D i n h i b i t i o n o f i o n u p t a k e i n m a i z e r o o t s by d e p o l a r i z a t i o n o f t h e t r a n s r o o t p o t e n t i a l ( t h e d i f f e r e n c e between t h e b a t h i n g s o l u t i o n and t h e x y l e m e x u d a t e o f e x c i s e d r o o t s ) a t pH 3.5. However, a t pH 5.5 o n l y s m a l l d e p o l a r i z a t i o n s were o b s e r v e d . Many r e p o r t s d e s c r i b e an i n c r e a s e o r d e c r e a s e i n u p t a k e and t r a n s l o c a t i o n a f t e r t r e a t m e n t w i t h p h e n o x y h e r b i c i d e s . U p t a k e and a c c u m u l a t i o n o f n a p h t h a l a m was i n c r e a s e d by 2,4-D t r e a t m e n t ( D e v l i n and Y a h l i c h , 1 9 7 2 ) , w h i l e p i c l o r a m t r e a t m e n t i n c r e a s e d t h e t r a n s l o c a t i o n o f 2,4-D ( A g b a k o d a and G o o d i n , 1 9 7 0 ) . MCPA p r e t r e a t m e n t r e d u c e d t r a n s p o r t o f r a d i o l a b e l l e d MCPA ( R o b e r t s o n and K i r k w o o d , 1 9 7 0 , ) . S i m i l a r l y , 2,4-D s i g n i f i c a n t l y r e d u c e d a b s o r p t i o n and t r a n s l o c a t i o n o f r a d i o l a b e l l e d 2,4-D i n y e l l o w n u t s e d g e (Bhan e t a l . 1970) as w e l l as r e d u c i n g t r a n s l o c a t i o n -23-o f TCA, c h l o r a m b e n , a m i t r o l e and d i c a m b a (Chow, 1 9 7 5 ) . R e d u c t i o n o f t r a n s l o c a t i o n by 2,4-D may r e s u l t f r o m h e r b i c i d e a c t i o n as an u n c o u p l e r o f t h e e l e c t r o n t r a n s p o r t c h a i n t h e r e b y r e d u c i n g m e t a b o l i c e n e r g y a v a i l a b l e f o r t r a n s l o c a t i o n and l o a d i n g m e c h a n i s m s . R o b e r t s o n and K i r k w o o d ( 1970) s u g g e s t e d t h a t d e c r e a s e d t r a n s l o c a t i o n f o l l o w i n g p h e n o x y t r e a t m e n t s r e s u l t e d f r o m p l u g g i n g o f phloem e l e m e n t s . Eames ( 1 9 5 0 ) o b s e r v e d d e s t r u c t i o n o f p h l o e m v e s s e l s and p r o l i f e r a t i n g p a r e n c h y m a c e l l s a f t e r t r e a t m e n t w i t h 2,4-D. -2k-MATERIALS AND METHODS PLANT MATERIAL Oat p l a n t s ( Avena s a t i v a L. ' C a s c a d e ' ) were grown i n a o g r o w t h chamber u n d e r i l l u m i n a t i o n o f 200 uE/m / s e c d u r i n g a 1 6 - h o u r p h o t o p e r i o d w i t h a 25 C d a y and 15 C n i g h t t e m p e r a t u r e r e g i m e . ISOLATION OF PROTOPLASTS Oat p r o t o p l a s t s were i s o l a t e d f r o m l e a f s e g m e n t s by e n z y m a t i c d i g e s t i o n o f c e l l w a l l s . F i v e grams o f 7-10 d a y o l d o a t s e e d l i n g s , a t t h e 1 t o 2 l e a f s t a g e , w e r e c u t i n t o s e g m e n t s 0.5mm t o 1.0mm i n l e n g t h . L e a f s e g m e n t s were i n c u b a t e d i n t h e d a r k a t 30 C f o r 3.5 h o u r s i n 25 ml o f enzyme s o l u t i o n m o d i f i e d f r o m Kao e t a l . ( 1 9 7 4) c o n s i s t i n g o f 2.0% (5000 u n i t s ) c e l l u l y s i n ( C a l b i o c h e m ) , 0.5% ( 3 7 5 u n i t s ) m a c e r a s e ( C a l b i o c h e m ) , 0.35M m a n n i t o l , 0 .35M s o r b i t o l , 6.0mM C a C l 2 .2H 20, 0 .7M NaH 2PO^.H 20 and 3.0mM Mes-NaOH (pH 5 . 7 ) . F o l l o w i n g c e l l w a l l d i g e s t i o n , p r o t o p l a s t s were p u r i f i e d by m o d i f i c a t i o n o f t h e p r o c e d u r e s o f Edwards e t a l . ( 1 9 7 9 ) . The d i g e s t i o n m i x t u r e was f i l t e r e d t h r o u g h two l a y e r s o f c h e e s e c l o t h , and t h e r e s i d u e r i n s e d t w i c e w i t h 25.0 ml s o r b i t o l b u f f e r ( 0.7M s o r b i t o l , 6 .OmM C a C l 2 .2H 20 and 5 .OmM Hepes-NaOH (pH 7 . 0 ) ) . The c o m b i n e d f i l t r a t e was c e n t r i f u g e d a t 300 X g f o r two m i n u t e s . The s u p e r n a t a n t was d i s c a r d e d and t h e p e l l e t s u s p e n d e d i n 5.0 m l o f b u f f e r c o n t a i n i n g 0 .7M s u c r o s e , 6.0 mM C a C l 2 .H 20 and 5.0 mM Hepes-NaOH (pH 7 . 0 ) . The s u s p e n s i o n was c a r e f u l l y o v e r l a i d w i t h 2.0 ml s o r b i t o l b u f f e r and c e n t r i f u g e d a t 300 X g -25-f o r 5.0 m i n u t e s . P u r i f i e d p r o t o p l a s t s c o l l e c t e d a t the i n t e r f a c e o f the s o r b i t o l and s u c r o s e l a y e r s , w h i l e damaged p r o t o p l a s t s and c e l l u l a r d e b r i s were p e l l e t e d . P r o t o p l a s t s were removed , r i n s e d i n s o r b i t o l b u f f e r and r e suspended i n s o r b i t o l b u f f e r . In a l l e x p e r i m e n t s the p r o t o p l a s t s u s p e n s i o n was s t a n d a r d i z e d to a c o n s t a n t c o n c e n t r a t i o n o f a p p r o x i m a t e l y one m i l l i o n p r o t o p l a s t s per m i l l i l i t e r b u f f e r . P r o t o p l a s t c o u n t s were made u s i n g a hemocy tomete r . P r o t e i n c o n t e n t o f the p r o t o p l a s t s u s p e n s i o n was d e t e r m i n e d by the Coomass ie b lue dye method o f B r a d f o r d ( 1 9 7 6 ) . T h i s a s s a y was chosen to a v o i d the Hepes b u f f e r i n t e r f e r e n c e w i t h c o l o u r deve lopment t h a t has been obse r ved w i t h the Lowry p r o t e i n d e t e r m i n a t i o n . B r a d f o r d (1976) has demons t r a t ed comparab le r e s u l t s between the Lowry a s s a y and the Coomasie dye d e t e r m i n a t i o n . The v i a b i l i t y o f the p r o t o p l a s t p r e p a r a t i o n s was e v a l u a t e d by the e x c l u s i o n o f non-permeant Evans b lue dye ( G a f f and O k o n g ' o - o g o l a , 1970) and the up take and c o n t a i n m e n t o f a permeant n e u t r a l r ed dye ( L i l l i e , 1969) . -26-MEMBRANE PERMEABILTY N e u t r a l r e d a s s a y The e f f e c t o f DM and d i c l o f o p on membrane p e r m e a b i l i t y o f i s o l a t e d o a t p r o t o p l a s t s was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l y by m o n i t o r i n g l e a k a g e o f n e u t r a l r e d dye f r o m damaged p r o t o p l a s t s . I s o l a t e d o a t p r o t o p l a s t s were i n c u b a t e d i n a 400:1 (V/W) s o l u t i o n o f n e u t r a l r e d dye f o r 5 m i n u t e s . P r o t o p l a s t s were r i n s e d i n 5 v o l u m e s s o r b i t o l b u f f e r and c e n t r i f u g e d a t 300 x g f o r 2 m i n u t e s . The p e l l e t was r e s u s p e n d e d i n 5 v o l u m e s s o r b i t o l b u f f e r and r e c e n t r i f u g e d . A f t e r 3 w a s h e s , t h e f i n a l p e l l e t was s u s p e n d e d i n one v olume s o r b i t o l b u f f e r . Two h u n d r e d u l o f d y e d p r o t o p l a s t s were added t o 1.0 ml o f t r e a t m e n t s o l u t i o n , made up o f s o r b i t o l b u f f e r and v a r y i n g c o n c e n t r a t i o n s o f d i c l o f o p m e t h y l , d i c l o f o p o r e t h a n o l . D i c l o f o p m e t h y l and d i c l o f o p t r e a t m e n t s were p r e p a r e d by d i s s o l v i n g t h e h e r i c i d e compounds i n 9 5 % e t h a n o l , t h e n a d d i n g m i c r o l i t r e amounts t o 1.0 ml s o r b i t o l b u f f e r . The f i n a l e t h a n o l c o n c e n t r a t i o n i n a l l DM and d i c l o f o p t r e a t m e n t s was m a i n t a i n e d a t 0.1% u n l e s s o t h e r w i s e s t a t e d . Dyed p r o t o p l a s t s were i n c u b a t e d i n t r e a t m e n t s o l u t i o n s a t room t e m p e r a t u r e f o r one h o u r and c e n t r i f u g e d a t 12,000 x g f o r 15 s e c o n d s . The s u p e r n a t a n t was d e c a n t e d f o r d e t e r m i n a t i o n o f a b s o r b a n c e a t 545 nm ( L i l l i e , 1 9 6 9 ) . -27-A t o m i c a b s o r p t i o n The v a l i d i t y o f t h e n e u t r a l r e d p e r m e a b i l i t y a s s a y was c o n f i r m e d by c o m p a r i s o n t o e x p e r i m e n t s i n w h i c h t h e l e a k a g e o f K+ i o n s f r o m p r o t o p l a s t s a f t e r t r e a t m e n t w i t h DM o r d i c l o f o p was d e t e r m i n e d by a t o m i c a b s o r p t i o n s p e c t r o s c o p y . Two h u n d r e d u l o f p r o t o p l a s t s were i n c u b a t e d i n v a r y i n g c o n c e n t r a t i o n s o f DM, d i c l o f o p , o r e t h a n o l . A l l h e r b i c i d e t r e a t m e n t s were p r e p a r e d as d e s c r i b e d a b o v e . A f t e r one h o u r i n c u b a t i o n , p r o t o p l a s t s were c e n t r i f u g e d a t 12,000 x g f o r 15 s e c o n d s . One h u n d r e d u l o f s u p e r n a t a n t were removed and d i l u t e d t o 10.0 ml w i t h d i s t i l l e d w a t e r . A l l s a m p l e s c o n t a i n e d 2.0 mg/ml N a C l t o r e d u c e i o n i z a t i o n o f K+ i o n s . The a t o m i c a b s o r p t i o n a t 766.5 nm was d e t e r m i n e d u s i n g a J a r r e l l Ash ( F i s h e r S c i e n t i f i c Co.) a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r w i t h an a i r - a c e t y l e n e f l a m e . The K+ c o n t e n t o f t r e a t m e n t s o l u t i o n s was c a l c u l a t e d by c o m p a r i s o n t o a s e r i e s o f s t a n d a r d K+ c o n c e n t r a t i o n s . -28-DICLOFOP METHYL AND DICLOFOP UPTAKE Uptake of DM and d ic lo fop by oat protoplasts was measured using 14C-diclofop methyl or 14C-dic lofop, uniformly l abe l l ed on the dioxyphenyl r i n g , with s p e c i f i c a c t i v i t i e s of 19.8mCi/g and 9.66 mCi/g respec t ive ly (Hoechst I n c . ) . Gas chromatographic analys is by Hoechst Inc. indicated the synthesized r a d i o l a b e l l e d herbic ide was pure. Thin layer chromatography in a benzene:methanol:acetic acid (85:10:5 V/V/V) solvent system (Gorbach et a l . 1977) indicated both r a d i o l a b e l l e d and nonradiolabel led mater ia l remained pure throughout the experiments. The r a d i o l a b e l l e d herbic ide was dissolved in 95% and ethanol then added in m i c r o l i t r e amounts to 200 u l of protoplast suspension. F i n a l concentration of ethanol was not permitted to exceed 1.0% ( v / v ) . The inf lux measurement was i n i t i a t e d by addit ion of r a d i o l a b e l l e d herbic ide to the protoplasts at room temperature and under normal room l i g h t . After the uptake per iod, protoplasts were separated by vacuum f i l t r a t i o n onto 2.4cm glass f ibre f i l t e r s (Whatman GF/C) . The f i l t e r s were r insed with s o r b i t o l buffer for 10 seconds (approximately equivalent to 15.0 ml b u f f e r ) . The f i l t e r s were placed in s c i n t i l l a t i o n v i a l s and 5.0ml s c i n t i l l a t i o n f l u i d (Handif luor , Mal l inckrodt Inc.) was added. Radioac t iv i ty was determined by s c i n t i l l a t i o n spectroscopy (Tracor Analy t i c Mark III 6881) with external s tandardizat ion . Background counts were determined by the addit ion of r a d i o l a b e l l e d herbic ide to 200ul s o r b i t o l buffer, without protoplas t s , followed by f i l t r a t i o n under vacuum onto glass f iber f i l t e r s . Radiolabel -29-r e t a i n e d on t h e s e f i l t e r s was d e t e r m i n e d and was u s e d t o c o r r e c t t h e v a l u e s f r o m t h e t r e a t m e n t s a m p l e s . A t i m e p r o f i l e o f DM and d i c l o f o p u p t a k e was d e t e r m i n e d f o r t h e i n c u b a t i o n p e r i o d s f r o m 5 s e c o n d s t o one h o u r , u s i n g a c o n s t a n t c o n c e n t r a t i o n o f 2.5uM 14C DM o r 14C d i c l o f o p . A c o n c e n t r a t i o n p r o f i l e f o r DM u p t a k e by p r o t o p l a s t s was d e t e r m i n e d . A m i x t u r e o f r a d i o l a b e l l e d and c o l d d i c l o f o p m e t h y l was p r e p a r e d and added t o 200 u l o f p r o t o p l a s t s u s p e n s i o n so t h a t 2.0 uM 14C DM was p r e s e n t i n e a c h t r e a t m e n t , and t h e c o n c e n t r a t i o n o f u n l a b e l l e d h e r b i c i d e v a r i e d f r o m 0.0 t o 48 .OuM g i v i n g a t o t a l DM c o n c e n t r a t i o n o f 2.0 t o 50.OuM. The u p t a k e o f DM and d i c l o f o p by i n t a c t and b r o k e n p r o t o p l a s t s was compared t o d i s t i n g u i s h u p t a k e i n t o p r o t o p l a s t s f r o m a d s o r p t i o n o n t o t h e p r o t o p l a s t e x t e r i o r . P r o t o p l a s t s were d i s r u p t e d by s u s p e n s i o n i n d i s t i l l e d w a t e r . Two h u n d r e d u l o f p r o t o p l a s t s were c e n t r i f u g e d a t 12,000 X g f o r 15 s e c o n d s . The s u p e r n a t a n t was removed and t h e p e l l e t r e s u s p e n d e d i n 2 0 0 u l d i s t i l l e d w a t e r and a g i t a t e d t o d i s r u p t t h e p r o t o p l a s t s . L i g h t m i c r o s c o p i c e x a m i n a t i o n o f t h e b r o k e n p r o t o p l a s t p r e p a r a t i o n s showed s w o l l e n c h l o r o p l a s t s and c e l l u l a r d e b r i s . U p t a k e o f 14C d i c l o f o p m e t h y l and 14C d i c l o f o p was d e t e r m i n e d a t 2.0, 4.0, and 8 .0 uM c o n c e n t r a t i o n s w i t h b r o k e n and i n t a c t p r o t o p l a s t p r e p a r a t i o n s . -39-DISTRIBUTION OF RADIOLABEL D e n s i t y g r a d i e n t f r a c t i o n a t i o n s S e p a r a t i o n o f p r o t o p l a s t s f r o m n o n - a b s o r b e d t r a c e r was c a r r i e d o u t i n a g r a d i e n t p r e p a r e d i n 1.5ml t u b e s a s f o l l o w s : 2 5 0 u l o f 5.0% F i c o l K Sigma) d i s s o l v e d i n s o r b i t o l b u f f e r s o l u t i o n were p l a c e d i n t h e b o t t o m o f t h e t u b e s w i t h s u c c e s s i v e l a y e r s c o n s i s t i n g o f 4 0 0 u l o f s i l i c o n e o i l ( d i m e t h y l d i p h e n y l p o l y s i l o x a n e , S i g m a ) , and 2 5 0 u l o f s o r b i t o l b u f f e r ( F i g u r e 3) F o u r h u n d r e d u l o f p r o t o p l a s t s u s p e n s i o n were i n c u b a t e d f o r one h o u r i n a 8 .OuM 14C DM s o l u t i o n a t room t e m p e r a t u r e w i t h o u t a g i t a t i o n . F o l l o w i n g i n c u b a t i o n , I 8 0 u l o f r a d i o l a b e l l e d p r o t o p l a s t s u s p e n s i o n were removed and l a y e r e d o n t o t h e t h r e e c omponent g r a d i e n t . Tubes were c e n t r i f u g e d a t 12,000 X g f o r 30 s e c o n d s . The p r o t o p l a s t s were p e l l e t e d , l e a v i n g t h e n o n - a b s o r b e d t r a c e r i n t h e u p p e r l a y e r s . . A l l t h r e e l a y e r s were removed by a s p i r a t i o n . The b o t t o m o f e a c h t u b e was c a r e f u l l y e x c i s e d and t h e c o n t a i n e d p e l l e t was d i s p e r s e d i n I 8 0 u l d i s t i l l e d w a t e r , f o l l o w e d by a d d i t i o n o f an e q u a l v o l u m e o f a c e t o n e added t o s t o p enzyme a c t i v i t y . T h i s f r a c t i o n i s r e f e r r e d t o as t h e p e l l e t . A s e c o n d 180 u l a l i q u o t o f t h e p r o t o p l a s t s s u s p e n s i o n was removed f r o m t h e t r e a t m e n t s o l u t i o n and c e n t r i f u g e d a t 12,000 X g f o r 30 s e c o n d s . The p e l l e t was d i s c a r d e d , 180 u l o f a c e t o n e was added t o t h e d e c a n t e d s u p e r n a t a n t , and d e s i g n a t e d t h e e x t e r n a l s o l u t i o n . ( F i g u r e 4.) One h u n d r e d m i c r o l i t r e s e a c h o f t h e p e l l e t and t h e e x t e r n a l s o l u t i o n s a m p l e s was t r a n s f e r r e d t o s c i n t i l l a t i o n v i a l s w i t h - 3 1 -Figure 3 . Density gradient fractionation tubes 0.7M sorbitol buffer silicone o i l 5$ F i c o l l in 0.7M sorbitol buffer -32-5.0 ml o f s c i n t i l a t i o n f l u i d ( H a n d i f l u o r , M a l l i n c k r o d t , I n c . ) An a d d i t i o n a l 100 u l s a m p l e was s e p a r a t e d by t h i n l a y e r c h r o m a t o g r a p h y (TLC) on p l a s t i c s i l i c a g e l 60 F 254 TLC p l a t e s (BDH C h e m i c a l ) . The p l a t e s were d e v e l o p e d t o 15cm i n b e n z e n e : m e t h a n o l : a c e t i c a c i d ( 8 5 : 1 0 : 5 , v / v / v ) ( G o r b a c h e t a l . 1977) • S e c t i o n s o f t h e TLC p l a t e s c o r r e s p o n d i n g t o t h e r e f e r e n c e s t a n d a r d s f o r d i c l o f o p m e t h y l , d i c l o f o p , and h y d r o x y d i c l o f o p a s o b s e r v e d u n d e r UV l i g h t , were c u t o u t and p l a c e d i n s c i n t i l l a t i o n v i a l s w i t h 5.0 m l o f s c i n t i l l a t i o n f l u i d . R a d i o a c t i v i t y o f a l l s a m p l e s was d e t e r m i n e d by s c i n t i l l a t i o n s p e c t r o s c o p y . The c o n v e r s i o n o f d i c l o f o p m e t h y l t o d i c l o f o p was c a l c u l a t e d f r o m t h e TLC d a t a . S o l u b i I t y f r a c t i o n a t i o n F o u r h u n d r e d m i c r o l i t r e s o f p r o t o p l a s t s u s p e n s i o n were i n c u b a t e d i n 8 .OuM 14C DM f o r 1.0 h o u r a t room t e m p e r a t u r e , t h e n f r a c t i o n a t e d i n t o t h e e x t e r n a l s o l u t i o n , w a t e r - s o l u b l e and w a t e r - i n s o l u b l e c o m p o n e n t s o f t h e p r o t o p l a s t s u s p e n s i o n . T h i s p r o c e d u r e i s o u t l i n e d i n F i g u r e 4. The d i s t r i b u t i o n o f r a d i o l a b e l i n t h e t h r e e f r a c t i o n s was d e t e r m i n e d by TLC and s c i n t i l l a t i o n s p e c t r o s c o p y a s d e s c r i b e d p r e v i o u s l y . P r o t e i n and p h o s p h o l i p i d c o n t e n t o f t h e w a t e r - s o l u b l e and i n s o l u b l e f r a c t i o n s were a l s o d e t e r m i n e d . P r o t e i n c o n t e n t was m e a s u r e d by t h e C o o m a s s i e b l u e p r o t e i n d e t e r m i n a t i o n d e s c r i b e d by B r a d f o r d ( 1 9 7 6 ) . T o t a l l i p i d was e x t r a c t e d f o l l o w i n g p r o c e d u r e s o f B l i g h and Dyer ( 1 9 5 9 ) f o l l o w e d by d i g e s t i o n i n 70% p e r c h l o r i c a c i d a t 200 C. I n o r g a n i c p h o s p h o r u s was d e t e r m i n e d by a m o d i f i e d p r o c e d u r e o f -33-Fixmre k Flow chart of protoplast f r a c t i o n a t i o n Figure 4 . * ^ o c t h e e x t e f n a i s o l u t i o n , water-soluble and water-insoluble f r a c t i o n s . protoplast suspension (J+OOul) I80ul centrifuge at 12,000 X g f o r 15' p e l l e t discard supernatant decant add acetone (1) I80ul centrifuge at 12,000 X g f o r 3 0 ' through the 3 layer gradient (Figure 3 ) pelljet disperse i n H 20 vortex centrifuge 12,000 X g for. 15' at p e l l e t disperse i n H 20 vortex add I80ul acetone (2) supernatant decant add I80ul acetone ( 3 ) (1) External s o l u t i o n (2) Water-insoluble f r a c t i o n ( 3 ) water-soluble f r a c t i o n - 3 4 -F i s k e and SubbaRow ( 1 9 2 5 ) as d e s c r i b e d i n a m a n u a l o f l a b o r a t o r y p r o c e d u r e s ( U B C - B i o c h e m i s t r y 5 0 1 ) . A 0.7 ml s a m p l e was added t o 14ml o f ammonium m o l y b d a t e s o l u t i o n ( 2 . 2 0 g ammonium m o l y d b a t e + 20ml c o n c e n t r a t e d s u l f u r i c a c i d ) and 0.6ml o f F i s k e - S u b b a R o w r e a g e n t ( 3 0 g NaHSO^ ; 1 g NaSO; 40.5g 1,2,4-a m i n o n a p h t h o l s u l f o n i c a c i d d i s s o l v e d i n 200ml d i s t i l l e d w a t e r ) . S a m p l e s were p l a c e d i n a b o i l i n g w a t e r b a t h f o r 15 m i n u t e s , a l l o w e d t o c o o l and t h e a b s o r b a n c e r e a d a t 660nm. -35-PH PROFILE The h y d r o l a s e enzymes r e s p o n s i b l e f o r t h e c o n v e r s i o n o f d i c l o f o p m e t h y l t o d i c l o f o p were e x t r a c t e d and p a r t i a l l y p u r i f i e d as d e s c r i b e d by H i l l e t a l . ( 1 9 8 0 ) . Ten mg. o f t h e l y o p h i l i z e d enzyme p r e p a r a t i o n were d i s s o l v e d i n 2.0ml o f 0.05M b u f f e r s o l u t i o n s a t pH 4.0, ( p o t a s s i u m p h t h a l a t e ) , pH 5.0 ( M e s ) , pH 6.0 ( M e s ) , pH 7.0 ( M o p s ) , pH 8.0 ( T r i c i n e ) , pH 9.0 ( C h e s ) , pH 10.0 ( p o t a s s i u m c a r b o n a t e / p o t a s s i u m b o r a t e - K O H ) . One ml o f e n z y m e - b u f f e r s o l u t i o n a t e a c h pH was p l a c e d i n a b o i l i n g w a t e r b a t h f o r 1.0 h o u r t o d e n a t u r e t h e h y d r o l a s e enzymes f o r use as c o n t r o l s . R a d i o l a b e l l e d DM was added t o 2 0 0 u l o f enzyme o r d e n a t u r e d enzyme s o l u t i o n t o a c o n c e n t r a t i o n o f 5.OuM and i n c u b a t e d a t 30 C f o r one h o u r . The r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 200 u l o f a c e t o n e . One h u n d r e d u l o f e a c h s o l u t i o n were a n a l y s e d by t h i n l a y e r c h r o m a t o g r a g h y and s c i n t i l l a t i o n s p e c t r o s c o p y as d e s c r i b e d p r e v i o u s l y . The p e r c e n t c o n v e r s i o n o f d i c l o f o p m e t h y l t o d i c l o f o p was d e t e r m i n e d a t e a c h pH and compared t o d e n a t u r e d enzyme c o n t r o l s . -36-RESULTS AND DISCUSSION PROTOPLAST VIABILITY The i n t e g r i t y of the protoplast membranes was shown by the exclusion of Evans blue dye and containment of neutral red dye. Gaff and Okang 1o-ogola (1970) demonstrated that Evans blue was excluded from most l i v e c e l l s due to the semi-permeable properties of the plasmalemma. Damaged or dead c e l l s were dyed by Evans blue indicating a loss of c e l l membrane i n t e g r i t y . Neutral red i s a non-toxic permeant dye widely used as a v i t a l s t a i n . In l i v e c e l l s neutral red i s taken up and contained by the tonoplast. Figures 5 and 6 show sample protoplast preparations stained with neutral red and Evans blue dye respectively. -37-Figure 5. Containment of neutral red dye in viable oat protoplasts. (X 300) -38-Figure 6. Exclusion of Evan's blue dye from viable oat protoplasts, (x 120) -39-MEMBRANE PERMEABILITY N e u t r a l r e d p e r m e a b i l i t y and K+ l e a k a g e e x p e r i m e n t s were t r e a t e d a s a r a n d o m i z e d b l o c k d e s i g n f o r s t a t i s t i c a l a n a l y s i s o f t h e d a t a . D i f f e r e n t p r o t o p l a s t p r e p a r a t i o n s were t r e a t e d a s b l o c k s t o p a r t i t i o n t h e v a r i a b l i t y i n h e r e n t i n p r e p a r a t i o n s c a r r i e d o u t a t d i f f e r e n t t i m e s . Under t h e e x p e r i m e n t a l c o n d i t i o n s u s e d , b o t h t h e n e u t r a l r e d p e r m e a b i l i t y and t h e K+ l e a k a g e d a t a showed s i g n i f i c a n t l i n e a r r e l a t i o n s h i p s between e t h a n o l , DM and d i c l o f o p c o n c e n t r a t i o n s and membrane p e r m e a b i l t y . T a b l e s 1a and 1b show t h e e f f e c t o f e t h a n o l on l e a k a g e o f n e u t r a l r e d dye and K+ f r o m p r o t o p l a s t s . A s i g n i f i c a n t l i n e a r r e l a t i o n s h i p was o b s e r v e d between i n c r e a s i n g e t h a n o l c o n c e n t r a t i o n s and l e a k a g e o f n e u t r a l r e d and K+ (ANOVA i n a p p e n d i x 1 and 2) . T h i s l i n e a r r e l a t i o n s h i p was n o t a r e s u l t o f h e t e r o g e n e o u s v a r i a n c e s s i n c e B a r t l e t t s T e s t f o r h o m o g e n e t i t y o f v a r i a n c e s showed t h a t t r e a t m e n t v a r i a n c e s were homogeneous. The d a t a p r e s e n t e d h e r e s u p p o r t a l i n e a r r e l a t i o n s h i p w i t h d e v i a t i o n s f r o m t h e l i n e a r n o t s i g n i f i c a n t . The r e g r e s s i o n c o e f f i c i e n t s c a l c u l a t e d f r o m t r e a t m e n t means a r e s m a l l , r e f l e c t i n g s h a l l o w s l o p e s . Duncan's m u l t i p l e r a n g e t e s t was u s e d t o compare t r e a t m e n t means. B e c a u s e o f t h e s m a l l r e g r e s s i o n c o e f f i c e n t , a s i g n i f i c a n t d i f f e r e n c e between c o n c e n t r a t i o n s o f 0.0%, 0.1% and 1.0% e t h a n o l was n o t d e t e c t e d . A t l o w e t h a n o l c o n c e n t r a t i o n s t h e r e was no d e t e c t a b l e s i g n i f i c a n t d i f f e r e n c e between t r e a t m e n t means. The e f f e c t s o f d i c l o f o p m e t h y l on membrane p e r m e a b i l i t y a r e - 4 0 -T a b l e 1 a, The e f f e c t o f e t h a n o l on l e a k a g e o f n e u t r a l r e d dye f r o m o a t p r o t o p l a s t s . e t h a n o l c o n e . A545 % i n c r e a s e o v e r P e r c e n t c o n t r o l 0.0 0.314a 0.00 0.1 0.313a -0.23 1.0 0 .330a 5.19 5 .0 0.412b 31 .18 10 .0 0.502c 59.96 E a c h t r e a t m e n t v a l u e i s a m r e p l i c a t i o n s p e r t r e a t m e n t V a l u e s f o l l o w e d by t h e same d i f f e r e n t a t t h e 5.0% l e v e l ean o f 3 e x p e r i m e n t s , w i t h 3 i n e a c h e x p e r i m e n t . l e t t e r a r e n o t s i g n i f i c a n t l y by Duncan's m u l t i p l e r a n g e t e s t . R e g r e s s i o n e q u a t i o n o f e t h a n o l c o n c e n t r a t i o n and A545 Y= .0191X + .3128. r= .9996 ' -kl-T a b l e s 1b, The e f f e c t o f e t h a n o l on t h e l e a k a g e o f K+ f r o m o a t p r o t o p l a s t s . e t h a n o l c o n e . ppm K+ % i n c r e a s e o v e r p e r c e n t c o n t r o l 0 .0 1 .02a 0.00 0.1 1.03a 0.98 1 .0 1 .00a -2.56 10.0 2.22b 118.28 T r e a t m e n t v a l u e s a r e t h e mean o f t h r e e e x p e r i m n e t s , w i t h 3 r e p l i c a t i o n s p e r t r e a t m e n t i n e a c h e x p e r i m e n t . V a l u e s f o l l o w e d by t h e same l e t t e r s a r e n o t s i g n i f i c a n t l y d i f f e r e n t by Duncan's m u l t i p l e r a n g e t e s t s a t t h e 5.0% l e v e l s i g n i f i c a n c e . R e g r e s s i o n e q u a t i o n o f e t h a n o l c o n c e n t r a t i o n and ppm K+ Y=.1220X + .9701 r= .9930 - 4 2 -shown in Table 2a and 2b. There was no s i g n i f i c a n t detectable e f fect of DM on membrane permeabilty at 10 or 50uM. Only 100uM DM resul ted in a s t a t i s t i c a l l y s i g n i f i c a n t d i f ference between treatment means. However, a s i g n i f i c a n t l inear re la t i onsh ip was observed between DM concentration and increased leakage of neutra l red dye and K+. As DM concentration was increased, permeabilty to neutral red dye and K+ increased ind ica t ing a gradual loss of membrane i n t e g r i t y (ANOVA in appendix 3 and 4). Addit ion of the a c i d , d i c l o f o p , resulted in a less pronounced effect on membrane permeabi l t iy . The resu l t s of the neutral red permeabi l i ty test and K+ leakage are presented in Tables 3a and 3b. A s i g n i f i c a n t l inear re la t ionsh ip was observed between d ic lo fop and leakage of neutral red dye and K+ (ANOVA in appendix 5 and 6 ) . However, no s i g n i f i c a n t d i f ference between d ic lo fop treatments was detected by Duncan's mul t ip le range test at the 5.0% leve l of s i g n i f i c a n c e . The re su l t s indicate DM and d ic lo fop have l imited ef fects on membrane permeabi l i ty under the experimental condit ions tes ted . S i g n i f i c a n t l inear re la t ionsh ips were observed with increas ing concentrat ions , but the small regression coe f f i c i en t s indicated only s l i g h t increases in membrane permeabi l i ty over the concentration range tes ted . The primary purpose of the membrane permeabil i ty tests was to e s tab l i sh concentration l i m i t s for ethanol , DM, and d ic lo fop which would re su l t in a detectable increase in membrane permeabi l i ty . A l l herbic ide solut ions were prepared in ethanol as described in the methods sec t ion . Precautions were taken to reduce ethanol concentrations below leve ls which caused -1+3-T a b l e 2 a . The e f f e c t o f d i c l o f o p m e t h y l on t h e l e a k a g e o f n e u t r a l r e d dye f r o m o a t p r o t o p l a s t s . DM c o n e . uM A545 % i n c r e a s e o v e r c o n t r o l 0 .0 0 . 3 0 0 a 0 .00 10 .0 0 .303a '.1.10 50 .0 0 .318a 6.10 100 .0 0 .338b 12 .65 A l l h e r b i c i d e t r e a t m e n t s c o n t a i n e d 0.1% e t h a n o l T r e a t m e n t v a l u e s a r e t h e mean o f 6 e x p e r i m e n t s , w i t h 3 r e p l i c a t e s p e r t r e a t m e n t i n e a c h e x p e r i m e n t . V a l u e s f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t by Duncan's m u l t i p l e r a n g e t e s t a t 5.0% l e v e l o f s i g n i f i c a n c e . R e g r e s s i o n e q u a t i o n o f DM c o n c e n t r a t i o n and A545 . Y=3 . 8 x 10 X + . 2 9 9 3 r= . 9 9 9 8 T a b l e 2b The e f f e c t s o f DM on l e a k a g e o f K+ f r o m o a t p r o t o p l a s t s DM c o n e . ppm K+ % i n c r e a s e o v e r uM c o n t r o l 0.0 0.89ab 0.00 10 .0 0 .88a -1 .41 50.0 1 .05bc 16.00 100 .0 1 .15c 26 .00 A l l h e r b i c i d e t r e a t m e n t s c o n t a i n e d 0.1% e t h a n o l T r e a t m e n t v a l u e s a r e t h e mean o f 3 e x p e r i m e n t s w i t h 3 r e p l i c a t e s p e r t r e a t m e n t i n e a c h e x p e r i m e n t . V a l u e s f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t by Duncan's m u l t i p l e r a n g e t e s t a t t h e 5.0% l e v e l s i g n i f i c a n c e . R e g r e s s i o n e q u a t i o n o f DM c o n c e n t r a t i o n and ppm K+ Y= .0028X + .8768 r= .9795 T a b l e s 3 a , The e f f e c t o f d i c l o f o p on t h e l e a k a g e o f n e u t r a l r e d dye f r o m o a t p r o t p o l a s t s . d i c l o f o p c o n e . uM A545 % i n c r e a s e o v e r c o n t r o l 0 .0 0 .220a 0 .00 10 .0 0 .229a 4 . 0 9 50 .0 0 . 2 3 4 a 6 .51 100 .0 0 .241a 9 .60 A l l h e r b i c i d e t r e a t m e n t s c o n t a i n 0.1% e t h a n o l . T r e a t m e n t v a l u e s a r e means o f 4 e x p e r i m e n t s , w i t h 3 r e p l i c a t e s p e r t r e a t m e n t i n e a c h e x p e r i m e n t . V a l u e s f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t by Du n c a n ' s m u l t i p l e r a n g e t e s t a t t h e 5.0% l e v e l o f s i g n i f i c a n c e . R e g r e s s i o n e q u a t i o n o f d i c l o f o p c o n c e n t r a t i o n and A545 Y=1 . 8 3 7 x 10 X + . 2 2 3 5 r= . 9 3 7 - 4 6 -T a b l e 3b, The e f f e c t o f d i c l o f o p on l e a k a g e o f K+ f r o m o a t p r o t o p l a s t s . d i c l o f o p c o n e . uM ppm K+ % i n c r e a s e o v e r c o n t r o l 0 .0 0 .78a 0 .00 10 .0 0 .80a 2 .04 50 .0 0.79a 1.14 100 .0 0 .85a 9 .25 A l l h e r b i c i d e t r e a t m e n t s c o n t a i n 0.1% e t h a n o l E a c h t r e a t m e n t v a l u e i s a mean o f 2 e x p e r i m e n t s , w i t h 3 r e p l i c a t e s p e r t r e a t m e n t i n e a c h e x p e r i m e n t . V a l u e s f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t a t t h e 5.0% Duncan's m u l t i p l e r a n g e t e s t . R e g r e s s i o n e q u a t i o n o f d i c l o f o p c o n c e n t r a t i o n and ppm K+ Y=6.36x 10 X + .7797 r= .8864 -47-d e t e c t a b l e membrane damage. B o t h t h e n e u t r a l r e d p e r m e a b i l i t y t e s t and a t o m i c a b s o r p t i o n o f K+ l e a k a g e gave s i m i l a r r e s u l t s , s u p p o r t i n g t h e v a l i d i t y o f t h e s e t e s t s . S e v e r a l h e r b i c i d e s , i n c l u d i n g g l y p h o s a t e , d a l a p o n , b r o m a c i l , p r o m e t y n e , l i n u r o n , ( C h r o w l e y and P r e n d e v i l l e , 1980) and d i c l o f o p m e t h y l ( C h r o w l e y and P r e n d e v i l l e , 1979) h a v e been r e p o r t e d t o i n c r e a s e c e l l membrane p e r m e a b i l i t y . I n t h o s e s t u d i e s , l o s s o f membrane i n t e g r i t y was d e t e r m i n e d by an i n c r e a s e i n c o n d u c t i v i t y o f a s o l u t i o n c o n t a i n i n g h e r b i c i d e - t r e a t e d l e a f s e g m e n t s . I n c r e a s e d c o n d u c t i v i t y o c c u r r e d w i t h i n 12 h o u r s o f a f o l i a r a p p l i c a t i o n o f DM a t f i e l d r a t e s s u g g e s t i n g t h a t DM i n c r e a s e d membrane p e r m e a b i l i t y i n i n t a c t p l a n t s . However, t h e s e s t u d i e s d i d n o t p r o v i d e d i r e c t i n f o r m a t i o n t o p r e d i c t DM e f f e c t s on p r o t o p l a s t s a t t h e c o n c e n t r a t i o n s and e x p o s u r e p e r i o d s u s e d i n t h e s e e x p e r i m e n t s . The e x p e r i m e n t a l c o n d i t i o n s o f t h e p e r m e a b i l i t y t e s t s d e s c r i b e d i n t h i s t h e s i s were c h o s e n t o e v a l u a t e s t a n d a r d c o n d i t i o n s f o r f u t u r e e x p e r i m e n t s . Under t h e s e c o n d i t i o n s , one ho u r i n c u b a t i o n , room t e m p e r a t u r e , no a g i t a t i o n , c o n c e n t r a t i o n s o f 50uM DM and 100uM d i c l o f o p o r l e s s ( i n 0.1% e t h a n o l ) r e s u l t e d i n no d e t e c t a b l e c h a n g e s i n membrane p e r m e a b i l i t y . L o n g e r i n c u b a t i o n p e r i o d s o r h i g h e r DM and d i c l o f o p c o n c e n t r a t i o n s may ha v e r e s u l t e d i n i n c r e a s e d membrane damage. B l e i n ( 1 9 8 1 , 1 9 8 2 ) s t u d i e d t h e e f f e c t s o f s e v e r a l h e r b i c i d e s on membrane p e r m e a b i l t i y o f A c e r p s e u d o p l a t a n u s c e l l c u l t u r e s . The r e l e a s e o f f l u o r e s c e i n dye f r o m c u l t u r e d c e l l s was m e a s u r e d a f t e r one ho u r i n c u b a t i o n s i n 100uM h e r b i c i d e -48-s o l u t i o n s . A l t h o u g h d i c l o f o p methyl was not t e s t e d i n t h i s s t u d y f l u o r o d i f e n , i o x i n i l , d i n o s e b , DNOC, desmetryne, m o n o l i n u r o n , c y c l u r o n , i s o p r o t u r o n and monuron were found t o i n c r e a s e f l u o r e s c e i n l e a k a g e . These r e s u l t s are c o n s i s t e n t w i t h the o b s e r v a t i o n s i n t h i s t h e s i s on DM and d i c l o f o p e f f e c t s on p r o t o p l a s t s a t s i m i l a r c o n c e n t r a t i o n s . -49-DICLOFOP METHYL AND DICLOFOP UPTAKE The t i m e p r o f i l e o f 14C-DM u p t a k e by o a t p r o t o p l a s t s f r o m a 2.5uM DM s o l u t i o n i s shown i n T a b l e 4. E x p e r i m e n t s c o n d u c t e d w i t h d i f f e r e n t p r o t o p l a s t p r e p a r a t i o n s were t r e a t e d a s b l o c k s t o remove v a r i a b i l i t y due t o d i f f e r e n c e s between p r o t o p l a s t p r e p a r a t i o n s . DM was r a p i d l y ( _< 5 s e c o n d s ) t a k e n up by p r o t o p l a s t s f o l l o w i n g a d d i t i o n o f t h e r a d i o l a b e l l e d h e r b i c i d e , and d i d n o t c h a n g e s i g n i f i c a n t l y between 5.0 s e c o n d s and 60.0 m i n u t e s . I n d i v i d u a l d e g r e e o f f r e e d o m c o n t r a s t s , c o m p a r i n g a l l t i m e p e r i o d s t o t h e c o n t r o l (no p r o t o p l a s t s ) i n d i c a t e d t h a t t h e c o n t r o l was s i g n i f i c a n t l y d i f f e r e n t f r o m u p t a k e a t a n y t i m e p e r i o d . D i f f e r e n c e s between t i m e p e r i o d s were n o t s i g n i f i c a n t (ANOVA i n a p p e n d i x 7) . U p t a k e v a l u e s a r e e x p r e s s e d as p i c o m o l e s DM. P o s s i b l e m e t a b o l i s m o f r a d i o l a b e l l e d DM wa 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 . The t i m e p r o f i l e o f d i c l o f o p u p t a k e i s p r e s e n t e d i n T a b l e 5. D i c l o f o p was a l s o t a k e n up by p r o t o p l a s t s t o a r e l a t i v e l y c o n s t a n t l e v e l w i t h i n 5.0 s e c o n d s . T r e a t m e n t t i m e p e r i o d s and t h e c o n t r o l were s i g n i f i c a n t l y d i f f e r e n t , b u t d i f f e r e n c e s b etween t i m e p e r i o d s were n o t s i g n i f i c a n t (ANOVA i n a p p e n d i x 8) . C o m p a r i n g DM and d i c l o f o p u p t a k e ( T a b l e s 4 and 5) t h e d a t a i n d i c a t e DM u p t a k e i s a p p r o x i m a t e l y 10 t i m e s t h a t o f d i c l o f o p . F o r t y p e r c e n t o f DM i n t h e i n c u b a t i o n s o l u t i o n was t a k e n up i n t o p r o t o p l a s t s i n 1.0 h o u r . I n c o n t r a s t , o n l y 4.0% o f d i c l o f o p was t a k e n up d u r i n g a s i m i l a r t i m e p e r i o d . The low u p t a k e v a l u e s f o r d i c l o f o p make i n t e r p r e t a t i o n o f t h e d a t a -50-T a b l e 4 Time p r o f i l e o f DM u p t a k e T r e a t m e n t p i c o m o l e s DM c o n t r o l 48.4 5 s e c o n d s 198 .9 10 s e c o n d s 202.6 30 s e c o n d s 181 .5 1 m i n u t e 199 .0 10 m i n u t e s 192.2 60 m i n u t e s 167.5 T r e a t m e n t v a l u e s a r e means o f 3 e x p e r i m e n t s w i t h 3 r e p l i c a t e s p e r t r e a t m e n t i n e a c h e x p e r i m e n t . The c o n t r o l r e p r e s e n t s p i c o m o l e s o f 14C-DM r e m a i n i n g on t h e f i l t e r p a p e r , when p r o t o p l a s t s w e r e o m i t t e d f r o m t h e p r o c e d u r e DM c o n c e n t r a t i o n i n t h e i n c u b a t i o n s o l u t i o n was 2.5uM One p i c o m o l e i s e q u i v a l e n t t o 14.8 DPM 14C-DM. -51-T a b l e 5 Time p r o f i l e o f d i c l o f o p u p t a k e . T r e a t m e n t p i c o m o l e s d i c l o f o p c o n t r o l 5.8 5 s e c o n d s 23.9 10 s e c o n d s 19.9 30 s e c o n d s 23 .5 1 m i n u t e 21 .6 10 m i n u t e s 17 .6 60 m i n u t e s 25 .0 T r e a t m e n t v a l u e s a r e t h e means o f 3 e x p e r i m e n t s w i t h 3 r e p l i c a t e s p e r t r e a t m e n t i n e a c h e x p e r i m e n t . The c o n t r o l r e p r e s e n t s p i c o m o l e s 1 4 C - d i c l o f o p r e m a i n i n g on t h e f i l t e r p a p e r when p r o t o p l a s t s were o m i t t e d . D i c l o f o p c o n c e n t r a t i o n i n t h e i n c u b a t i o n s o l u t i o n was 2,5uM One p i c o m o l e i s e q u i v a l e n t t o 6.95 DPM 1 4 C - d i c l o f o p . -52-d i f f i c u l t . F o r e x a m p l e , a 2 ,5uM d i c l o f o p s o l u t i o n c o n t a i n i n g 3490 DPM r e s u l t e d i n 40 DPM i n c o n t r o l t r e a t m e n t s and 150DPM i n p r o t o p l a s t . T h i s low u p t a k e by p r o t o p l a s t s may be t h e r e s u l t o f r a d i o l a b e l a d s o r b i n g t o t h e s u r f a c e o f t h e p r o t o p l a s t s . D i c l o f o p i s a p p a r e n t l y n o t e f f e c t i v e l y t a k e n up by p r o t o p l a s t s . The c o n c e n t r a t i o n p r o f i l e o f DM u p t a k e f r o m 2.OuM t o 50 .OuM i s shown i n F i g u r e 7. A s i g n i f i c a n t l i n e a r r e l a t i o n s h i p between DM c o n c e n t r a t i o n and DM u p t a k e was o b s e r v e d ( a p p e n d i x 9 ) . The amount o f DM t a k e n up by p r o t o p l a s t s i s p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f DM i n t h e i n c u b a t i o n s o l u t i o n . No s a t u r a t i o n o f DM u p t a k e a t h i g h c o n c e n t r a t i o n s was o b s e r v e d i n d i c a t i n g t h a t DM u p t a k e was n o t l i k e l y t o be c a r r i e r - m e d i a t e d a t c o n c e n t r a t i o n s u s e d i n t h e s e e x p e r i m e n t s . The t e r m ' u p t a k e ' h a s been u s e d i n t h i s t h e s i s t o d e s c r i b e t h e r a d i o l a b e l r emoved f r o m t h e i n c u b a t i o n s o l u t i o n by t h e p r o t o p l a s t s . T h e r e i s no e x p e r i m e n t a l e v i d e n c e t o d i s t i n g u i s h b e t w een a d s o r p t i o n o r a b s o r t i o n t o membranes o r e n t r y i n t o t h e c y t o p l a s m o f t h e p r o t o p l a s t s . The u p t a k e o f DM and d i c l o f o p i n t o i n t a c t and b u r s t p r o t o p l a s t i s p r e s e n t e d i n T a b l e 6. I n d i v i d u a l d e g r e e o f f r e e d o m c o n t r a s t s w e r e u s e d t o compare c o n t r o l v a l u e s ( w i t h o u t p r o t o p l a s t s ) t o t r e a t m e n t s ( b u r s t o r i n t a c t p r o t o p l a s t s ) . A s i g n i f i c a n t d i f f e r e n c e between c o n t r o l s and t r e a t m e n t s was o b s e r v e d f o r b o t h d i c l o f o p and DM u p t a k e . However, t h e r e was no s i g n i f i c a n t d i f f e r e n c e between DM u p t a k e i n t o i n t a c t p r o t o p l a s t s o r b u r s t p r o t o p l a s t f r a g m e n t s , i n d i c a t i n g DM u p t a k e d o e s n o t d epend on i n t a c t v i a b l e p r o t o p l a s t s . I n c o n t r a s t d i c l o f o p u p t a k e i n t o i n t a c t p r o t o p l a s t s was s i g n i f i c a n t l y - 5 3 -Figure 7. Concentration p r o f i l e of d i c l o f o p methyl uptake i n oat protoplasts. -54-T a b l e 6. U p t a k e o f DM and d i c l o f o p by i n t a c t and b u r s t p r o t o p l a s t s . c o n c e n t r a t i o n t r e a t m e n t p i c o m o l e s p i c o m o l e s DM d i c l o f o p 2 .OuM c o n t r o l 41 .0 7.3 2 .0 b u r s t 201 .2 14.3 2 .0 i n t a c t 210.4 24 .1 4 .0 c o n t r o l 62 .4 9 .7 4 .0 b u r s t 396 .9 28 .1 4 .0 i n t a c t 432 .6 36 .8 8 .0 c o n t r o l 67 .0 11 .2 8 .0 bur s t 753 .3 36 .2 8 .0 i n t a c t 785 .9 42 .9 T r e a t m e n t v a l u e s a r e t h e means o f 3 e x p e r i m e n t s w i t h 3 r e p l i c a t e s p e r t r e a t m e n t i n e a c h e x p e r i m e n t . C o n t r o l v a l u e s r e p r e s e n t t h e p i c o m o l e s o f h e r b i c i d e r e m a i n i n g on t h e f i l t e r p a p e r when p r o t o p l a s t s were o m i t t e d . DM and d i c l o f o p e x p e r i m e n t s were n o t c o n d u c t e d w i t h t h e same p r o t o p l a s t p r e p a r a t i o n . One p i c o m o l e i s e q u i v a l e n t t o 14.8 DPM 14C-DM o r 6.95 DPM 1 4 C - d i c o l f o p . -55-g r e a t e r t h a n u p t a k e by b u r s t c e l l s (ANOVA i n a p p e n d i x 10) . T h i s d i f f e r e n c e may i n d i c a t e t h a t d i c l o f o p u p t a k e i s a p r o c e s s i n v o l v i n g i n t a c t v i a b l e p r o t o p l a s t s . Such a c o n c l u s i o n , h o w e v e r , must be c o n s i d e r e d s p e c u l a t i v e i n v i e w o f t h e v a r i a b l e and l ow u p t a k e c o u n t s a s s o c i a t e d w i i t h d i c l o f o p m e a s u r e m e n t s . I t c a n be c o n c l u d e d t h a t DM i s r a p i d l y t a k e n up i n t o p r o t o p l a s t s . DM u p t a k e i s 10 t i m e s g r e a t e r t h a n t h a t o f d i c l o f o p . The amount o f DM t a k e n up i s d e p e n d e n t upon t h e e x t e r n a l c o n c e n t r a t i o n and t h e r e f o r e i s n o t c a r r i e r m e d i a t e d . S i n c e DM u p t a k e d oes n o t d epend on i n t a c t v i a b l e p r o t o p l a s t s , i t i s p r o b a b l y a p a s s i v e p r o c e s s . Under t h e c o n d i t i o n s t e s t e d i n t h i s t h e s i s , d i c l o f o p i s a p p a r e n t l y t a k e n up i n t o p r o t o p l a s t s a t v e r y l o w r a t e s . DM and d i c l o f o p a r e b o t h l i p o p h i l i c m o l e c u l e s . H a l l (1981) f o u n d t h e l o g o f t h e o c t a n o l / w a t e r c o e f f i c e n t s ( l o g P) o f DM and d i c l o f o p t o be 3-11 and 2 . 34 a t pH 5 . 3 . 2 ,4-D, a r e l a t i v e l y n o n - l i p o p h i l i c h e r b i c i d e , had a p a r t i t i o n c o e f f i c e n t w i t h l o g P=0.29 . I t was c o n c l u d e d t h a t DM and d i c l o f o p were v e r y l i p o p h i l i c h e r b i c i d e s and p r e f e r e n t i a l l y p a r t i t i o n i n t o l i p o p h i l i c compounds. I n p r o t o p l a s t s DM may p a r t i t i o n i n t o t h e l i p i d c o m p o n e n t s o f c e l l membranes. T h i s w o u l d e x p l a i n b o t h t h e r a p i d u p t a k e o f DM and t h e e q u a l u p t a k e i n t o i n t a c t p r o t o p l a s t s and b u r s t p r o t o p l a s t f r a g m e n t s Vacuum f i l t r a t i o n o f p r o t o p l a s t s t o remove u n a b s o r b e d t r a c e r i s a u s e f u l t e c h n i q u e i n u p t a k e s t u d i e s , a s t h e t i m e r e q u i r e d t o p e r f o r m t h i s o p e r a t i o n i s l e s s t h a n 5 .0 s e c o n d s . M e t h o d s , s u c h as r e p e a t e d c e n t r i f u g a t i o n ( R u b i n s t e i n and T a t t a r 1980) and c e n t r i f u g a t i o n t h r o u g h d e n s i t y g r a d i e n t s ( L i n 1980) r e q u i r e -56-l o n g e r time p e r i o d s . The r a p i d uptake o f DM and d i c l o f o p make th e s e l a t t e r methods i n a p p r o p r i a t e f o r s t u d y i n g DM u p t a k e . There are d i s a d v a n t a g e s to the f i l t r a t i o n method. P r o t o p l a s t s cannot be removed from the f i l t e r papers to a s s e s s damage r e s u l t i n g from the vacuum f i l t r a t i o n p r ocedure or to determine the c h e m i c a l form o f the h e r b i c i d e i n the p r o t o p l a s t s . -57-DISTRIBUTION OF DM AND METABOLITES IN OAT PROTOPLASTS The d i s t r i b u t i o n o f r a d i o l a b e l l e d h e r b i c i d e i n t h e p r o t o p l a s t s u s p e n s i o n was d e t e r m i n e d . The r e c o v e r y r a t e o f t h e a p p l i e d r a d i o l a b e l was a p p r o x i m a t e l y 50%. The r e l a t i v e l y l ow r e c o v e r y may h a v e been due t o l o s s o f r a d i o l a b e l d u r i n g t h e c e n t r i f u g a t i o n p r o c e d u r e s and a b s o r p t i o n o f r a d i o l a b e l on t h e P y r e x t u b e s u s e d i n t h e e x p e r i m e n t s . E f f o r t s t o r e d u c e t h e amount o f a d s o r p t i o n on g l a s s w a r e were u n s u c e s s f u l . N o n e t h e l e s s , a l t h o u g h r e c o v e r y was l o w , i t was f o u n d t o be c o n s i s t e n t and r e p r o d u c i b l e . The p e r c e n t o f r e c o v e r e d r a d i o l a b e l i n t h e e x t e r n a l s o l u t i o n and p r o t o p l a s t p e l l e t i s shown i n T a b l e 7a. S i x t y p e r c e n t o f t h e r e c o v e r e d r a d i o l a b e l was f o u n d i n t h e p r o t o p l a s t p e l l e t . The c h e m i c a l f o r m o f t h e r e c o v e r e d r a d i o l a b e l i s p r e s e n t e d i n T a b l e 7 b . E i g h t y t h r e e p e r c e n t o f t h e r a d i o l a b e l i n t h e p r o t o p l a s t p e l l e t r e m a i n s i n t h e m e t h y l e s t e r f o r m . I n t h e e x t e r n a l s o l u t i o n , o n l y 19.44 p e r c e n t r e m a i n s as t h e e s t e r DM. S e v e n t y s e v e n p e r c e n t o f t h e r e c o v e r e d r a d i o l a b e l i n t h e e x t e r n a l s o l u t i o n i s e n z y m a t i c a l l y c o n v e r t e d t o d i c l o f o p . O n l y s m a l l amounts o f t h e n o n - p h y t o t o x i c m e t a b o l i t e , h y d r o x y d i c l o f o p were f o u n d i n e i t h e r t h e e x t e r n a l s o l u t i o n o r t h e p r o t o p l a s t s a f t e r a 1.0 h o u r i n c u b a t i o n p e r i o d . The h i g h p e r c e n t a g e o f r a d i o l a b e l as DM i n t h e p r o t o p l a s t s u p p o r t s t h e r e s u l t s o f t h e f i l t r a t i o n u p t a k e e x p e r i m e n t s d e m o n s t r a t i n g t h e e a s e a t w h i c h DM i s t a k e n u p . D i c l o f o p was t a k e n up a t a much l o w e r r a t e ; o f t h e r e c o v e r e d r a d i o l a b e l i n t h e p r o t o p l a s t s , o n l y 14.3% was d i c l o f o p , e v e n t h o u g h a n a l y s i s -58-T a b l e 7a. D i s t r i b u t i o n o f r a d i o l a b e l i n p r o t o p l a s t s u s p e n s i o n s . Component p e r c e n t o f r e c o v e r e d r a d i o l a b e l P r o t o p l a s t p e l l e t 60.6 + 2.30 E x t e r n a l s o l u t i o n 39.4 + 2.30 V a l u e s a r e t h e mean + s t a n d a r d e r r o r o f f o u r e x p e r i m e n t s w i t h 3 r e p e a t e d d e t e r m i n a t i o n s f o r e a c h component i n e a c h e x p e r i m e n t . T a b l e 7b. P e r c e n t c o m p o s i t i o n o f r a d i o l a b e l i n t h e p r o t o p l a s t s and t h e e x t e r n a l s o l u t i o n a f t e r 1.0 h o u r i n c u b a t i o n . C h e m i c a l f o r m P r o t o p l a s t p e l l e t E x t e r n a l s o l u t i o n DM 83 .4 + 1 .06 19 .4 + 1 .44 D i c l o f o p 14 .3 + 1 .06 77.7 + 1 .50 H y d r o x y d i c l o f o p 2 .3 + 0.33 2.8 + 0 .30 V a l u e s a r e t h e means + s t a n d a r d e r r o r o f 4 e x p e r i m e n t s w i t h 3 r e p e a t e d d e t e r m i n a t i o n s i n e a c h e x p e r i m e n t . -59-o f the e x t e r n a l s o l u t i o n showed t h a t a l a r g e p e r c e n t a g e o f the r a d i o l a b e l i n the e x t e r n a l s o l u t i o n was i n the a c i d f o r m . These da t a a re a l s o c o n s i s t e n t w i t h the r e s u l t s o f f i l t r a t i o n e x p e r i m e n t s wh i ch showed r e l a t i v e l y low up take o f d i c l o f o p . In a s i m i l a r s t u d y Dusky e t a l . ( 1 9 8 0 ) found 55% o f the r e c o v e r e d r a d i o l a b e l i n a m e t h a n o l - s o l u b l e c e l l e x t r a c t a f t e r a one hour i n c u b a t i o n w i t h r a d i o l a b e l l e d DM. D i c l o f o p was found t o be the main m e t a b o l i t e i n both the c e l l e x t r a c t and the c e l l medium. These r e s u l t s c o n f l i c t w i t h the f i n d i n g s o f t h i s t h e s i s where DM was the main m e t a b o l i t e found i n the p r o t o p l a s t s . A major d i f f e r e n c e between c e l l c u l t u r e s and p r o t o p l a s t s u s p e n s i o n s i s the p r e sence o f the c e l l w a l l i n t he f o r m e r . D i c l o f o p may be a s s o c i a t e d w i t h c e l l w a l l c o n s t i t u e n t s i n t h e c e l l c u l t u r e s , r e s u l t i n g i n the a s s o c i a t i o n o f a l a r g e p r o p o r t i o n o f r a d i o l a b e l as d i c l o f o p i n the m e t h a n o l - s o l u b l e c e l l e x t r a c t s . The p e l l e t was d i s p e r s e d i n d i s t i l l e d water t o r u p t u r e the p r o t o p l a s t s , and then c e n t r i f u g e d to d i v i d e the p r o t o p l a s t s i n t o two f r a c t i o n s ; w a t e r - s o l u b l e components and the w a t e r - i n s o l u b l e components ( F i g u r e 4) P r o t e i n a n a l y s i s o f the two f r a c t i o n s showed t h a t the bu l k o f p r o t e i n was a s s o c i a t e d w i t h the w a t e r - s o l u b l e f r a c t i o n ( T a b l e 8) The w a t e r - i n s o l u b l e f r a c t i o n c o n t a i n e d o n l y s m a l l amounts o f p r o t e i n but a l a r g e p e r c e n t a g e o f the t o t a l p h o s p h o l i p i d . T h i s o b s e r v a t i o n would s u g g e s t the p r e sence o f l a r g e q u a n t i t i e s o f l i p i d membranes p r e s e n t i n the i n s o l u b l e f r a c t i o n . L i g h t m i c r o s c o p i c e x a m i n a t i o n o f the i n s o l u b l e m a t e r i a l showed the p r e sence o f p r o t o p l a s t f r agmen t s and s w o l l e n c h l o r o p l a s t s . I t was assumed -60-T a b l e 8 P r o t e i n and p h o s p h o r u s c o n t e n t o f t h e w a t e r s o l u b l e and i n s o l u b l e p r o t o p l a s t f r a c t i o n s . s o l u b l e f r a c t i o n i n s o l u b l e f r a c t i o n P r o t e i n mg/ml 1.64 0.46 P h o s p h o r u s ug/ml 1.01 5.27 P r o t e i n c o n t e n t was d e t e r m i n e d on t h e w a t e r - s o l u b l e and i n s o l u b l e f r a c t i o n s o f t h e p r o t o p l a s t s . P h o s p h o r u s c o n t e n t was d e t e r m i n e d on t h e l i p i d e x t r a c t o f t h e w a t e r - s o l u b l e and i n s o l u b l e f r a c t i o n s o f t h e p r o t o p l a s t s . -61-t h a t the w a t e r - i n s o l u b l e f r a c t i o n i n c l u d e d p r o t o p l a s t fragments and o r g a n e l l e s w i t h s u b s t a n t i a l amounts o f l i p i d membrane s t r u c t u r e s , whi l e the water s o l u b l e f r a c t i o n c o n t a i n e d p r e d o m i n a n t l y c y t o p l a s m i c p r o t e i n s and low m o l e c u l a r weight c o n s t i t u e n t s . T a b l e 9 shows the d i s t r i b u t i o n o f r a d i o l a b e l w i t h i n the p r o t o p l a s t p e l l e t and the e x t e r n a l s o l u t i o n . In the p r o t o p l a s t p e l l e t , two t h i r d s o f the r a d i o l a b e l was i n the w a t e r - i n s o l u b l e f r a c t i o n . N i n e t y one percent o f t h i s r a d i o l a b e l was i n the e s t e r form, DM. In the w a t e r - s o l u b l e f r a c t i o n a p p r o x i m a t e l y one h a l f the r a d i o l a b e l was present as d i c l o f o p a c i d and one h a l f as DM. The two l a r g e s t f r a c t i o n s o f r a d i o l a b e l were d i s t r i b u t e d as d i c l o f o p i n the e x t e r n a l s o l u t i o n , and DM i n the i n s o l u b l e f r a c t i o n o f the p r o t o p l a s t p e l l e t . The r e c o v e r y o f l a r g e q u a n t i t i e s o f DM in the w a t e r - i n s o l u b l e f r a c t i o n , which c o n t a i n e d l a r g e q u a n t i t i e s o f p h o s p h o l i p i d s , i s not s u r p r i s i n g c o n s i d e r i n g the h i g h l i p i d s o l u b i l i t y o f DM ( H a l l , 1981). These r e s u l t s suppor t the c o n t e n t i o n t h a t DM may p a r t i t i o n i n t o the l i p i d membranes i n the p r o t o p l a s t s . Most o f the d i c l o f o p r e c o v e r e d from the p r o t o p l a s t s was i n the water s o l u b l e f r a c t i o n . There are s e v e r a l p o s s i b l e mechanism t h a t c o u l d e x p l a i n the presence o f d i c l o f o p i n the e x t e r n a l s o l u t i o n ( T a b l e 7b); 1) l eakage o f d i c l o f o p out o f p r o t o p l a s t s a f t e r i n t r a c e l l u l a r h y d r o l y s i s , 2) l eakage o f i n t r a c e l l u l a r h y d r o l a s e enzymes i n t o the e x t e r n a l s o l u t i o n , or 3) h y d r o l a s e enzymes on the e x t e r n a l s u r f a c e o f the plasmalemma. I t has been shown tha t DM i s r a p i d l y conver ted to d i c l o f o p i n p l a n t s by a h y d r o l a s e enzyme -62-T a b l e 9 The d i s t r i b u t i o n o f r a d i o l a b e l w i t h i n t h e p r o t o p l a s t s and t h e e x t e r n a l s o l u t i o n as a p e r c e n t o f t o t a l r e c o v e r a b l e r a d i o l a b e l . E x t e r n a l ( 1 ) S o l u b l e ( 2) I n s o l u b l e ( 3) DM d i c l o f o p H y d r o x y d i c l o f o p T o t a l 9.1 + 0 .94 30 .5 + 2 .31 1.2 + 0 .07 40.7 + 3.87 8 .6 + 0 .77 8 - 9 + 1 .41 0 .9 + 0.13 1 8 . 3 + 1 .81 37.5 + 2.56 2 .7 + 0 .74 0.8 + 0 .08 41 .0 j _ 2 .80 ( 1 ) P e r c e n t c o m p o s i t i o n o f t h e e x t e r n a l s o l u t i o n s u r r o u n d i n g t h e p r o t o p l a s t s . ( 2 ) P e r c e n t c o m p o s i t i o n o f t h e w a t e r - s o l u b l e c o m p o n e n t s o f t h e p r o t o p l a s t s . See F i g u r e 4. ( 3 ) P e r c e n t c o m p o s i t i o n o f t h e w a t e r - i n s o l u b l e c o m p o n e n t s o f t h e p r o t o p l a s t s . See F i g u r e 4. V a l u e s a r e t h e means + s t a n d a r d e r r o r o f f o u r e x p e r i m e n t s w i t h 3 r e p e a t e d d e t e r m i n a t i o n s f o r e a c h c h e m i c a l component i n e a c h e x p e r i m e n t . -63-T a b l e 10 D e t e r m i n a t i o n o f h y d r o l a s e a c t i v i t y i n p r o t o p l a s t i n c u b a t i o n s o l u t i o n . C h e m i c a l f o r m E x t e r n a l ( 1) E x t e r n a l (2) S o r b i t o l c o n t r o l ( 3 ) DM D i c l o f o p H y d r o x y d i c l o f o p 1 9 . 4 + 1 .44 77 .7 + 1 .50 2.3 + 0.33 79.3 + 2.14 19 .7 + 2 .09 1 .0 + 0 .07 96 .9 + 1 .83 2.3 + 0.14 0.8 + 0 .06 (1) P e r c e n t c o m p o s i t i o n o f r a d i o l a b e l i n t h e e x t e r n a l s o l u t i o n a f t e r one h o u r i n c u b a t i o n s w i t h p r o t o p l a s t s . ( 2 ) P e r c e n t c o m p o s i t i o n o f r a d i o l a b e l i n t h e e x t e r n a l s o l u t i o n a f t e r a one h o u r i n c u b a t i o n w i t h t h e e x t e r n a l s o l u t i o n f r o m w h i c h p r o t o p l a s t s had been removed a f t e r one h o u r . (3) P e r c e n t c o m p o s i t i o n o f t h e r a d i o l a b e l i n s o r b i t o l b u f f e r V a l u e s a r e means + s t a n d a r d e r r o r o f 4 e x p e r i m e n t s w i t h 3 r e p e a t e d d e t e r m i n a t i o n s f o r e a c h measurement i n e a c h e x p e r i m e n t . - 6 4 -( H i l l e t a l . 1 9 8 0 ) . However, t h e l o c a t i o n o f t h i s enzyme w i t h i n t h e p l a n t i s n o t known. The r e s u l t s p r e s e n t e d i n T a b l e 10 compare t h e c o n v e r s i o n o f 14C-DM t o d i c l o f o p and h y d r o x y d i c l o f o p i n s o r b i t o l b u f f e r t o t h a t i n e x t e r n a l s o l u t i o n s c o n t a i n i n g p r o t o p l a s t s , o r w i t h p r o t o p l a s t s removed a f t e r a one h o u r i n c u b a t i o n b u t b e f o r e DM a d d i t i o n . The d e t e c t i o n o f 20% o f t h e l a b e l as d i c l o f o p i n t h e e x t e r n a l s o l u t i o n when DM was a d d e d a f t e r p r o t o p l a s t r e m o v a l s u p p o r t s t h e a r g u m e n t f o r DM c o n v e r s i o n v i a e x t r a c e l l u l a r a c t i v i t y o f an i n t r a c e l l u l a r o r membrane a s s o c i a t e d h y d r o l a s e w h i c h had l e a k e d i n t o t h e e x t e r n a l s o l u t i o n . The c o n v e r s i o n o f DM t o d i c l o f o p i n t h e e x t e r n a l s o l u t i o n a f t e r p r o t o p l a s t s were removed i s l o w e r t h a n when p r o t o p l a s t s w e r e p r e s e n t ( T a b l e 10) I f t h e h y d r o l a s e enzymes were i n t r a c e l l u l a r , b u t l e a k e d o u t o f t h e p r o t o p l a s t d u r i n g t h e 1.0 h o u r i n c u b a t i o n p e r i o d s , t h e p e r c e n t c o n v e r s i o n o f DM t o d i c l o f o p s h o u l d be c o m p a r a b l e i n e x t e r n a l s o l u t i o n s w i t h and w i t h o u t p r o t o p l a s t s . I n b o t h c a s e s , p r o t o p l a s t s were i n c u b a t e d f o r one h o u r t o a l l o w f o r l e a k a g e o f i n t r a c e l l u l a r e n z y m e s . However, t h e l e v e l o f c o n v e r s i o n i n t h e a b s e n c e o f p r o t o p l a s t s a r g u e s f o r t h e p r e s e n c e o f s u r f a c e enzymes l o c a t e d on t h e p l a s m a l e m m a . T h i s a s s o c i a t i o n w o u l d r e s u l t i n a h i g h d e g r e e o f c o n v e r s i o n o f DM o n l y when p r o t o p l a s t s were p r e s e n t . The l o w e r r a t e o f c o n v e r s i o n a f t e r p r o t o p l a s t r e m o v a l may r e a s o n a b l y be a c o n s e q u e n c e o f d i s s o c i a t i o n o f some h y d r o l a s e m o l e c u l e s i n t o t h e e x t e r n a l s o l u t i o n . D u s ky e t a l . ( 1 9 8 0 ) a l s o r e p o r t e d h y d r o l a s e a c t i v i t y i n t h e c e l l - f r e e medium f r o m 4-day o l d c u l t u r e s . I t was s u g g e s t e d t h a t t h i s was due t o l e a k a g e o f -65-h y d r o l a s e enzymes f r o m c e l l s o r f r o m l y z e d c e l l s i n t h e medium. An a l t e r n a t i v e e x p l a n a t i o n f o r t h e s e r e s u l t s may be t h e i n c r e a s e d membrane p e r m e a b i l i t y c a u s e d by DM. I n c r e a s e d p e r m e a b i l i t y may p r o d u c e an i n c r e a s e i n l e a k a g e o f i n t r a c e l l u l a r enzymes i n t o t h e e x t e r n a l s o l u t i o n when DM i s p r e s e n t . However, s i n c e t h e c o n c e n t r a t i o n s o f DM and e t h a n o l i n t h e s e e x p e r i m e n t s were much l o w e r t h a n t h o s e w h i c h r e s u l t e d i n d e t e c t a b l e i n c r e a s e s i n membrane p e r m e a b i l i t y h e r b i c i d e - i n d u c e d l e a k a g e d o e s n o t seem a v a l i d i n t e r p r e t a t i o n o f t h e s e d a t a . The r e s u l t s p r e s e n t e d i n d i c a t e u p t a k e o f DM i n t o t h e w a t e r i n s o l u b l e c o m p o n e n t s o f t h e p r o t o p l a s t s u c h a s t h e c e l l membranes. C o m p a r a t i v e l y , o n l y s m a l l q u a n t i t i e s o f d i c l o f o p w ere p r e s e n t i n t h e p r o t o p l a s t s . However, s i g n i f i c a n t amounts o f d i c l o f o p were f o u n d i n t h e e x t e r n a l s o l u t i o n . I t i s l o g i c a l t o p r o p o s e t h a t t h i s d i s t r i b u t i o n may be a r e s u l t o f t h e a c t i v i t y o f h y d r o l a s e enzymes a s s o c i a t e d w i t h t h e p r o t o p l a s t membranes. A l t h o u g h some i n t r a c e l l u l a r h y d r o l a s e a c t i v i t y may c o n v e r t DM t a k e n up by t h e p r o t o p l a s t s t o d i c l o f o p w h i c h r e m a i n s i n s i d e t h e p r o t o p l a s t s , most o f t h e h y d r o l a s e a c t i v i t y a p p e a r s t o be a s s o c i a t e d w i t h t h e e x t e r n a l s o l u t i o n . The l a r g e s t a c c u m u l a t i o n s o f a p p l i e d r a d i o l a b e l a r e l o c a t e d i n t h e w a t e r - i n s o l u b l e f r a c t i o n as DM and i n t h e e x t e r n a l s o l u t i o n as d i c l o f o p . -66-2,4-D EFFECTS ON DM UPTAKE AND METABOLISM The u p t a k e o f 14C-DM and l 4 C - d i c l o f o p i n t o p r o t o p l a s t s was n o t a f f e c t e d by t h e a d d i t i o n o f t h e h e r b i c i d e 2,4-D d u r i n g a one h o u r i n c u b a t i o n p e r i o d ( T a b l e s 11 and 1 2 ) . R a d i o l a b e l n o t t a k e n up by t h e p r o t o p l a s t s was removed by t h e vacuum f i l t r a t i o n p r o c e d u r e s d e s c r i b e d p r e v i o u s l y . The a d d i t i o n o f 2,4-D t o t h e p r o t o p l a s t s c h a n g e d t h e i n i t i a l pH o f t h e i n c u b a t i o n s o l u t i o n . To a v o i d s u c h pH e f f e c t s , 2,4-D was p r e p a r e d i n a 5.OmM Hepes b u f f e r s o l u t i o n f o r e x p e r i m e n t s o f w h i c h d a t a a r e p r e s e n t e d i n T a b l e s 11a, 12a and 1 3 a . A t h i g h e r 2,4-D c o n c e n t r a t i o n s t h e Hepes b u f f e r c o n c e n t r a t i o n i n t h e i n c u b a t i o n s o l u t i o n was 10.0 mM, and t h e i n i t i a l pH o f e a c h t r e a t m e n t v i a l was a d j u s t e d t o 7.0 w i t h 0.5N NaOH ( T a b l e s 11b, 12b, 13b). I n d i v i d u a l d e g r e e o f f r e e d o m c o n t r a s t s c o m p a r i n g c o n t r o l v a l u e s w i t h o u t p r o t o p l a s t s t o t r e a t m e n t v a l u e s showed a s i g n i f i c a n t d i f f e r e n c e between c o n t r o l and t r e a t m e n t means (ANOVA i n a p p e n d i x 11 and 1 2 ) . However, c o n t r a s t s c o m p a r i n g t r e a t m e n t means, showed no s i g n i f i c a n t e f f e c t o f 2,4-D a d d i t i o n . DM and d i c l o f o p u p t a k e i n t o p r o t o p l a s t s was n o t s i g n i f i c a n t l y d i f f e r e n t a t 2,4-D c o n c e n t r a t i o n s o f 10uM t o 2.OmM f r o m t r e a t m e n t s w i t h o u t 2,4-D. The a d d i t i o n o f 2,4-D a t c o n c e n t r a t i o n s o f 0 t o 50uM d i d n o t h a v e a s i g n i f i c a n t e f f e c t on t h e c o n v e r s i o n o f DM t o d i c l o f o p i n t h e p r o t o p l a s t o r t h e e x t e r n a l s o l u t i o n ( T a b l e 1 3 a ) . However, a t h i g h e r 2,4-D c o n c e n t r a t i o n s ( T a b l e 13b) a s i g n i f i c a n t i n v e r s e l i n e a r r e l a t i o n s h i p between 2,4-D -6?-T a b l e 11a The e f f e c t o f 2,4-D on DM u p t a k e T r e a t m e n t DM u p t a k e p i c o m o l e s c o n t r o l 34 .1 0 2,4-D 175 .4 10uM 210 .4 25uM 177 .6 50uM 206 .0 T a b l e 11b T r e a t m e n t DM u p t a k e p i c o m o l e s c o n t r o l 21 .8 0.0 2,4-D 179 .1 100uM 2 0 3 .4 500uM 194 .3 1mM 181.1 2mM 185 . 3 V a l u e s i n T a b l e s a r e t h e r e s u l t s o f 2 d i f f e r e n t e x p e r i m e n t s . T r e a t m e n t v a l u e s a r e t h e means o f 3 r e p l i c a t e s p e r t r e a t m e n t . C o n t r o l v a l u e s r e p r e s e n t r a d i o l a b e l r e m a i n i n g on t h e f i l t e r p a p e r when p r o t o p l a s t s were o m i t t e d f r o m t h e i n c u b a t i o n s o l u t i o n . -68-T a b l e 12a The e f f e c t o f 2,4-D on d i c l o f o p u p t a k e T r e a t m e n t D i c l o f o p u p t a k e p i c o m o l e s c o n t r o l 9.8 0.0 2,4-D 16 .4 10.OuM 17.1 25 .OuM 20.0 50.OuM 17.8 T a b l e 12b T r e a t m e n t D i c l o f o p u p t a k e p i c o m o l e s c o n t r o l 4.1 0.0 2,4-D 1 3 . 9 100uM 12.2 500uM 9 .9 1 .OmM 10 .6 2.OmM 12.4 V a l u e s i n T a b l e s a r e t h e r e s u l t s o f 2 d i f f e r e n t e x p e r i m e n t s . V a l u e s a r e t h e means o f 3 r e p l i c a t e s p e r t r e a t m e n t . C o n t r o l s r e p r e s e n t r a d i o l a b e l r e m a i n i n g on t h e f i l t e r p a p e r when p r o t o p l a s t s were o m i t t e d f r o m t h e i n c u b a t i o n medium. -69-T a b l e 13a The e f f e c t o f 2,4-D on t h e c o n v e r s i o n o f DM t o d i c l o f o p a c i d . P e r c e n t o f r e c o v e r e d r a d i o l a b e l as d i c l o f o p E x t e r n a l P r o t o p l a s t s o l u t i o n p e l l e t 0 .OuM 81 .5 10 .9 10 .OuM 83 .0 10 .5 25 .OuM 82 .0 11 .3 50 .OuM 81 .2 10.3 T a b l e 13 b P e r c e n t o f r e c o v e r e d r a d i o l a b e l as d i c l o f o p 2,4-D E x t e r n a l P r o t o p l a s t s o l u t i o n p e l l e t 0 .0 uM 79 .0 12 .4 100.0 uM 78 .3 10 .4 500 .0 uM 77 .0 9 .5 1 .OmM 66 .7 8.3 2 .OmM 74 .3 8.0 The v a l u e s i n T a b l e s 13a and 13b a r e t h e r e s u l t s o f d i f f e r e n t e x p e r i m e n t s . V a l u e s a r e t h e means o f 3 r e p l i c a t e s p e r t r e a t m e n t . - 7 0 -c o n c e n t r a t i o n and c o n v e r s i o n to d i c l o f o p i n the p e l l e t and a s i g n i f i c a n t i n v e r s e q u a d r a t i c r e l a t i o n s h i p to c o n v e r s i o n i n the e x t e r n a l s o l u t i o n was (ANOVA i n append ix 1 3 ) . I t i s l i k e l y t h a t the q u a d r a t i c e f f e c t i s due to an e x p e r i m e n t a l e r r o r and i s u n l i k e l y to r e f l e c t any i m p o r t a n t b i o l o g i c a l r e l a t i o n s h i p . The d e c r e a s e i n DM c o n v e r s i o n may be a r e s u l t o f the p h y s i o l o g i c a l a c t i o n o f 2,4-D i n t e r f e r e n c e w i t h h y d r o l a s e a c t i v i t y . I t may be t h a t the d e c r e a s e i n DM c o n v e r s i o n a t h i g h e r 2,4-D c o n c e n t r a t i o n s i s the r e s u l t o f i n c r e a s e d H+ i o n e x t r u s i o n s t i m u l a t e d by 2,4-D (Mar re e t a l . 1 9 7 3 ) . T h i s e f f e c t would g e n e r a t e a lower pH i n the e x t e r n a l s o l u t i o n and a p o s s i b l e d e c r e a s e i n DM h y d r o l y s i s s i n c e the pH optimum o f an e x t r a c t f rom o a t s w i t h h y d r o l a s e a c t i v i t y was found t o l i e between 7.0 and 8.0 ( F i g u r e 8 ) . However, measurements o f the pH o f the e x t e r n a l s o l u t i o n showed no change f rom the s t a r t i n g pH o f 7.0 d u r i n g the 1.0 hour i n c u b a t i o n . When DM a p p l i c a t i o n to w i l d o a t s was combined w i t h 2,4-D (Todd and S t o b b e , 1980) or MCPA ( Q u e r e s h i and Vanden B o r n , 1 9 7 9 ) , a h i g h e r p e r c e n t a g e o f the r e c o v e r e d r a d i o l a b e l was i n the e s t e r form than when DM was a p p l i e d a l o n e . I t was s u g g e s t e d t h a t the phenoxy h e r b i c i d e s i n t e r f e r e d w i t h a c t i v i t y o f the h y d r o l a s e enzyme. Q u e r e s h i and Vanden Born (1979) found t h a t DM was c o n v e r t e d t o d i c l o f o p i n a c rude s u p e r n a t a n t e x t r a c t i n d i c a t i n g the p r e sence o f a c t i v e h y d r o l a s e enzymes i n  v i t r o . T h i s h y d r o l a s e a c t i v i t y c o u l d be i n h i b i t e d by MCPA. However , H i l l e t a l . (1980) i s o l a t e d and p a r t i a l l y p u r i f i e d an enzyme f r a c t i o n w i t h h y d r o l a s e a c t i v i t y f rom w i l d oa t p l a n t s . - 7 1 -Figure 8. pH profile of hydrolase activity. Hydrolase a c t i v i t y i the percent radiolabel as diclofop in the enzyme extract + standard error Controlsi the percent radioabel as diclofop in enzyme extracts boiled for 1.0 hour. -72-The i s o l a t e d enzyme was n o t i n h i b i t e d by 2,4-D ( 1 0 u M ) . The d a t a p r e s e n t e d i n t h i s t h e s i s a r e c o n s i s t e n t w i t h t h e r e s u l t s o f H i l l e t a l . ( 1 9 8 0 ) . O n l y v e r y h i g h c o n c e n t r a t i o n s o f 2,4-D c a u s e d any d e c r e a s e i n DM h y d r o l y s i s t o d i c l o f o p . R e s e a r c h i n t o t h e a c t i v i t y o f 2,4-D o f t e n p r o d u c e s c o n f l i c t i n g r e s u l t s i n t h e l i t e r a t u r e b e c a u s e o f v a r y i n g p l a n t r e s p o n s e t o d i f f e r e n t 2,4-D c o n c e n t r a t i o n r a n g e s . F o r t h i s r e a s o n a w i d e r a n g e o f 2,4-D c o n c e n t r a t i o n s was t e s t e d i n t h i s t h e s i s . 2,4-D c o n c e n t r a t i o n s o f 1mM and 2mM, where d e c r e a s e s i n h y d r o l a s e a c t i v i t y were o b s e r v e d , a r e u n r e a l i s t i c a l l y h i g h c o m p a r e d t o f i e l d r a t e s and c o n c e n t r a t i o n s t e s t e d i n g r o w t h r e s p o n s e s t u d i e s . A s h t o n e t a l . ( 1 9 7 7 ) f o u n d most p h y s i o l o g i c l a l r e s p o n s e s ( p h o t o s y n t h e s i s , r e s p i r a t i o n , RNA s y n t h e s i s , p r o t e i n s y n t h e s i s and l i p i d s y n t h e s i s ) w e re i n h i b i t e d a t 100uM 2,4-D i n e t i o l a t e d l e a f c e l l s o f r e d k i d n e y beans . -73-GENERAL DISCUSSION The u s e o f p l a n t p r o t o p l a s t s t o s t u d y h e r b i c i d e u p t a k e e l i m i n a t e s p r o b l e m s a s s o c i a t e d w i t h h e r b i c i d e p e n e t r a t i o n i n t h e c u t i c l e and p a r t i t i o n i n g i n t o i n t r a c e l l u l a r s p a c e s and t h e a p o p l a s t w h i l e f a c i l i t a t i n g r a p i d e s t a b l i s h m e n t o f known c o n c e n t r a t i o n s o f h e r b i c i d e a t t h e c e l l u l a r l e v e l . I n a p r o t o p l a s t s y s t e m , c e l l u l a r i n t e g r i t y i s d e p e n d e n t upon t h e s e m i - p e r m e a b l e c h a r a c t e r i s t i c s o f c e l l membranes. S i n c e h e r b i c i d e s , i n c l u d i n g d i c l o f o p m e t h y l ( C h r o w l e y and P r e n d e v i l l e , 1979) h a v e a d v e r s e e f f e c t s on p l a n t membranes, i t i s p o s s i b l e t h a t t h e s e h e r b i c i d e s may i n t e r f e r e w i t h membrane u p t a k e p r o c e s s e s . The r e s u l t s i n t h i s t h e s i s i n d i c a t e d t h a t DM and d i c l o f o p had l i m i t e d e f f e c t s on p r o t o p l a s t membrane i n t e g r i t y . O b s e r v e d c h a n g e s i n membrane p e r m e a b i l i t y were s m a l l w i t h o n l y t h e h i g h e s t DM c o n c e n t r a t i o n (100uM) i n d u c i n g a n y s i g n i f i c a n t d i f f e r e n c e f r o m u n t r e a t e d p r o t o p l a s t s . Any d i r e c t i n f l u e n c e o f DM o r d i c l o f o p on membrane p e r m e a b i l i t y was n e g l i g i b l e a t t h e c o n c e n t r a t i o n s u s e d i n t h e s e e x p e r i m e n t s t o e x a m i n e u p t a k e . B o t h DM and d i c l o f o p were t a k e n up by p r o t o p l a s t s . However t h e r a t e o f DM u p t a k e was t e n t i m e s g r e a t e r t h a n t h a t o f d i c l o f o p . W h i l e DM u p t a k e was n o t s a t u r a b l e a t t h e c o n c e n t r a t i o n s t e s t e d i n t h e s e e x p e r i m e n t s s u g g e s t i n g a p a s s i v e u p t a k e p r o c e s s , t h e low r a t e o f d i c l o f o p u p t a k e r e f l e c t s a low membrane p e r m e a b i l i t y . The l a t t e r o b s e r v a t i o n may i n d i c a t e a p o t e n t i a l p r o b l e m f o r d i c l o f o p t o r e a c h t h e s i t e o f a c t i o n i n t h e s y m p l a s t . DM and d i c l o f o p a r e b o t h p h y t o t o x i c b u t may -74-i n d u c e d i f f e r e n t p h y s i o l o g i c a l r e s p o n s e s i n p l a n t s ( S h i m a b u k u r o e t a l . 1979; T o d d , 1979). The d i f f e r e n t i a l r a t e o f u p t a k e may be r e f l e c t e d i n d i f f e r e n t s i t e s o f a c t i o n i n p l a n t s t h u s c a u s i n g d i f f e r e n t p l a n t r e s p o n s e s . DM and d i c l o f o p a r e v e r y l i p o p h i l i c m o l e c u l e s w i t h t h e l o g o f t h e p a r t i t i o n c o e f f i c i e n t s ( l o g P) o f 3.11 and 2.34 r e s p e c t i v e l y ( H a l l 1981). I t i s p r o b a b l e t h a t DM u p t a k e o c c u r s v i a a p a s s i v e a b s o r p t i o n i n t o t h e l i p i d p h a s e o f p r o t o p l a s t s membranes. T h i s w o u l d e x p l a i n t h e r a p i d and r e l a t i v e l y h i g h r a t e o f DM u p t a k e by b o t h b u r s t and i n t a c t p r o t o p l a s t s and l a r g e a c c u m u l a t i o n s o f DM i n p r o t o p l a s t f r a c t i o n s c o n t a i n i n g a h i g h p r o p o r t i o n o f p h o s p h o l i p i d s . However, l i p i d p e r m e a b i l i t y c a n n o t be t h e o n l y f a c t o r d e t e r m i n i n g u p t a k e . The r a t e o f d i c l o f o p u p t a k e i s low ev e n t h o u g h d i c l o f o p i s a r e l a t i v e l y l i p o p h i l i c h e r b i c i d e . H owever, t h e a c i d g r o u p on d i c l o f o p may be d i s s o c i a t e d a t p h y s i o l o g i c a l pH g i v i n g a c h a r g e d m o l e c u l e w h i c h w o u l d be l e s s p e r m e a b l e t o p l a n t membranes and r e s u l t i n u p t a k e l e v e l s l e s s t h a n t h o s e p r e d i c t e d on t h e b a s i s o f l i p o p h i l i c i t y a l o n e . I f DM r a p i d l a d s o r b e d o n t o membranes i n p l a n t s i t may become i m m o b i l i z e d i n t h e l i p i d p h a s e a s was p r o p o s e d by P e n n i s t o n (1969) f o r l i p o p h i l i c compounds. The DM a b s o r b e d i n p l a n t membranes w o u l d be u n a b l e t o p a r t i t i o n o u t o f t h e l i p i d p h a s e and i n t o t h e a q u e o u s a p o p l a s t and i n t e r c e l l u l a r s p a c e s . T h i s e f f e c t w o u l d e x p l a i n t h e r a p i d u p t a k e i n t o o a t p r o t o p l a s t s r e p o r t e d h e r e and t h e low t r a n s l o c a t i o n r a t e o f DM e s t e r o u t o f t h e t r e a t m e n t z o n e n o t e d by Todd (1979) and S h i m a b u k u r o e t a l . (1979) . DM i s c o n v e r t e d t o d i c l o f o p by n o n s p e c i f i c h y d r o l a s e -75-e n z y m e s . L a r g e a c c u m u l a t i o n s o f d i c l o f o p were i d e n t i f i e d i n t h e e x t e r n a l s o l u t i o n w h i c h c o u l d r e s u l t f r o m e i t h e r e f f l u x o f d i c l o f o p p r o d u c e d i n t h e c y t o p l a s m by i n t r a c e l l u l a r DM h y d r o l y s i s o r d i c l o f o p p r o d u c e d i n t h e e x t e r n a l s o l u t i o n by h y d r o l a s e enzymes l e a k e d f r o m t h e c y t o p l a s m , o r a s s o c i a t e d w i t h t h e e x t e r n a l s u r f a c e o f t h e p r o t o p l a s t s . A n a l y s i s o f t h e d a t a p r e s e n t e d i n t h i s t h e s i s i n d i c a t e s t h a t h y d r o l a s e a c t i v i t y was a s s o c i a t e d w i t h t h e e x t e r n a l s u r f a c e o f t h e p r o t o p l a s t s , c o n v e r t i n g DM t o d i c l o f o p i n t h e e x t e r n a l s o l u t i o n . I n i n t a c t p l a n t s , DM w o u l d be c o n v e r t e d t o d i c l o f o p i n t h e a p o p l a s t s o r i n t e r c e l l u l a r s p a c e s o f t h e p l a n t . The r e s u l t s o f t h i s t h e s i s have shown t h a t d i c l o f o p i s n o t r e a d i l y t a k e n up by o a t p r o t o p l a s t s . T h e r e f o r e , i t m i g h t be e x p e c t e d t h a t most o f t h e d i c l o f o p p r e s e n t i n t h e a p o p l a s t w o u l d r e m a i n i n t h e i n t e r c e l l u l a r s p a c e s r a t h e r t h a n m o v i n g i n t o t h e s y m p l a s t . Whether t h e a p o p l a s t i c d i c l o f o p c a n e n t e r t h e p h l o e m and be t r a n s l o c a t e d i s n o t known. However l i m i t e d b a s i p e t a l and a c r o p e t a l t r a n s p o r t o f d i c l o f o p and d i c l o f o p c o n j u g a t e s h a v e been o b s e r v e d ( T o d d , 1979) . I n p l a n t s , d i c l o f o p i s c o n v e r t e d t o g l y c o s y l e s t e r c o n j u g a t e s . G e i g e r ( 1 9 7 4 ) and G i a q u i n t a ( 1 9 7 6 ) o b s e r v e d t h a t s u g a r s a r e l o a d e d d i r e c t l y f r o m t h e a p o p l a s t i n t o s p e c i a l i z e d p a r e n c h y m a c e l l s a s s o c i a t e d w i t h p h l o e m t i s s u e . D i c l o f o p o r i t s g l y c o s y l c o n j u g a t e s may be l o a d e d i n t o p h l o e m i n a s i m i l a r f a s h i o n and t h e n t r a n s l o c a t e d t o t h e s i t e o f a c t i o n i n p l a n t m e r i s t e m s . C o n j u g a t e d d i c l o f o p may be t a k e n up i n p l a n t s a t a f a s t e r r a t e t h a n d i c l o f o p a s a r e s u l t o f t h e s u g a r m o i e t y . I n t h i s t h e s i s c o n j u g a t e s o f DM and d i c l o f o p r e p r e s e n t e d o n l y a -76-s m a l l p e r c e n t a g e o f t h e r e c o v e r e d r a d i o l a b e l . T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h o t h e r r e p o r t s where o n l y 1 .0% o f r e c o v e r e d r a d i o l a b e l was c o n j u g a t e d a f t e r 1.0 h o u r ( T o d d , 1979; D u s k y e t a l . 1980) . A n a l y s i s o f t h e r a d i o l a b e l i n t h e p r o t o p l a s t s i n d i c a t e d t h a t w h i l e most o f t h e r a d i o l a b e l was i n t h e e s t e r f o r m , 14.27% o f t h e r e c o v e r e d r a d i o l a b e l i n t h e p r o t o p l a s t was d i c l o f o p . T h i s s m a l l f r a c t i o n may be t h e r e s u l t o f t h e s l o w u p t a k e o f d i c l o f o p f r o m t h e e x t e r n a l s o l u t i o n o r b e c a u s e o f i n t r a c e l l u l a r h y d r o l y s i s o f DM a b s o r b e d t o p r o t o p l a s t membranes. The r a t e o f i n t r a c e l l u l a r h y d r o l y s i s i s a p p a r e n t l y much l e s s t h a n t h a t i n t h e e x t e r n a l s o l u t i o n , a s t h e amount o f d i c l o f o p i n t h e p r o t o p l a s t s i s much l e s s t h a n t h a t f o u n d i n t h e e x t e r n a l s o l u t i o n . Combined a p p l i c a t i o n s o f t h e h e r b i c i d e s 2,4-D and DM r e s u l t i n a l o s s o f h e r b i c i d a l a c t i v i t y o f DM and l o s s o f c o n t r o l o f g r a s s y w eeds. P r e v i o u s r e s e a r c h h a s shown t h a t 2,4-D a p p l i c a t i o n s r e d u c e t h e amount o f 1 4 C - d i c l o f o p r e c o v e r e d f r o m p l a n t s e c t i o n s ( Q u e r e s h i and Vanden B o r n , 1979; Todd, 1979) and r e d u c e t h e t r a n s l o c a t i o n o f 1 4 C - d i c l o f o p f o u n d i n r o o t and s h o o t m e r i s t e m s . I t h a s been s u g g e s t e d t h a t 2,4-D may i n h i b i t t h e c o n v e r s i o n o f DM t o d i c l o f o p and t h u s a f f e c t t h e u p t a k e o f DM o r d i c l o f o p i n t o o a t p r o t o p l a s t s . However, t h e a d d i t i o n o f 2,4-D t o t h e i n c u b a t i o n medium d i d n o t a l t e r t h e p r e v i o u s l y o b s e r v e d p a t t e r n o f u p t a k e o f DM o r d i c l o f o p i n t o o a t p r o t o p l a s t s . The c o n v e r s i o n o f DM t o d i c l o f o p was a l s o u n a f f e c t e d by 2,4-D c o n c e n t r a t i o n s up t o 50 uM. However, a t h i g h e r 2,4-D c o n c e n t r a t i o n s (1.0mM and 2.OmM) t h e r e was a -77-d e c r e a s e i n DM c o n v e r s i o n . T h i s d e c r e a s e was n o t due t o d e c r e a s i n g pH o f t h e e x t e r n a l s o l u t i o n as t h e pH r e m a i n e d u n c h a n g e d t h r o u g h o u t t h e 1.0 h o u r i n c u b a t i o n . A l t h o u g h s i g n i f i c a n t , t h e d e c r e a s e i n h y d r o l a s e a c t i v i t y was v e r y s l i g h t , and t h e 2,4-D c o n c e n t r a t i o n s i n d u c i n g t h i s d e c r e a s e were u n r e a l i s t i c a l l y h i g h compared t o t h o s e a s s o c i a t e d w i t h n o r m a l p h y s i o l o g i c a l p l a n t r e s p o n s e s ( A s h t o n e t a l . 1977). I t was c o n c l u d e d t h a t 2,4-D does n o t a f f e c t u p t a k e o f DM o r d i c l o f o p o r DM h y d r o l y s i s i n t h e o a t p r o t o p l a s t s y s t e m . I n i n t a c t p l a n t s t h e a p p l i c a t i o n o f 2,4-D and DM r e s u l t e d i n an i n c r e a s e i n t h e p e r c e n t r a d i o l a b e l as DM r e c o v e r e d f r o m t h e p l a n t s ( Q u e r e s h i and Vanden B o r n , 1979; Todd, 1979). However t h e a c t i v i t y o f i s o l a t e d h y d r o l a s e enzyme f r a c t i o n s were u n a f f e c t e d by 2,4-D. The r e a s o n f o r t h e d i s c r e p a n c y b e tween 2,4-D e f f e c t s i n i n t a c t p l a n t s and t h o s e i n t h e p r o t o p l a s t s y s t e m i s unknown b u t may be t h e r e s u l t o f d i f f e r i n g b u f f e r i n g c a p a c i t i e s o f t h e s e s y s t e m s . -78-CONCLUSION The u s e o f o a t p r o t o p l a s t s p r o v e d t o be a s u c e s s f u l t e c h n i q u e t o s t u d y DM and d i c l o f o p u p t a k e and m e t a b o l i s m . I n s t u d i e s where i n t a c t p l a n t s o r p l a n t s e g m e n t s h a v e been u s e d , t h e l i p o p h i l i c c u t i c l e a b s o r b s l a r g e q u a n t i t i e s o f t h e h e r b i c i d e due t o t h e l i p o p h i l i c n a t u r e o f DM. T h i s w o u l d c a u s e d i f f i c u l t i e s s e p a r a t i n g u p t a k e i n t o c e l l s w i t h t h a t o f s u r r o u n d i n g t i s s u e s . P r o t o p l a s t s u s p e n s i o n s a r e r e l a t i v e l y e a s y t o h a n d l e , however t e c h n i c a l d i f f i c u l t i e s may a r i s e as a r e s u l t o f t h e s m a l l v o l u m e s a v a i l a b l e f o r use a t any one t i m e and t h e t i m e r e q u i r e d f o r t h e i s o l a t i o n p r o c e d u r e s . V a r i a b i l i t y b e t w een d i f f e r e n t p r o t o p l a s t s p r e p a r a t i o n s was c o n s i d e r a b l e , r e q u i r i n g e x p e r i m e n t s t o be r e p e a t e d and e x p e r i m e n t a l d e s i g n t o r e d u c e t h e e f f e c t s o f s u c h d i f f e r e n c e s . E x t r a p o l a t i o n o f t h e DM u p t a k e r e s u l t s f r o m p r o t o p l a s t s t o i n t a c t t i s s u e may n o t a l w a y s be a p p r o p r i a t e . However t h e b e n e f i t s and s i m p l i c i t y a s s o c i a t e d w i t h t h e p r o t o p l a s t s y s t e m o u t w e i g h t h e s e d i s a v a n t a g e s . F u r t h e r e x p l o r a t i o n o f t h e s y s t e m t o e x a m i n e h e r b i c i d e m e t a b o l i s m i n p l a n t c e l l s i s c e r t a i n l y w a r r a n t e d . -79-BIBLIOGRAPHY Agbakoda , C . S . O . and J . R . G o o d i n . 1970 . A b s o r p t i o n and t r a n s l o c a t i o n o f 1 4 C - l a b e l e d 2,4-D and p i c l o r a m i n F i e l d B i n d w e e d . Weed S c i . 18 :168 A n d e r s o n , R .N . 1976 . Response o f monoco t y l edons to HOE 22870 and HOE 2 3 4 0 8 . Weed S c i . 24 :266-269-A s h t o n , F . M . , O .T . De M i l l i e r s , R.K. G lenn and W.B. Dueck . 1 9 7 7 . The l o c a l i z a t i o n o f m e t a b o l i c s i t e s o f a c t i o n o f h e r b i c i d e s . P e s t i c . B i o chem. P h y s i o l . 7 :122 Bhan, V . M . , E . W . S t o l l e r , and F.W. S l i f e . 1970 . T o x i c i t y , a b s o r p t i o n , t r a n s l o c a t i o n and m e t a b o l i s m o f 2,4-D i n Y e l l o w N u t s e d g e . 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M o d i f i c a t i o n o f i o n t r a n s p o r t i n l i p i d b i l a y e r membranes i n t h e p r e s e n c e o f 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d . B i o p h y s , J . 2 6 : 4 4 1 . S m i t h , A.E. 1977. D e g r a d a t i o n o f t h e h e r b i c i d e d i c l o f o p m e t h y l i n p r a i r i e s o i l s . J . A g r i c . Food Chem. 2 5 : 8 9 3 - 8 9 9 . Todd, B.G.and E.H. S t o b b e . 1 9 7 7 . S e l e c t i v i t y o f d i c l o f o p m e t h y l among w h e a t , b a r l e y w i l d o a t and g r e e n f o x t a i l . Weed S c i . 25:382-385 . T o d d , B.G. 1979. s e l e c t i v i t y and m e t a b o l i s m o f d i c l o f o p m e t h y l i n w h e a t , b a r l e y , w i l d o a t , and g r e e n f o x t a i l . PhD. T h e s i s . U n i v e r s i t y o f M a n i t o b a , W i n n i p e g . 1 8 7 pp. Wu, G.H. and P.W. S a n t e l m a n n . 1976. P h y t o t o x i c i t y and s o i l a c t i v i t y o f Hoe 2 3 4 0 8 . Weed S c i 2 4 : 6 0 1 - 6 0 4 . -86-Appendix 1. Analysis of variance with individual degree of freedom contrasts for data presented in Table l a . The effect of ethanol on the leakage of neutral red dye from oat protoplasts Treatments 1-5 represent ethanol concentrations of 0.0% 0.1%, 1.0%, 5.0% and 10.0% respectively ANALYSIS OF VARIANCE - DENSITY SOURCE BLOCK TREAT BXT ERROR TOTAL * * * * * * * * DF 2 4 8 30 44 » • * * * * * * < SUM SO MEAN SO ERROR F-VALUE PR0B 0 .72009 0 .36005 8 7 9 . 5 9 0 .24382 0.60956E-01 BXT 11.823 0.41246E-01 0 .51557E-02 12.595 0.12280E-01 0 .40933E-03 1 .0174 .************************************************** 0.23280E-26 0.19376E-02 O.10141E-06 ********* TREAT MN DENSITY 1 . 0 . 3 1 4 0 CONTRAST 1 2 CONTRAST 1.OOOOO -0.OOOOO -0.00000 -0.00001 . 2 . 0 .3133 3 1.OOOOO -0.OOOOO -O.OOOOO 1 - OOOOO -o.OOOOO . 3 . 0 .3303 4 1 .OOOOO .4 . 0 .4119 . 5 . 0 .5023 CONTRAST 0-SOUARED F-VALUE PROBABILITY 1 LINEAR 0 .243636 47 .255 0 .00013 2 QUAD 0 .100932E-03 0.19577E-01 0 .89218 3 CUB 0 .621993E-04 0.12064E-01 0 .91524 4 DEV 0 .239995E-04 0 .46549E-02 0 .94728 TREATMENT SUM OF SQUARES 0 .243824 SUM OF Q SQUARED 0 .243823 DIFFERENCE OR RESIDUAL 0 .279808E-06 AVERAGE F-RATIO 11.8229 DIFFERENCE IS 0 .11476E-03 PERCENT OF TREATMENT SUM OF SQUARES TIME FOR CONTRASTS IS 0 .8945E-02 SECONDS. DUNCAN'S MULTIPLE RANGE TEST . RANGES FOR ALPHA=0.05 3 .2570 3 .3960 3.4775 3 .5179 THERE ARE 3 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT S IZE ) WHICH ARE LISTED AS FOLLOWS ( 2, 1. 3) ( 4) ( 5) -67-Appendix 2. Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table l b . The e f f e c t s of ethanol on the leakage of K from oat protoplasts. Treatment numbers 1-4 represent ethanol concen-tr a t i o n s of 0.0%, 0.1%, 1.0% and 10.0% respectively. SOURCE BLOCK TREAT BXT ERROR TOTAL ************ ANALYSIS OF VARIANCE - DENSITY OF SUM SO MEAN SO ERROR F-VALUE PROB 2 3 6 24 1 .8345 7.5708 0.45425 0.22557 0.91727 2.5236 0.75708E-01 0.93986E-02 BXT 97.596 33.333 8.0552 0.29690E-11 0.38821E-03 0.78078E-04 35 10.085 *************.****************************************************** TREAT 1 . FREQUENCIES 9 MN DENSITY 0.9050 CONTRAST 1 2 CONTRAST 1 1.00000 2 0.OOOOO 1•OOOOO 3 O.OOOOO O.OOOOO .2. 9 0.9054 9 0.9114 1.OOOOO .4. 9 1.966 CONTRAST 1 LINEAR 2 QUAD 3 DEV Q-SQUARED 7.51287 0.578681E-01 0.316594E-05 F-VALUE 99.235 0.76436 0.41818E-04 PROBABILITY 0.OOOO6 0.41559 0.99505 TREATMENT SUM OF SQUARES 7.57076 SUM OF 0 SQUARED 7.57074 DIFFERENCE OR RESIDUAL 0.168373E-04 AVERAGE F-RATIO 33.3331 DIFFERENCE IS 0.22240E-03 PERCENT OF TREATMENT SUM OF SQUARES TIME FOR CONTRASTS IS 0.8633E-02 SECONDS. DUNCAN'S MULTIPLE RANGE TEST, RANGES FOR ALPHA«0.05 3.4605 3.5867 3.6452 THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBStT OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ' ( 1. 2. 3) ( 4 ) -88-Am^ndix 3 Analysis of variance with individual degree Appendix 3. An * c o n t r a s t s f o r d a t a presented in _ . . - r r , i . . ~ + « «-p nwt r>n 1 palraffe of Table 2a. The effects of DM on leakage of neutral red dye from oat protoplasts. Treatment numbers 1-4 represent DM concentrations of O.OuM, lOuM, 50uM, and lOOuM respectively. SOURCE BLOCK TREAT BXT ERROR TOTAL DF 5 3 15 44 67 ANALYSIS OF VARIANCE - DENSITY SUM SO MEAN SO ERROR F-VALUE 0.76937 0.15292E-01 0.95965E-02 0.86710E-02 0.80293 0. 15387 0.50973E-02 0.63977E-03 0.19707E-03 BXT 780.82 7.9675 3.2464 PROB 0.27756E-16 0.20709E-02 O.11854E-02 .****»»*******»». * TREAT MN DENSITY 1 . 0.2996 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 -O.OOOOO 1.OOOOO 3 -O.OOOOO -O.OOOOO 2. 0.3029 3 .3. 0.3179 1.OOOOO .4. 0.3375 CONTRAST 1 LINEAR 2 OUAD 3 DEV Q-SQUARED 0.152867E-01 0.442057E-05 0.791969E-06 F-VALUE 23.894 069097E-02 O.12379E-02 PROBABILITY 0.00020 0.93485 0.97240 TREATMENT SUM OF SQUARES SUM OF 0 SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS O.152919E-01 O.152919E-01 0.307532E-07 7.96744 0.20111E-03 PERCENT OF TREATMENT SUM OF SQUARES TIME FOR CONTRASTS IS 0.8789E-02 SECONDS. DUNCAN'S MULTIPLE RANGE TEST. RANGES FOR ALPHA-0.05 3.0090 3.1564 3.2575 THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ' ( 1 . 2 . 3 ) ( 4) -89-Appendix 4. Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table 2b. The e f f e c t s of DM on leakage of K from oat protoplasts. Treatments numbered 1-4 represent DM concentrations of O.OuM, 10uM, 50uM and lOOuM respectively. SOURCE B L O C K T R E A T BXT ERROR T O T A L DF 2 3 6 24 3 5 « * » « * » * * * » * * * • TREAT MN D E N S I T Y . 1 A N A L Y S I S OF V A R I A N C E - D E N S I T Y SUM SO MEAN SO ERROR F-VALUE PROB 1 8 3 3 2 0 9 1 6 5 9 9 1 . 9 8 0 0 . 5 5 8 2 0 E - 1 1 0 4 4 0 . 1 5 0 7 5 BXT 7 . 6 1 4 7 ° - ^ O B S E - O l 0 . 1 1 8 7 8 0 . 1 9 7 9 7 E - 0 1 1 . 9 8 6 6 0 . 1 0 7 4 4 0 . 2 3 9 1 6 0 . 9 9 6 5 1 E - 0 2 ,.,*•?:??*?•*•••••**•••* ......».»**» *«** * 0 . 8 9 9 0 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 O.OOOOO 1.OOOOO 3 0 . 0 -O.OOOOO . 2 . 0 . 8 7 4 9 3 . 3 . 1 . 0 4 9 1 .OOOOO 1 . 149 CONTRAST 1 L I N E A R 2 QUAD 3 DE V 0-SOUARED 0 . 4 3 0 3 8 8 0 . 3 4 2 1 0 4 E - 0 2 0 . 1 8 4 2 6 8 E - 0 1 F-VALUE 2 1 . 7 4 0 0 . 17281 0 . 9 3 0 8 0 P R O B A B I L I T Y 0 . 0 0 3 4 6 0 . 6 9 2 0 9 0 . 3 7 1 9 1 TREATMENT SUM OF SQUARES SUM OF 0 SQUARED D I F F E R E N C E OR R E S I D U A L AVERAGE F - R A T I O 0 . 4 5 2 2 3 7 0 . 4 5 2 2 3 6 0 . 5 5 0 7 3 4 E - 0 6 7 . 6 1 4 6 7 D I F F E R E N C E I S 0 . 1 2 1 7 8 E - 0 3 PERCENT OF TREATMENT SUM OF SQUARES T I M E FOR CONTRASTS I S 0 . 8 3 5 9 E - 0 2 S ECONDS . D U N C A N ' S M U L T I P L E RANGE T E S T . RANGES FOR A L P H A = 0 . 0 5 3 . 4 6 0 5 3 . 5 8 6 7 3 . 6 4 5 2 THERE ARE 3 HOMOGENEOUS S U B S E T S ( S U B S E T S OF E L E M E N T S . NO P A I R OF WHICH D I F F E R BY MORE THAN THE SHORTEST S I G N I F I C A N T RANGE FOR A S U B S E T OF THAT S I Z E ) WHICH ARE L I S T E D A S FOLLOWS ( 2 . 1) ( 1 . 3 ) ' ( 3 . 4 ) -90-Appendix 5« Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table 3a. The e f f e c t s of di c l o f o p on leakage of neutral red dye from oat protoplasts. Treatment numbers 1-4 represent d i c l o f o p concentrations of O.OuM, 10uM,50uMt and lOOuM respectively. S O U R C E B L O C K T R E A T B X T E R R O R T O T A L ANALYSIS OF VARIANCE - DENSITY DF 3 3 9 28 43 S U M S O O.19333 O.26304E-02 0.23G06E-02 0.40703E-02 0.20239 MEAN SO 0.64444E-01 0.87679E-03 026229E-03 O.14537E-03 ERROR B X T F - V A L U E 443.32 3.3428 1.8043 PROB 0.69389E-16 0.69599E-01 O.11203 „».*,*.***************** »...».»...**..*************** * » * • * * * * * * * * * * * * * * * * * * * * * * ' TREAT MN DENSITY . 1 . 0.2198 CONTRAST 1 2 .2. 0.2285 3 .3 . 0.2341 CONTRAST 1 2 3 1 .OOOOO 0.OOOOO 1•OOOOO 0.OOOOO -0.00000 1.ooooo CONTRAST LINEAR QUAD DEV 0-SOUARED 0.234263E-02 0.108916E-03 O. 178784E-03 .4 . 0.2409 F-VALUE 8.9313 0.41525 0.68162 PROBABILITY 0.01524 0.53539 0.43037 TREATMENT SUM OF SQUARES g^gg"^ SUM OF Q SQUARED ° / S i o E-07 DIFFERENCE OR RESIDUAL VXSK ^EFFERlNCE"rSTIS.13171E-02 PERCENT OF TREATMENT SUM OF SQUARES -91-Appendix 6. Analysis of variance with individual degree of freedom contrasts for data presented in Table 3b. The effects of diclofop on leakage of K from oat protoplasts. Treatment numbers represent diclofop concentrations of O.OuM, lOuM, 50uM, and lOOuM respectively. SOURCE BLOCK TREAT BXT ERROR TOTAL ANALYSIS OF VARIANCE - DENSITY DF SUM SO MEAN SO ERROR F-VALUE 2 3 € 24 35 1 .5869 0.28757E-01 0.20840E-01 0 .20526 1 .8417 0 .79345 0 .95858E-02 0 .34734E-02 0 .85524E-02 BXT 92 .775 2.7598 0 .40613 . .«.»..*.*.... .*. PROB 0.50944E-1 1 O. 13407 0 .86760 TREAT . 1 . 2 . FREQUENCIES 9 9 MN DENSITY 0 . 7 8 0 9 0 .7968 CONTRAST 1 2 3 CONTRAST 1 1.OOOOO 2 O.OOOOO 1.OOOOO 3 o.O -0.OOOOO 1.OOOOO . 3 . 9 0 .7898 9 0.8531 CONTRAST 1 LINEAR 2 QUAD 3 DEV Q-SQUARED 0.225978E-01 O.430441E-02 0 .185507E-02 F-VALUE 6 .5059 1 .2392 0 .53408 PROBABILITY 0 .04345 0 .30822 0 .49244 TREATMENT SUM OF SOUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO 0.287574E-01 0.287573E-01 O. 142516E-06 2 .75975 DIFFERENCE IS 0 .49558E-03 PERCENT OF TREATMENT SUM OF SQUARES - 9 2 -Appendix ?. Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table 4. Time p r o f i l e of DM uptake. Treatment numbers 1-7 represent controls, 5.0 seconds, 10 seconds, 3 0 seconds, 1.0 minute, 10 minutes, and 60 minutes resp e c t i v e l y . SOURCE DATS TREAT D X T ERROR TOTAL DF 2 6 12 42 62 ANALYSIS OF VARIANCE - YIELD SUM SO MEAN SO 476 .27 O.16339E+06 25682. 37499. 0.22705E+06 238 .14 27232. 2140.2 892 .84 STANDARD DEVIATION OF VARIABLE 1 IS 6 0 . 5 1 5 ERROR F-VALUE D X T 0 .26672 12.724 2 .3970 PROB 0.76718 . 0 .13735E-03 O.18272E-01 * TREAT 1. FREQUENCIES 9 MN YIELD 1 48 .42 SD YIELD 1 16.12 CONTRAST 1 CONTRAST 1 1.OOOOO . 2 . 9 198 .9 3 2 . 3 9 9 202 .6 29 .56 . 4 . 9 181.5 31 .95 9 199.0 40 .84 9 192.2 39.01 9 167.5 39 .55 CONTRAST CONTROL / TREAT Q-SQUARED 155283. F-VALUE 72 .556 PROBABILITY 0.OOOOO TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 4 . 9 6 1 5 163390. 155283. 8106.52 72.5562 PERCENT OF TREATMENT SUM OF SQUARES TEST OF DIFFERENCE GIVES AN F-VALUE OF 0 .75756 AND FPROB OF 0.59691 -93-Appendix 8. Analysis of variance with individual degree of freedom contrasts for data presented i n Table 5. Time profile of diclofop uptake. Treatment numbers 1-7 represent control, 5 seconds, 10 seconds, 30 seconds, 1.0 minute 10 minutes, and 60 minutes respectively. SOURCE DAYS TREAT D X T ERROR TOTAL DF 2 6 12 35 55 ANALYSIS OF VARIANCE - PICAMOLE SUM SO MEAN SO ERROR F-VALUE 5388.1 2 0 9 5 . 9 1377.9 3221 .5 12083. 2 6 9 4 . 0 349 .32 114 .82 92 .044 D X T 29 .269 3 .0422 1 .2475 PROB 0 .33799E-07 0 .47765E-01 0 .29198 FREQUENCIES. MEANS. STANDARD DEVIATIONS * * * * * * * * * * * * TREAT ,**************************' ********************** ****************** . 1 . FREQUENCIES 8 MN PICAMOLE 5 .772 SD PICAMOLE 2.291 CONTRAST 1 CONTRAST 1 1 .OOOOO 8 23 .87 18.91 . 3 . 8 19.87 12 .60 .4 . 8 23 .54 17.25 . 5 . 8 21 .60 1 1 .49 . 6 . 8 17 .64 8 .64 1 . 7 . CONTRAST TREAT/CONT Q-SQUARED 1787.94 F-VALUE 15.571 PROBABILITY 0 .00194 TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 14.694 2095 .93 1787.94 307 .986 15 .5710 PERCENT OF TREATMENT SUM OF SQUARES TEST OF DIFFERENCE GIVES AN F-VALUE OF 0 .53644 AND FPROB OF 0 .74533 -9k~ Appendix 9. Analysis of variance with individual degree of freedom contrasts for the DM concentration profile shown in Figure 7* Treatment numbers 1-6 represent DM concentrations of 2.0, k.0, 8.0, 15.0, 25.0, and 50.0uM DM respectively. ANALYSIS OF VARIANCE - PICAMOLE SOURCE BLOCK TREAT BXT ERROR TOTAL DF 4 5 20 54 83 SUM SO 0.43989E+07 0.75B59E+08 0.86616E+07 0.46198E+07 0.93539E+08 MEAN SO O.10997E+07 0 . 15172E+08 0.43308E+06 85551 . ERROR F-VALUE BXT 12.854 35 .032 5.0622 PROB 0.20291E-06 0.31295E-08 0 .99872E-06 FREQUENCIES. MEANS. STANDARD DEVIATIONS * » » » * « » • » * * * • * * » * * * » * • • • * * * * * * * * * * * * * * * ' TREAT FREQUENCIES MN PICAMOLE SD PICAMOLE . 1 . 14 115.1 31 .94 CONTRAST 1 CONTRAST . 2 . 14 221 .0 71 . 16 1 1.OOOOO 2 O OOOOO 1•OOOOO 3 0 OOOOO -O.OOOOO 1•OOOOO 4 -0 ooooo -0.00000 -0.00000 5 -O.OOOOO O.OOOOO O.OOOOO » » * « » • * • * • » • * * * • * * * * * * * * * * * * * * . 3 . 14 4 7 9 . 7 138.8 14 894 .4 209 .7 1 .OOOOO -O.OOOOO 1.OOOOO 14 1493 . 397 .8 « » » » » » « * » * . 6 . 14 2872. 1064. CONTRAST LINEAR OUAD CUB QUART DEV Q-SQUARED 0.758166E+08 39214.4 851 .021 6 .84923 2390 .84 F-VALUE 175.06 0.9054BE-01 0 . 19650E-02 O.15815E-04 0 .55206E-02 PROBABILITY O.OOOOO 0 .76659 0 .96508 0 .99687 0.94151 TREATMENT SUM OF SQUARES SUM OF 0 SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 0 .87425E-04 0.758591E+08 0.758590E+08 66 .3195 35 .0324 PERCENT OF TREATMENT SUM OF SQUARES -95-Appendix 10, Analysis of variance with individual degree of freedom contrasts for data presented in Table 6. Uptake of DM and diclofop by burst and intact protoplasts. Treatment numbers 1-3 represent DM uptake in control, burst and intact protoplasts at 2.OuM DM, 4-6 at 4.OuM DM and 7-9 at 8.OuM DM. SOURCE BLOCK TREAT BXT ERROR TOTAL ANALYSIS OF VARIANCE - PICAMOLE DF SUM SO MEAN SO ERROR F-VALUE 2 8 16 49 75 O. 33825E+06 0.56107E+07 85657 . 0 .78791E+06 0.68225E+07 0.16912E+06 0.70134E+06 5353 .6 16O80. BXT 10.518 131 .00 0 .33294 PROB O .15830E-03 0 . 4 1 4 2 8 E - 1 2 0 .99054 FREQUENCIES. MEANS, STANDARD DEVIATIONS ,..........**.*.*•*«»**»»»**.»*»»*»•**«***•*«*****»*******«******•**,,******' TREAT . 1 . . 2 . . 3 . MN PICAMOLE 6 2 . 4 2 3 9 6 . 9 4 3 2 . 6 . 7 . . 8 . . 9 . 4 0 . 9 9 201 .2 210 .4 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 O . O 1.OOOOO 67 .02 . 5 . 7 5 3 . 3 785 .9 CONTRAST 1 CONTROL/TREAT 2 BURST/ INTACT Q-SQUARED F-VALUE 0.281901E+07 526 .57 8 3 0 9 . 5 9 1.5522 PROBABILITY O.OOOOO 0 .23075 TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 4 9 . 6 0 B 0.561070E+07 0.282732E+07 0.27833BE+07 264 .059 PERCENT OF TREATMENT SUM OF SQUARES TEST OF DIFFERENCE GIVES AN F-VALUE OF 86 .652 , AND FPROB OF 0.OOOOO -96-Treatment numbers 1-3 represent uptake of diclofop by control, burst and intact at 2.OuM diclofop, 4-6 at 4.OuM diclofop and 7-9 at 8.OuM diclofop. ANALYSIS OF VARIANCE - PICAMOLE SOURCE DF SUM SO MEAN SQ BLOCK 2 1929.3 964 .66 TREAT 8 14804. 1850.5 BXT 16 3060 .8 191 .30 ERROR 54 4928 .4 9 1 . 2 6 6 TOTAL 80 24722. FREQUENCIES . MEANS. STANDARD DEVIATIONS * * * * * * * * * * * * * * * * * * * * * * * * * * * * » • « « * » » * » » » * TREAT . 1 . . 2 . . 3 . MN PICAMOLE 7 .338 14 .23 24 .7 . . 8 . . 9 . 1 1 . 16 36 . 19 47 CONTRAST 1 2 ERROR F-VALUE BXT 10 .570 9 .6729 2.0961 PROB 0 . 13371E-03 0 . 7 4 7 4 0 E - 0 4 0 .22311E-01 CONTRAST 1 2 1 .OOOOO 0 . 0 1.00000 . 4 . 9 .667 . 5 . 2 8 . 0 9 . 6 . 3 6 . 8 0 CONTRAST 1 CONTROL/TREAT 2 BURST/ INTACT Q-SQUARED F-VALUE 8585 .36 44 .878 13B2.41 7 .2263 PROBABILITY 0 .00001 0 .01616 TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 32 .667 14803.6 9967 .77 4835 .84 26 .0524 PERCENT OF TREATMENT SUM OF SQUARES - 9 7 -Appendix 11 Analysis of variance with individual degree of freedom contrasts for data presented in Table 11. The effect of 2,4-D on DM uptake. Treatment numbers 1-5 represent control, O.OuM, lOuM, 25uM, and 50uM 2,4-D respectively. SOURCE TREAT BLOCK ERROR TOTAL DF 4 2 8 14 ANALYSIS OF VARIANCE - PICAMOLE SUM SO 63295 . 9 4 9 . 7 6 4177 .2 68422 . MEAN SO 15824. 474 .88 522 . 15 ERROR F-VALUE 30 .305 0 .90947 STANDARD DEVIATION OF VARIABLE 1 IS 6 9 . 9 0 9 PROB 0 .69818E-04 0 .44066 TREAT 1. FREQUENCIES 3 MN PICAMOLE 34 . 13 SD PICAMOLE 11.04 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 -O.OOOOO 1.OOOOO 3 175.4 3 9 . 8 0 3 2 1 0 . 9 2 6 . 5 6 4 . 3 177 .6 12.04 »*****«•*•**' 5. 3 2 0 6 . 0 2 .603 CONTRAST 1 TREAT/CONT 2 24D Q-SQUARED 60179 .4 1171.93 F-VALUE 115.25 2 .2444 PROBABILITY O.OOOOO 0 . 17248 TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL AVERAGE F -RATIO DIFFERENCE IS 3 .0706 63294 .8 6 1 3 5 1 . 3 1943.55 5 8 . 7 4 8 3 PERCENT OF TREATMENT SUM OF SQUARES TEST OF DIFFERENCE GIVES AN F-VALUE OF 1.8611 AND FPROB OF 0 .21693 - 9 8 -A . l l . cont'd Treatment numbers 1-6 represent control, O.OuM, lOOuM, 500uM, l.OmM, and 2.OmM 2,4-D. respectively. ANALYSIS OF VARIANCE - PERCENT SOURCE OF SUM SQ MEAN SO ERROR F -VALUE PROB TREAT 5 70769. 14154. 16.941 0. 13413E-03 BLOCK 2 1631.0 815.51 0.97608 0.40999 ERROR 10 8355.0 835.50 TOTAL 17 80755. FREQUENCIES, MEANS, STANDARD DEVIATIONS * * * * » » » » » » • » * * » » » * * * » » » » » • * * * * * * » * * * * * * » * * » * * * * * » * * * * * * * ft*****.***************** TREAT 1 . 2. 3. 4 . 5. 6. FREQUENCIES 3 3 3 3 3 3 MN PERCENT 21 .83 179. 1 203.4 194.3 181.1 185.3 SD PERCENT 6.673 29.07 17.55 6. 162 14.71 59.50 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 O.OOOOO 1.OOOOO CONTRAST 1 CONT/TREAT 2 24D Q-SQUARED 69544.4 342.249 F-VALUE .83.237 0.40964 PROBABILITY 0. OOOOO 0.53654 TREATMENT SUM OF SQUARES SUM OF 0 SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATI0 DIFFERENCE IS 1.2469 70769.0 69886.6 882.419 41 .8235 PERCENT OF TREATMENT SUM OF SQUARES TEST OF DIFFERENCE GIVES AN F-VALUE OF 0.35205 . AND FPROB OF 0.78872 -99-Appendix 12. Analysis of variance with individual degre< of freedom contrasts for data presented in Table 12. The effect of 2,4-D on diclofop uptake. Treatment numbers 1-5 represent control, O.OuM, lOuM, 25uM, and 50uM 2,4-D respectively. SOURCE TREAT BLOCK ERROR TOTAL ANALYSIS OF VARIANCE - PICAMOLE DF SUM SO MEAN SO ERROR F-VALUE 4 2 8 14 183.29 27.604 327.71 538.60 45.823 13.802 40.964 1 . 1186 0.33693 PROB 0.41184 0.72362 FREQUENCIES. MEANS. STANDARD DEVIATIONS TREAT 1. 2. 3. 4. 5. FREQUENCIES 3 3 3 3 3 MN PICAMOLE 9.833 16.37 17.07 20.37 17.77 SD PICAMOLE 0.7505 3.798 5.636 9.075 6.967 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 -O.OOOOO 1.OOOOO CONTRAST Q-SQUARED ^y/LUE P R 0 ^ ^ Y 1 TREAT/CONT 155.848 3.8045 0.08692 1 TREAT/CONT 9.30257 0.22709 0.64643 TREATMENT SUM OF SQUARES 183.291 SUM OF Q SQUARED 165.151 DIFFERENCE OR RESIDUAL 18.1401 A V F R A G E F-RATIO 2.01582 DIFFERENCE IS 9.8969 PERCENT OF TREATMENT SUM OF SQUARES TEST OF DIFFERENCE GIVES AN F-VALUE OF 0.22142 . AND FPROB OF 0.80613 -100-.12. cont'd Treatment numbers 1-6 represent control, O.OuM, lOOuM, 500uM, l.OmM, and 2.OmM 2,4-D respectively. SOURCE DF SUM SO TREAT 5 180.04 BLOCK 2 8. 1674 ERROR 10 67.395 TOTAL 17 255.61 FREQUENCIES, MEANS, STANDARD «.».*•»..***.*»»»»»*»»»»**»* TREAT 1 . 2. FREQUENCIES 3 MN PERCENT 4.053 13 SD PERCENT 0.7563 1.1 ANALYSIS OF VARIANCE - PERCENT MEAN SO 36.009 4.0837 6.7395 ERROR F-VALUE 5.3430 0.60593 PROB 0. 11962E-01 0.56443 3 12.22 1 .683 CONTRAST 1 2 CONTRAST 1 1.00000 2 0.OOOOO 1.OOOOO 4 . 3 9.903 1 .887 3 10.58 4 .605 6. 3 12.37 2.451 CONTRAST 1 CONT/TREAT 2 24D Q-SQUARED 149.872 16.8116 F-VALUE 22.238 2.4945 PROBABILITY 0.00082 O.14533 TREATMENT SUM OF SQUARES SUM OF 0 SOUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 7.4209 180.045 166.684 13.3609 12 3662 PERCENT OF TREATMENT SUM OF SQUARES TEST OF DIFFERENCE GIVES AN F-VALUE OF 0.66082 AND FPROB OF 0.59468 -101-Appendix 13. Analysis of variance with individual degree Appendix ^ freedom contrasts for data presented in Table 13. The effects of 2,4-D on the conversion of DM to diclofop. Treatment numbers 1-4 represent the percent radiolabel as diclofop in the external solution in O.OuM, lOuM, 25uM and 50uM 2,4-D respectively. SOURCE TREAT BLOCK ERROR TOTAL DF 3 2 6 11 ANALYSIS OF VARIANCE - PERCENT SUM SO 5.7339 20.520 8.8621 35.116 MEAN SO 1 .9113 10.260 1.4770 ERROR F-VALUE 1 .2940 6.9465 PROB 0.35935 0.27438E-01 FREQUENCIES, MEANS. STANDARD DEVIATIONS * TREAT FREQUENCIES MN PERCENT SD PERCENT 1 . 3 81.51 1 .889 CONTRAST 1 CONTRAST 1 1.OOOOO 2 -0.00000 3 -0.00000 2. 3 83 .00 2.294 1 . OOOOO 0. OOOOO 3. * * *»**•* .»**»»** * * * ' 4 . 3 82.01 1 .676 1.OOOOO 3 81 . 16 1 .744 CONTRAST 1 LINEAR 2 OUAD 3 CUB Q-SQUARED 1 .29355 2.14727 2.29316 F-VALUE 0.87578 1.4538 1 .5526 PROBABILITY 0.38548 0.27331 O. 25920 TREATMENT SUM OF SQUARES SUM OF 0 SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO . DIFFERENCE IS 5.73393 5.73397 -0.401697E-04 1 .29404 0.70056E-03 PERCENT OF TREATMENT SUM OF SQUARES -102-13. cont'd Treatment numbers 1-5 " V " " ^ . ^ ^ ^ S a ^ J » " o . S » ^ SoJSTKS- an, 2.OmM 2,4-D respectively. A N A L Y S I S O F V A R I A N C E - P E R C E N T S O U R C E D F S U M S O T R E A T B L O C K E R R O R T O T A L 4 2 8 14 3 0 2 . 9 3 9 . 7 3 3 3 1 4 0 . 2 7 4 5 2 . 9 3 M E A N S O 7 5 . 7 3 3 4 . 8 6 G 7 1 7 . 5 3 3 E R R O R F - V A L U E 4 . 3 1 9 4 0 . 2 7 7 5 7 P R O B 0 . 3 7 4 G 3 E - 0 1 0 . 7 8 4 6 3 .«**»«•»*< T R E A T F R E Q U E N C I E S M N P E R C E N T S D P E R C E N T 2 . 3 . 4 . 3 7 9 . 0 0 4 . 3 5 9 3 7 8 . 3 3 2 . 5 1 7 3 7 7 . O O 1 . 7 3 2 3 6 6 . 6 7 6 . 6 5 8 C O N T R A S T 1 C O N T R A S T 1 . 0 0 0 0 0 0 . O O O 0 1 0 . 0 0 0 0 0 0 . 0 0 0 0 0 1 . O O O O O - 0 . O O O O O - 0 . O O O O O C O N T R A S T L I N E A R Q U A D C U B D E V 1 . O O O O O 0 . O O O O O 1 . O O O O O Q - S Q U A R E D 8 2 . 6 4 7 9 1 4 2 . 4 8 3 7 3 . 4 3 0 4 4 . 3 7 0 6 3 — V A L U E 4 . 7 1 3 8 8 . 1 2 6 4 4 . 1 8 8 0 0 . 2 4 9 2 8 3 7 4 . 3 3 1 . 5 2 8 P R O B A B I L I T Y 0 . 0 6 1 7 1 0 . 0 2 1 4 6 0 . 0 7 4 9 2 0 . 6 3 1 0 2 T R E A T M E N T S U M O F S Q U A R E S ^ 2 " 9 3 1 S U M O F Q S Q U A R E D n 1 Q 3 6 8 5 E - 0 2 D I F F E R E N C E OR R E S I D U A L VSSaS S I F F E R E N C E " I S T I 0 . 6 3 9 3 6 E - 0 3 P E R C E N T O F T R E A T M E N T S U M O F S Q U A R E S ]03> -104-A.13- cont'd Treatment numbers 1-4 represent the percent r a d i o l a b e l as di c l o f o p i n the Protoplast p e l l e t i n O.OuM, 10uM, 25uM and 50uM 2,4-D respectively. SOURCE TREAT BLOCK ERROR TOTAL DF 3 2 6 11 ANALYSIS OF VARIANCE - PERCENT SUM SO MEAN SO ERROR F-VALUE 1.7699 0.55772 4.7452 7.0728 0.58997 0.27886 0.79086 0.74598 0.35260 PROB 0.56288 0.71650 »•*»****»*»**»*»*** ...................... TREAT FREQUENCIES 3 MN PERCENT 10.86 SD PERCENT 0.6902 CONTRAST 1 2 CONTRAST 1 1.00000 2 -O.OOOOO 1.00000 3 -O.OOOOO O.OOOOO CONTRAST 1 LINEAR 2 QUAD 3 CUB 3 10.47 1 .320 3. 3 11 .29 0.5220 1.OOOOO O-SQUARED 0.229803 0.573051 0.967047 3 10.29 0.3999 F-VALUE 0.29057 0.72459 1 .2228 PROBABILITY 0.60926 0.42730 0.31117 TREATMENT SUM OF SOUARES 1 • 76990 SUM OF Q SQUARED 1.76990 DIFFERENCE OR RESIDUAL ~ ° ™ 1 Q O O DIFFERENCE-IST-0.18191E-04 PERCENT^  TREATMENT SUM OF SOUARES -105-A.13. cont'd ^ , Treatment numbers 1-5 represent the percent radiolabel as diclofop in the protoplast pellet in O.OuM, lOOuM, 500uM, l.OmM and 2.OmM 2,4-D respectively. SOURCE TREAT BLOCK ERROR TOTAL ANALYSIS OF VARIANCE - PERCENT DF SUM SO MEAN SO ERROR F-VALUE 4 2 8 14 38.631 O. 18545 18.656 57.472 9.6577 0.92727E-2.3320 01 4. 1413 0.39762E-01 PROB 0.41588E-01 0.96121 FREQUENCIES. MEANS, STANDARD DEVIATIONS TREAT 1. 2. 3. FREQUENCIES 3 3 3 MN PERCENT 12.44 10.37 9.520 SD PERCENT 1.744 2.470 0.2777 CONTRAST 1 2 3 4 CONTRAST 1 1 .OOOOO 2 0.O0001 1.OOOOO 3 -O.OOOOO -0.OOOOO 1.OOOOO 4 -O.OOOOO -O.OOOOO O.OOOOO 1.ooooo 3 8.320 0.2858 3 7.967 0.3453 CONTRAST LINEAR QUAD CUB DEV O-SQUARED 27.5656 7.32224 0.718416 3.02438 F-VALUE 11.820 3. 1398 0.30806 1.2969 PROBABILITY 0.00885 0.11434 0.59405 0.28773 TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO 38.6308 38.6306 0.144766E-03 4.14130 DIFFERENCE IS 0.37474E-03 PERCENT OF TREATMENT SUM OF SQUARES -106-Appendix 14. Regression analysis of protein content and protoplasts number. ANALYSIS OF VARIANCE OF 2.ABSORBAN N= 4 OUT OF 4 SOURCE DF SUM SQRS MEAN SOR F-STAT SIGNIF REGRESSION 1 4084.1 4084.1 180.48 .0055 ERROR 2 45.259 22.630 TOTAL 3 4129.4 MULT R= .99450 R-SQR= .98904 SE= 4.7571 VARIABLE PARTIAL COEFF STD ERROR T-STAT SIGNIF CONSTANT . 13853 -1 3.7694 .36751 -2 .9974 CONC . .99450 .86695 .64533 -1 13.434 .0055 Standard curve of protein content and protoplast number. E o u p. 1.0 0.8 0.6 J O.k A 0.2 4 i i 1 i i 0 2 .0 4 .0 6.0 8.0 10.0 protoplasts (x 10^) per ml Regression equations Y 0.86?X 0.014, -107-Appendix 15. Analysis of variance with i n d i v i d u a l degree of freedom contrasts showing a l i n e a r r e l a t i o n s h i p between K concentration and atomic absorption at nM. Treatment numbers represent K concentration 0.0, 1.0, 2.0, 3.0, and k.0 ppm K . SOURCE BLOCK TREAT BXT ERROR TOTAL »»»•*»*»»»*' TREAT FREQUENCIES DF 2 4 8 30 44 ANALYSIS OF VARIANCE - DENSITY SUM SO MEAN SO ERROR 0.25973E-01 0.62300 O.11416E-01 0.63333E-03 0.66102 0.12987E-01 O.15575 0.14269E-02 0.21111E-04 BXT F-VALUE 615.16 109.15 67.592 PROB 0.44630E-24 0.51662E-06 0.45305E-16 ».»«»**»**«»*»*»*»..•** . . 1 . 9 MN DENSITY 0.0 CONTRAST 1 2 CONTRAST .2. 9 O. 1011 9 0.1822 9 0.2656 .5. 9 0.3328 1 2 3 4 1 .OOOOO 0.0 0.0 0.0 1.00000 0.0 O.OOOOO 1 .OOOOO 0.0 1.ooooo CONTRAST LINEAR QUAD CUB DEV 0-SOUARED 0.620010 0.276269E-02 O.136108E-04 0.211470E-03 F-VALUE 434.50 1.9361 0.95384E-02 O. 14820 PROBABILITY O.OOOOO 0.20156 0.92460 0.71030 TREATMENT SUM OF SQUARES SUM OF 0 SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 0.12297E-04 0.622998 0-622998 0.766097E-07 109.149 PERCENT OF TREATMENT SUM OF SQUARES -108-A.15. cont'd Standard curve for atomic absorption of potassium. - 1 0 9 -Appendix 1 6 . Glossary Herbicides 3 - a m i n o - l , 2 , 4 - t r i a z o l e 3-amino-2 ,5-dichlorobenzoic acid N-cyclo-octyl-N,N-dimethylurea 2-methyl- 4 , 6-dinitrophenol 2 , 2 - d i c h l o r o p r o p i o n i c acid amitrole: chloramben: cycluron: DNOC: dalapon: desmetryne: dicamba: 2 , 4-D 4-isopropylamino-6-methylamino-2-methylthio 1 , 3 , 5 - t r i a z i n e 3,6-dichloro-2-methoxybenzoic acid 2,4-dichlorophenoxy acetic acid d i c l o f o p methyl: methyl (2- (4- (2 ,4-dichlorophenoxy)phenoxy) propanoate dinoseb: 2 - ( 1-methylpropyl)- 4 , 6-dinitrophenol f l u o r o n i t r o f e n : 2 , 4-dichloro - 6-fluorophenyl - 4-nitrophenyl ether glyphosate: N-(phosphonomethyl)plycine i o x y n i l : 4-hydroxy- 3 , 5-di-iodobenzonitrile isoproturon: N'-(4-isopropylphenyl)-N,N-dimethyl urea linuron: N« -3,Zj.-dichlorophenyl)-N-methoxy-N-methylurea MCPA: 4-chloro-2-methylphenoxy a c e t i c acid monolinuron: N'-(4-chlorophenyl)-N-methoxy-N-methylurea monuron: N'-(4-chlorophenyl)-N;N-dimethylurea napthalam: N-l-naphthylphtalamic acid picloram: 4 - a m i n o - 3 , 5 , 6 - t r i c h l o r o p i c o l i n i c acid prometryne: 4 ,6-bisisopropylamino-2-methylthio - 1 , 3 , 5 - t r i a z i n e TCA: t r i c h l o r o a c e t i c acid - 1 1 0 -Buffers Ches: 2-(Cyclohexylamino)ethanesulphonic acid Hepes: N-2 Hydroxyethylpiperazine-N'-2-ethane sulphonic acid Mes: 2(N-Morpholinino)ethane sulphonic acid Mops: 3-(N-Morpholino)propane sulphonic acid T r i c i n e : N-(Tris-hydroxymethyl)methylglycine 

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