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Phenolic metabolism in plants: intracellular localization of some enzymes Camm, Edith Ellen 1972

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PHENOLIC METABOLISM IN PLANTS: INTRACELLULAR LOCALIZATION OF SOME ENZYMES by E D I T H E L L E N CAMM B.Sc. ( H o n . ) , Queen's U n i v e r s i t y , 1 ° 6 6 M . S c , 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 , 1 9 6 8 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF THE R E Q U I R E M E N T S FOR T H E D E G R E E OF DOCTOR OF P H I L O S O P H Y i n t h e d e p a r t m e n t o f B O T A N Y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e 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 OF B R I T I S H C O L U M B I A S e p t e m b e r , 1 9 7 2 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h 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 , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e 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 a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g 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 . D e p a r t m e n t o f T h 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 V a n c o u v e r 8, C a n a d a D a t e /f), 10 .17 ABSTRACT Two aspects of i n t r a c e l l u l a r o r g a n i z a t i o n of phenol metabol ism in p l a n t s were i n v e s t i g a t e d . The f i r s t i n v o l v e d the l o c a l i z a t i o n of some r e a c t i o n s i n the convers ion of t y r o s i n e to pi a s t o q u i n o n e , which i s accumulated in the c h l o r o p l a s t . More evidence i s presented f o r the fo rmat ion of p_-hydroxyphenyl py ruv i c a c i d and homogent is ic a c i d from t y r o s i n e by h igher p l a n t s , a l though n e i t h e r r e a c t i o n seems to occur in the c h l o r o p l a s t . The second par t of t h i s work i n v o l v e d the i n t r a -c e l l u l a r l o c a l i z a t i o n of s e v e r a l enzymes i n v o l v e d in the p h e n o l i c metabol ism of p o t a t o . Separa t ion of c e l l f r a c t i o n s by d i f f e r e n t i a l c e n t r i f u g a t i o n and sucrose d e n s i t y g r a d i e n t s e p a r a t i o n showed t h a t s h i k i m a t e dehydrogenase and prephenate dehydrogenase are who l l y s o l u b l e enzymes. In the cases of p h e n y l a l a n i n e ammonia l yase and an 0-methyl t r a n s f e r a s e a c t i n g on c a f f e i c a c i d , 90% of the enzyme was s o l u b l e . The remaining 10% of the enzymes t h a t was a s s o c i a t e d w i t h p a r -t i c u l a t e f r a c t i o n s d id not appear to be l o c a t e d i n the same f r a c t i o n s . M o s t o f t h e c i n n a m i c a c i d h y d r o x y l a s e i n ag e d p o t a t o was f o u n d i n t h e m i c r o s o m a l f r a c t i o n , b u t some o f t h i s enzyme a p p e a r e d t o be a s s o c i a t e d w i t h o t h e r p a r t i c l e s . A l l t h e enzymes s t u d i e d i n c r e a s e d t w o - f o l d o r more i n a c t i v i t y a f t e r t h e p o t a t o t u b e r was s l i c e d and a g e d i n t h e l i g h t . C i n n a m i c a c i d h y d r o x y l a s e i n c r e a s e d i n a c t i v i t y and a l s o c h a n g e d f r o m a m a i n l y s o l u b l e enzyme i n d o r m a n t p o t a t o t o t h e m i c r o s o m a l enzyme c h a r a c t e r i s t i c o f a c t i v e l y m e t a b o l i z i n g t i s s u e . i i i TABLE OF CONTENTS Page ABSTRACT i i L I S T OF T A B L E S i x L I S T OF FIGURES . x i ACKNOWLEDGEMENTS. . x i 1 1 INTRODUCTION. . .- 1 MATERIALS AND METHODS 15 C h e m i c a l s 15 B i o l o g i c a l M a t e r i a l s . . . . . . . . . 16 S p i n a c h . . . . . 16 B a r l e y . . 1 7 T o m a t o 17 T i s s u e c u l t u r e ^ P o t a t o . 1 9 F r a c t i o n a t i o n P r o c e d u r e s . 2 0 P r e p a r a t i o n o f c h l o r o p l a s t s 20 N o n - a q u e o u s c e l l f r a c t i o n a t i o n . 20 i v Page P r e p a r a t i o n o f p o t a t o c e l l f r a c t i o n s by d i f f e r e n t i a l c e n t r i f u g a t i o n 21 P r e p a r a t i o n o f c e l l f r a c t i o n s by s u c r o s e d e n s i t y g r a d i e n t s e p a r a t i o n 22 E x t r a c t i o n and C h r o m a t o g r a p h y P r o c e d u r e s . . 23 E x t r a c t i o n and c h r o m a t o g r a p h y o f p h e n o l i c a c i d s 23 F o r m a t i o n , e x t r a c t i o n and c h r o m a t o g r a p h y o f 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e s (DPNH) 24 E x t r a c t i o n and c h r o m a t o g r a p h y o f l i p i d s ( i n c l u d i n g p i a s t o q u i n o n e s ) . . . . . . 25 The P r e p a r a t i o n o f p - H y d r o x y p h e n y l p y r u v i c A c i d f r o m L - T y r o s i ne- ''C u s i n g L-Amino A c i d O x i d a s e . . . 26 R a d i o t r a c e r M e t h o d s 26 L i q u i d s c i n t i l l a t i o n c o u n t i n g . . . . . . . . . . . 26 E x a m i n a t i o n o f c h r o m a t o g r a m s . . . . . . . . . . . . 27 P r o t e i n E s t i m a t i o n . . . . . . . . . . . . . . . . . . 27 P r e p a r a t i o n o f P o t a t o P h e n y l a l a n i n e Ammonia-L y a s e ( P A L ) 28 Enzyme A s s a y s 29 C a t a l a s e . . . . . . . . . . . . . . . . . 29 Fu m a r a s e 29 PAL 30 0 - m e t h y l t r a n s f e r a s e a c t i n g on c a f f e i c a c i d . . . . 30 C i n n a m i c a c i d 4 - h y d r o x y l a s e 31 p_-Coumaric a c i d h y d r o x y l a s e 32 v Page S h i k i m i c a c i d : N A D P o x i d o r e d u c t a s e 33 P r e p h e n i c a c i d : N A D P o x i d o r e d u c t a s e . . . . . . . . . 34 P r e p a r a t i o n o f p r e p h e n i c a c i d f r o m t h e b a r i u m s a l t 34 E s t i m a t i o n o f p r e p h e n i c a c i d . . . 34 A l c o h o l d e h y d r o g e n a s e s . . . . . . . . 35 A l k y l a l c o h o l d e h y d r o g e n a s e . . . . . . . . . . . 35 A r o m a t i c a l c o h o l d e h y d r o g e n a s e 35 D - G l y c e r a l d e h y d e - 3 - p h o s p h a t e : N A D P o x i d o -r e d u c t a s e ( P h o s p h o r y l a t i n g ) . . . . . 35 PART I METABOLISM OF TYROSINE BY ISOLATED CHLOROPLASTS RESULTS . . . . . . . . 36 E x p e r i m e n t s w i t h S p i n a c h C h l o r o p l a s t s . . . . 36 E x p e r i m e n t s w i t h G r e e n i n g B a r l e y . . . . . . 38 E x p e r i m e n t s w i t h T o m a t o S e e d l i n g s . . . . . . . . . . . 42 E x p e r i m e n t s w i t h Ruta T i s s u e C u l t u r e . . . . . . . . . 47 DISCUSSION 52 v i PART I I INTR A C E L L U L A R L O C A L I Z A T I O N OF ENZYMES OF PHENYLPROPANOID METABOLISM I N POTATO TUBER Page RESULTS 54 G e n e r a l O b s e r v a t i o n s . . . . . 54 N a t u r e o f t h e b a n d s o b s e r v e d a f t e r d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . . . . 54 P o t a t o . . . . . . . . . . . 54 S p i n a c h . . 61 E f f e c t o f a n t i b i o t i c s on t h e p r e s e n c e o f enzymes 61 P h e n y l a l a n i n e A m m o n i a - L y a s e 63 C i n n a m i c A c i d 4 - H y d r o x y l a s e 67 O - M e t h y l T r a n s f e r a s e . . . . . . . . 71 S h i k i m i c A c i d r N A D P O x i d o r e d u c t a s e and P r e p h e n i c A c i d r N A D P O x i d o r e d u c t a s e . . . . . . . . . 73 NADP O x i d o r e d u c t a s e 73 p_-Coumaric A c i d H y d r o x y l a s e 75 A l c o h o l D e h y d r o g e n a s e s . . . . . . . 75 B i o s y n t h e t i c C a p a b i l i t i e s o f S u b c e l l u l a r F r a c t i o n s S u p p l i e d w i t h R a d i o a c t i v e P h e n y l a l a n i n e and T y r o s i n e 77 DISCUSSION 79 D i s t r i b u t i o n o f PAL i n S u b c e l l u l a r F r a c t i o n s f r o m Aged P o t a t o T u b e r . 79 v i i Page D i s t r i b u t i o n o f C i n n a m i c A c i d 4 - H y d r o x y l a s e f r o m S u b c e l l u l a r F r a c t i o n s o f Aged P o t a t o T u b e r 83 T h e E f f e c t o f A g e i n g on Enzymes o f P h e n y l -p r o p a n o i d M e t a b o l i s m o f P o t a t o 83 T y r o s i n e M e t a b o l i s m by Aged P o t a t o T i s s u e . . 88 G e n e r a l C o n c l u s i o n s . . 88 REFERENCES 93 vi i i LIST OF TABLES T a b l e Page 1 D i s t r i b u t i o n o f PAL i n g l y o x y s o m e s o f Ricinus communis e n d o s p e r m „ 10 2 R a d i o a c t i v i t y i n 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e d e r i v a t i v e s o f compounds f r o m t y r o s i n e - 1 f e d t o c e l l f r a c t i o n s o f y o u n g b a r l e y 41 3 R a d i o a c t i v e compounds r e c o v e r e d a f t e r a d m i n -i s t r a t i o n o f t y r o s i n e - 1 4 C t o t o m a t o s e e d l i n g s 43 4 D i s t r i b u t i o n o f PAL i n s u b c e l l u l a r f r a c t i o n s o f p o t a t o a g e d i n t h e l i g h t 64 5 T h e e f f e c t o f a g e i n g i n t h e l i g h t on t h e p r o d u c t i o n o f c i n n a m i c a c i d 4 - h y d r o x y l a s e i n p o t a t o t u b e r t i s s u e . . . . . . . . . 69 6 S u b c e l l u l a r d i s t r i b u t i o n o f c i n n a m i c a c i d 4 - h y d r o x y l a s e i n d o r m a n t p o t a t o t u b e r s and i n t i s s u e a g e d i n t h e l i g h t 70 7 D i s t r i b u t i o n o f c i n n a m i c a c i d 4 - h y d r o x y l a s e i n t h e 1 0 , 0 0 0 x £ p e l l e t o f s p i n a c h 71 8 S u b c e l l u l a r d i s t r i b u t i o n o f 0 - m e t h y l t r a n f e r a s e i n d o r m a n t p o t a t o t u b e r and i n t i s s u e a g e d i n t h e l i g h t 72 i x T a b l e Page 9 S u b c e l l u l a r d i s t r i b u t i o n o f s h i k i m i c a c i d : NADP o x i d o r e d u c t a s e (SOR) and p r e p h e n i c a c i d : NADP o x i d o r e d u c t a s e (POR) i n d o r m a n t p o t a t o t u b e r and i n t i s s u e a g e d i n t h e l i g h t . . . . . . 74 10 P o t a t o a l c o h o l d e h y d r o g e n a s e s as d e s c r i b e d by D a v i e s et al. , and r e d u c t i o n o f a l d e h y d e s by e x t r a c t s o f a g e d ' N e t t e d Gem' s l i c e s . . 76 11 R a t i o o f PAL and 0 - m e t h y l t r a n s f e r a s e a c t i v i t i e s i n s u b c e l l u l a r f r a c t i o n s o f ag e d p o t a t o . . . . . . . . . 81 12 T h e e f f e c t o f a g e i n g i n t h e l i g h t on some enzymes o f p h e n o l i c m e t a b o l i s m i n d i s k s o f p o t a t o t i s s u e . 84 x LIST OF FIGURES F i g u r e Page 1. M a i n l o c a l i z e d p h e n o l i c r e a c t i o n o f p l a n t c e l l s . 6 2. F o r m a t i o n o f u b i q u i n o n e and p i a s t o g u i n o n e f r o m p h e n y l a l a n i n e and t y r o s i n e . 12 3. C h r o m a t o g r a m o f l i p i d e x t r a c t f r o m b a r l e y s h o o t s a d m i n i s t e r e d t y r o s i n e - U L - 1 39 4. I n c r e a s e w i t h t i m e o f r a d i o a c t i v i t y i n t h e p r o t e i n o f s u b c e l l u l a r f r a c t i o n s o f t o m a t o p l a n t s a f t e r t h e a d m i n i s t r a t i o n o f t y r o s i n e -U L - l l f C 45 5. I n c r e a s e w i t h t i m e o f r a d i o a c t i v i t y i n t h e e t h e r e x t r a c t o f s u b c e l l u l a r f r a c t i o n s o f t o m a t o s e e d l i n g s a f t e r a d m i n i s t r a t i o n o f t y r o s i n e - U L - ^ C . . 46 6. I n c r e a s e w i t h t i m e o f r a d i o a c t i v i t y f r o m t y r o s i n e - U L - 1 i n t h e p r o t e i n o f s u b c e l l u l a r f r a c t i o n s o f Ruta c u l t u r e s g r o w i n g on B5 o r B5C2 medium . 48 7. Change w i t h t i m e o f r a d i o a c t i v i t y i n homo-g e n t i s i c a c i d f r o m Ruta c u l t u r e s a d m i n i s t e r e d t y r o s i n e - U L - ^ C 50 8. G r a d i e n t p r o f i l e o f enzymes f r o m t h e 1 0 , 0 0 0 x £ p e l l e t f r o m 100 gm o f p o t a t o d i s k s a g e d i n t h e l i g h t 55 x i F i g u r e Page 9. G r a d i e n t p r o f i l e o f enzymes f r o m t h e 10,000xg_ p e l l e t f r o m 100 gm o f d o r m a n t p o t a t o d i s k s . . . . . . . 56 10. G r a d i e n t p r o f i l e o f enzymes f r o m t h e 10,000xg_ p e l l e t f r o m 70 gm o f p o t a t o d i s k s a g e d i n t h e l i g h t 57 11. G r a d i e n t p r o f i l e o f enzymes f r o m t h e 10,000xg_ p e l l e t o f 45 gm o f d i s k s f r o m d o r m a n t p o t a t o 58 12. G r a d i e n t p r o f i l e o f enzymes f r o m t h e 10,000xg_ p e l l e t f r o m 80 gm o f p o t a t o d i s k s a g e d i n t h e l i g h t i n s t r e p t o m y c i n . . . . . 59 13. G r a d i e n t p r o f i l e o f enzymes f r o m t h e 10,000xg_ p e l l e t f r o m 50 gm o f s p i n a c h . . . . . . 62 14. A s s e s s m e n t o f e r r o r s i n t h e PAL a s s a y due t o o v e r n i g h t i n c u b a t i o n 66 15. G r a d i e n t p r o f i l e o f p a r t i a l l y p u r i f i e d PAL m i x e d w i t h o r g a n e l l e s f r o m d o r m a n t p o t a t o . . . . 68 16. L o c a l i z a t i o n o f p h e n y l p r o p a n o i d r e a c t i o n s i n d o r m a n t p o t a t o 89 17. L o c a l i z a t i o n o f p h e n y l p r o p a n o i d r e a c t i o n s i n d i s k s o f p o t a t o a g e d i n t h e l i g h t . . . . . . 90 x i i ACKNOWLEDGEMENTS T h e a u t h o r w i s h e s t o t h a n k D r . B. G r e e n , D r . B. Bohm, D r . D.M. Camm and t h e members o f t h e B o t a n y D e p a r t m e n t f o r h e l p f u l a d v i c e a nd d i s c u s s i o n s . M i s s S. S h i n o f t h i s d e p a r t m e n t d e s e r v e s t h a n k s f o r e x p e r t h e l p i n t h e p r e p a r a t i o n and i n t e r p r e t a t i o n o f e l e c t r o n m i c r o g r a p h s . S p e c i a l t h a n k s a r e due D r . G.H.N. T o w e r s f o r many i n t e r e s t i n g s u g g e s t i o n s a nd s t i m u l a t i n g d i s c u s s i o n s . T h i s work was c a r r i e d o u t u n d e r N a t i o n a l R e s e a r c h C o u n c i l S t u d e n t s h i p s f o r 1968-69 and 1969-70 and K i l l a m P r e d o c t o r a l F e l l o w s h i p s 1970-71 and 1 9 7 1 - 7 2 . x i i i INTRODUCTION T h e p h e n o l i c m e t a b o l i s m o f t h e p l a n t i s e s p e c i a l l y s e n s i t i v e t o e n v i r o n m e n t a l s t i m u l i [ Y o s h i d a 1 9 6 9 ] . In b r i g h t l i g h t , f o r e x a m p l e , t h e l e v e l s o f f l a v o n o i d s a r e much h i g h e r t h a n i n d i m , and i n j u r y o r i n f e c t i o n c a n r e s u l t i n t h e f o r m a t i o n o f n e c r o t i c a r e a s h i g h i n p h e n o l i c com-p o u n d s . T h i s s t u d y i s an i n v e s t i g a t i o n o f some r e a c t i o n s and enzymes o f p h e n o l i c m e t a b o l i s m , t h e i r l o c a t i o n w i t h i n t h e c e l l , and how t h e y a r e a f f e c t e d by e n v i r o n m e n t a l c h a n g e s . One a p p r o a c h has b e e n t h e i n v e s t i g a t i o n o f c h a n g e s o f l e v e l s o f enzymes i n v o l v e d i n t h e f o r m a t i o n o f p h e n o l i c c o m p o u n d s . C o n c o m i t a n t i n c r e a s e s i n l e v e l s o f p h e n y l a l a n i n e a m m o n i a - l y a s e ( P A L ) and p h e n y l p r o p a n o i d s h a v e b e e n demon-s t r a t e d i n many p l a n t s and p l a n t t i s s u e s . In g h e r k i n s e e d l i n g s , c h a n g e s i n PAL l e v e l s w h i c h o c c u r a f t e r t r e a t -ment w i t h b l u e l i g h t , l o n g t e r m r e d l i g h t , w o u n d i n g o r t e m p e r a t u r e c h a n g e s a r e a l l r e f l e c t e d i n t h e l e v e l s o f a c c u m u l a t e d h y d r o x y c i n n a m i c a c i d s [ E n g e l s m a 1 9 6 7 , 1 9 6 7 a , 1 9 6 8 , 1 2 1 9 6 9 ] . F r e s h p o t a t o t i s s u e , d e v o i d o f PAL o r c h l o r o g e n i c a c i d , d e v e l o p s b o t h on i n c u b a t i o n i n l i g h t [ Z u c k e r 1 9 6 5 ] . S i m i l a r i n c r e a s e s i n PAL and c h l o r o g e n i c a c i d h a v e b e e n d e s c r i b e d i n e t h y l e n e - t r e a t e d s w e e t p o t a t o t i s s u e [ I m a s e k i e t a l _ . 1 9 6 8 ] . F l a v o n o i d s , t o o , a r e f o r m e d i n i n c r e a s e d amounts a f t e r e x p o s u r e o f t h e p l a n t t o l i g h t [ S c h e r f and Zenk 1 9 6 7 ] . In b u c k w h e a t , PAL p r o d u c t i o n and a n t h o c y a n i n p r o d u c t i o n a r e a f f e c t e d t h e same way by r e d and f a r - r e d l i g h t [ S c h e r f and Z e nk 1 9 6 7 ] . A p i i n and g r a v e o b i o s i d e B f o r m a t i o n , a f t e r i l l u m i n a t i o n o f p a r s l e y c e l l c u l t u r e s , c o i n c i d e s w i t h a p e a k i n PAL a c t i v i t y [ H a h l b r o c k e_t a l _ . 1 9 7 1 ] . C i n n a m a t e h y d r o x y l a s e , a n o t h e r enzyme o f p h e n y l -p r o p a n o i d m e t a b o l i s m , a l s o shows an i n c r e a s e on e x p o s u r e o f p a r s l e y c e l l c u l t u r e s t o l i g h t [ H a h l b r o c k e_t a l _ . 1 9 7 1 ] , and a f t e r l i g h t and w o u n d i n g i n b u c k w h e a t s e e d l i n g s [ A m h r e i n and Z e nk 1 9 7 0 ] , In p a r s l e y , PAL, c i n n a m a t e h y d r o x y l a s e and p_-c o u m a r a t e : C o A l i g a s e i n c r e a s e i n p a r a l l e l a f t e r t h e c e l l c u l t u r e has b e e n e x p o s e d t o l i g h t [ H a h l b r o c k e_t a l _ . 1 9 7 1 ] . T h e s e enzymes c a t a l y s e t h e f i r s t t h r e e s t e p s i n t h e c o n v e r s i o n o f p h e n y l a l a n i n e t o t h e f l a v o n o i d g r a v e o b i o s i d e B. T h e l e v e l s o f o t h e r enzymes c o n c e r n e d m a i n l y w i t h t h e c a r b o h y d r a t e p o r t i o n o f t h e m o l e c u l e i n c r e a s e i n p a r a l l e l a l s o , b u t w i t h 3 d i f f e r e n t k i n e t i c s f r o m t h e f i r s t t h r e e . Enzymes o f b a s i c c e l l m e t a b o l i s m s u c h as a c e t a t e : C o A l i g a s e a r e u n a f f e c t e d d u r i n g a s i m i l a r l i g h t s t i m u l a t i o n o f c e l l c u l t u r e s o f s o y b e a n [ H a h l b r o c k e_t aJL 1 9 7 1 a ] . O t h e r enzymes c o n c e r n e d w i t h p h e n o l i c m e t a b o l i s m a r e a l s o s e n s i t i v e t o s t i m u l a t i o n . The t h r e e p h e n o l a s e s i n p o t a t o t u b e r s i n c r e a s e 3 - f o l d i n 24 h o u r a g e i n g o f t i s s u e s l i c e s [ R u i s 1 9 7 2 ] . In p a r s l e y c e l l c u l t u r e s , t h e enzyme m e t h y l a t i n g t h e m - h y d r o x y l o f £ - d i h y d r i c p h e n o l s ( a n 0 - m e t h y l t r a n s f e r a s e ) i s a l s o s t i m u l a t e d by l i g h t and a g e i n g [ H a h l b r o c k e t aj_. 1 9 7 1 , E b e l et_ aj_. 1 9 7 2 ] . The k i n e t i c s o f s t i m u l a t i o n o f t h i s enzyme a r e n o t t h e same as t h o s e e x h i b i t e d by PAL. The a p p r o a c h u s e d i n t h e s e s t u d i e s h a s b e e n t h e t r a d i t i o n a l one o f t h e b i o c h e m i s t - i n v e s t i g a t i o n o f s o l u -b i l i z e d enzymes f r o m a c e l l b r e i . B u t w i t h i n t h e p l a n t c e l l , enzymes a r e f r e q u e n t l y f o u n d a s s o c i a t e d w i t h s p e c i f i c o r g a n e l l e s . In some c a s e s , an i n t r a c e l l u l a r t r a n s p o r t s y s t e m a p p e a r s t o o p e r a t e b e t w e e n d i f f e r e n t o r g a n e l l e s [ K i s a k i and T o l b e r t 1 9 6 9 ] . S t u d i e s o f t h e c o n t r o l o f p l a n t p h e n o l i c m e t a b o l i s m must t a k e i n t o a c c o u n t t h i s i n t r a c e l l u l a r o r g a n i z a t i o n . In t h i s s t u d y , c h l o r o p l a s t s f r o m l e a f t i s s u e , and v a r i o u s p a r t i c u l a t e f r a c t i o n s f r o m d o r m a n t and a g e d p o t a t o 4 h a v e b e e n e x a m i n e d . The p a r t i c u l a t e f r a c t i o n s i n c l u d e d t h e m i c r o b o d y - r i c h , m i t o c h o n d r i o n - r i c h , and m i c r o s o m a l f r a c t i o n s . The m i c r o b o d y i s f o u n d i n many t y p e s o f p l a n t t i s s u e [ F r e d e r i c k and Newcomb 1 9 6 9 ] . I t u s u a l l y has a b u o y a n t d e n s i t y o f 1.25 t o 1.26 and c o n t a i n s a l l t h e g l y c o l i c a c i d o x i d a s e , u r i c a c i d o x i d a s e and c a t a l a s e o f t h e c e l l [ T o l b e r t e_t a l _ . 1968, V i g i l 1 9 6 9 ] . M i c r o b o d i e s f r o m t i s s u e o x i d i z i n g f a t t y a c i d s c o n t a i n a l l t h e p - o x i d a t i o n enzymes and enzymes o f t h e g l y o x y l a t e p a t h w a y . T h i s t y p e o f m i c r o -b o d y has b e e n t e r m e d t h e g l y o x y s o m e [ B e e v e r s 1 9 6 9 ] . M i c r o -b o d i e s f r o m p h o t o s y n t h e t i c t i s s u e a r e c a l l e d p e r o x i s o m e s , and a r e i n v o l v e d i n t h e g l y c o l a t e m e t a b o l i s m o f p h o t o -s y n t h e s i s and p h o t o r e s p i r a t i on [ T o l b e r t and Y a m a z a k i 1 9 6 9 ] . T i s s u e s s u c h as c u c u m b e r c o t y l e d o n s f i r s t m e t a b o l i z e f a t t y a c i d s t h e n become p h o t o s y n t h e t i c [ T r e l e a s e ejt aj_. 1 9 7 1 ] . I t a p p e a r s t h a t p e r o x i s o m a l enzymes m e r e l y r e p l a c e g l y o x y -s o m a l enzymes i n t h e same p o p u l a t i o n o f m i c r o b o d i e s . P o t a t o t u b e r t i s s u e ( u s e d i n t h i s s t u d y ) i s n e i t h e r p h o t o s y n t h e t i c n o r d o e s i t n o r m a l l y m e t a b l i z e f a t t y a c i d s . M i c r o b o d i e s f r o m t h i s t i s s u e have n e i t h e r g l y o x y l a t e n o r g l y c o l a t e c y c l e enzymes [ R u i s 1 9 7 1 ] . H o w e v e r , t h e s e p a r t i c l e s a r e o f t h e e x p e c t e d b u o y a n t d e n s i t y and c o n t a i n c a t a l a s e and u r i c a c i d o x i d a s e . 5 M i c r o s o m a l f r a c t i o n s w e re a l s o e x a m i n e d i n t h i s s t u d y . T h e d e f i n i t i o n o f t h i s f r a c t i o n i s a f u n c t i o n a l o n e , f o r i t i n c l u d e s t h o s e p a r t i c l e s w h i c h do n o t s e d i m e n t a f t e r c e n t r i f u g a t i o n a t 10,000 t i m e s g r a v i t y , b u t f o r m a p e l l e t a f t e r 1 h o u r a t 1 0 8 , 0 0 0 x £ . T h i s f r a c t i o n i n c l u d e s r i b o s o m e s and f r a g m e n t s o f t h e e n d o p l a s m i c r e t i c u l u m , a l t h o u g h o t h e r p a r t i c l e s a r e a l m o s t c e r t a i n l y p r e s e n t . W h i l e m o s t o f t h e work on l o c a l i z a t i o n o f enzymes has b e e n c o n c e r n e d w i t h o x i d a t i v e and p h o t o s y n t h e t i c e n z y m e s , t h e r e has b e e n some i n t e r e s t i n t h e l o c a l i z a t i o n o f enzymes c o n c e r n e d w i t h p h e n o l i c b i o s y n t h e s i s ( s u m m a r i z e d i n F i g u r e l ) . C h l o r o p l a s t s f r o m S a x i f r a g a a r e r e p o r t e d t o c o n v e r t p _ - c o u m a r i c a c i d t o c a f f e i c a c i d [ S a t o 1 9 6 6 ] , and s e c o n d l y , c i s - c a f f e i c a c i d t o e s c u l e t i n [ S a t o 1 9 6 7 ] . T h e s e c h l o r o -p l a s t s and t h o s e o f s e v e r a l o t h e r a r b u t i n - c o n t a i n i n g p l a n t s c o n t a i n £ - d i p h e n o l o x i d a s e - l i k e enzymes w h i c h a r e q u i t e s p e c i f i c f o r a r b u t i n , c o n v e r t i n g i t t o 3 , 4 - d i h y d r o x y p h e n y l -$ - D - g 1 u c o s i d e , and t h e n t o t h e £ - q u i n o n e [ H a t t o r i and S a t o 1 9 6 3 ] . H o w e v e r , t h e s e c h l o r o p l a s t p r e p a r a t i o n s m i g h t a l s o h a v e c o n t a i n e d p e r o x i s o m e s . S p i n a c h b e e t c h l o r o p l a s t s c o n t a i n a p h e n o l a s e w h i c h h y d r o x y l a t e s p _ - c o u m a r i c a c i d . S p i n a c h b e e t s t e m s , w h i c h a r e n o n - p h o t o s y n t h e t i c , l a c k t h i s enzyme [ P a r i s h 1 9 7 2 ] . 6 F i g u r e 1 ( f a c i n g ) M a i n l o c a l i z e d r e a c t i o n s o f p h e n o l i c m e t a b o l i s m o f p l a n t c e l l s . I, p h e n y l a l a n i n e ; 2, c i n n a m i c a c i d ; 3 , p_-"coumaric a c i d ; 4 , c a f f e i c a c i d ; 5, b e n z o i c a c i d ; 6 , s a l i c y l i c a c i d ; 7 , d i h y d r o c i n n a m i c a c i d ; 8 , 3 , 4 1 - d i h y d r o s t i I b e n e - 2 - c a r b o x y I i c a c i d ; 9 , o - c o u m a r i c a c i d ; 1 0 , umbeI I i f e r o n e ; I I , ^ - h y d r o x y -b e n z o i c a c i d ; 1 2 , f e r u l i c a c i d ; 1 3 , s c o p o l e t i n ; 1 4 , e s c u l e t i n ; 1 5 , t y r o s i n e ; 1 6 , p_-hyd r o x y p h e n y p y r u v i c a c i d ; 1 7 , h o m o g e n -t i s i c a c i d ; 1 8 , p - h y d r o x y p h e n y I a c e t i c a c i d ; 1 9 , a r b u t i n ; 2 0 , 3 - h y d r o x y a r b u t i n . M a j o r s i t e s o f enzyme: S, s o l u b l e ; G, g l y o x y s o m a l ; P, p e r o x i s o m a l ; C, c h l o r o p l a s t i d i c; M, m i t o c h o n d r i a l ; Ms, m i c r o s o m a l . 8 O t h e r r e a c t i o n s w h i c h o c c u r i n t h e c h l o r o p l a s t i n c l u d e t h e o _ - h y d r o x y l a t i on o f c i n n a m i c a c i d , p _ - c o u m a r i c a c i d , f e r u l i c a c i d and b e n z o i c a c i d [ K i n d l 1 9 7 1 ] . In s e v e r a l c a s e s , t h e c o r r e s p o n d i n g c o u m a r i n s a r e f o r m e d . In H y d r a n g e a  m a c r o p h y l 1 a , t h e i n t a c t c h l o r o p l a s t s a r e a b l e t o c a t a l y z e t h e c o n d e n s a t i o n o f p _ - c o u m a r i c a c i d w i t h t h r e e a c e t a t e u n i t s t o f o r m t h e s t i l b e n e , 3 , 4 1 - d i h y d r o x y s t i 1 b e n e - 2 -c a r b o x y l i c a c i d , a l t h o u g h t h e amounts f o r m e d a r e e x c e e d i n g l y low [ K i n d l . 1 9 7 1 a ] . C h l o r o p l a s t s a r e a l s o a b l e t o c a t a l y z e t h e f o r m a -t i o n o f p h e n y l a c e t a l d e h y d e and p h e n y l a c e t i c a c i d f r o m p h e n y l a l a n i n e [ K i n d l 1 9 7 2 ] . T h o s e f r o m p e a and b a r l e y c a n make i n d o l e a c e t i c a c i d and i n d o l y l a c r y l i c a c i d f r o m L -t r y p t o p h a n [ K i n d l 1 9 7 2 ] . M i t o c h o n d r i a do n o t seem t o be t h e s i t e o f many enzymes c o n c e r n e d w i t h p h e n o l i c m e t a b o l i s m , a l t h o u g h c i n -n a m i c a c i d i s r e p o r t e d t o be r e d u c e d t o d i h y d r o c i n n a m i c a c i d h e r e [ K i n d l 1 9 7 2 ] , In s p i n a c h b e e t , DOPA and c a t e c h o l o x i d a s e s a r e f o u n d i n t h e m i t o c h o n d r i a [ P a r i s h 1 9 7 2 ] . T he g l y o x y s o m e s a r e t h e s i t e o f c o n s i d e r a b l e p h e n o l i c m e t a b o l i s m , R i c i n u s communis g l y o x y s o m e s a r e c a p a b l e o f c o n v e r t i n g t y r o s i n e i n t o n _ - h y d r o x y p h e n y l a c e t i c a c i d and h o m o g e n t i s i c a c i d [ K i n d l and R u i s 1 9 7 1 ] . T h e g l y o x y s o m e s a r e a l s o a s i t e o f c h a i n s h o r t e n i n g o f t h e 9 c i n n a m i c a c i d d e r i v a t i v e s t o b e n z o i c a c i d d e r i v a t i v e s [ K i n d ! and R u i s 1 9 7 1 ] . P r e p a r a t i o n s o f g l y o x y s o m e s f r o m c a s t o r b e a n e n d o s p e r m h a v e t h e h i g h e s t s p e c i f i c a c t i v i t i e s o f PAL, t y r o s i n e a m m o n i a - l y a s e ( T A L ) and h i s t i d i n e a m m o n i a - l y a s e , a l t h o u g h a l l t h e p a r t i c l e s s e p a r a t e d on a s u c r o s e d e n s i t y g r a d i e n t showed a c t i v i t y . T h e a u t h o r s r e p o r t t h i s f i n d i n g i n unwashed o r g a n e l l e f r a c t i o n s as a l o c a l i z a t i o n o f t h e ammonia l y a s e s i n t h e g l y o x y s o m e s , b u t t h i s s t a t e m e n t s h o u l d be t r e a t e d w i t h c a u t i o n . S p e c i f i c a c t i v i t y d e p e n d s on t h e amount o f p r o t e i n p r e s e n t , and a h y p o t h e t i c a l o r g a n e l l e c o n t a i n i n g 90% o f t h e c e l l c o m p l e m e n t o f enzyme x, p l u s q u a n t i t i e s o f s t o r a g e p r o t e i n , s a y , w o u l d h a v e a v e r y l o w s p e c i f i c a c t i v i t y . T h e f o l l o w i n g d a t a a r e f r o m R u i s and K i n d ! 1971. T h e p e r c e n t a g e t o t a l a c t i v i t y has b e e n c a l c u l a t e d f r o m t h e i r d a t a ( T a b l e 1 ) . T h e s e w o r k e r s d i d n o t i n v e s t i g a t e t h e m i c r o s o m a l f r a c t i o n b u t e v e n s o , 9 5 % o f t h e t o t a l a c t i v i t y i s f o u n d i n t h e s o l u b l e f r a c t i o n . T h u s , w h i l e t h e g l y o x y s o m e s do a p p e a r t o c o n t a i n a m m o n i a - l y a s e s , t h e s e enzymes a r e e i t h e r v e r y r e a d i l y s o l u b i l i z e d o r a r e m a i n l y s o l u b l e i n v i v o . S i m i l a r work has b e e n c a r r i e d o u t on t h e p e r o x i -somes o f s u n f l o w e r and s p i n a c h [ R u i s and K i n d l 1 9 7 1 ] . H i g h 10 T a b l e I D i s t r i b u t i o n o f P A L in c e l l f r a c t i o n s o f Ri c i n us commun i s e n d o s p e r m f r a c t i on a c t i v i t y (mil) s p e c i f i c a c t i v i t y (mU/mg p r o t e i n) % a c t i v i t y 1 0 8 , 0 0 0 x g s u p e r n a t a n t 2 0 . 0 0 . 0 1 4 90 mi t o c h o n d r i a 0 . 5 0 . 0 4 2 2 . 2 p r o p 1 a s t i ds 0 . 7 0 . 2 3.2 g 1 y o x y s o m e s 1 .0 0 . 3 3 4 . 5 s p e c i f i c a c t i v i t i e s o f P A L , T A L , a n d t h e e n z y m e s f o r m i n g <*, p - u n s a t u r a t e d c a r b o x y l i c a c i d s f r o m a s p a r t i c a c i d , h i s t i d i n e a n d t r y p t o p h a n w e r e p r e s e n t i n t h e p e r o x i s o m e s . W h i l e a g a i n m o s t o f t h e a c t i v i t y was i n t h e s o l u b l e p h a s e , p u r i f i e d p e r o x i s o m a l f r a c t i o n s h a d h i g h e s t s p e c i f i c a c t i v i t y . T h e m i c r o b o d i e s o f p o t a t o , u n l i k e t h o s e o f c a s t o r b e a n e n d o s p e r m a n d s p i n a c h , h a v e no PAL a c t i v i t y [ R u i s 1 9 7 1 ] . T h e s e m i c r o b o d i e s d o , h o w e v e r , c o n t a i n £ - d i p h e n o l o x i d a s e a c t i v i t y . PAL a c t i v i t y i s a l s o f o u n d i n t h e m i c r o s o m a l f r a c t i o n f r o m S o r g h u m [ S t a f f o r d 1 9 6 9 ] . A m h r e i n a n d Z e n k 11 [ 1 9 7 1 ] , w o r k i n g w i t h e t i o l a t e d b u c k w h e a t , f o u n d t h a t some PAL a c t i v i t y was l o c a t e d i n t h e m i c r o s o m e s o f t h i s p l a n t , t o o , and t h a t i t c o u l d n o t be r e m o v e d by w a s h i n g . T h e y f o u n d t h a t t h i s p a r t i c l e was n o t a r i b o s o m e , n o r was i t i d e n t i c a l t o t h e p a r t i c l e s w i t h w h i c h c i n n a m i c a c i d 4-h y d r o x y l a s e i s a s s o c i a t e d . I n t h e work o f R u s s e l l and Conn [ 1 9 6 6 ] and R u s s e l l [ 1 9 7 1 ] t h e c i n n a m a t e h y d r o x y l a s e o f pea was l o c a t e d i n t h e m i c r o s o m e s . In y o u n g Sorghum a l l t h e c i n n a m a t e h y d r o x y l a s e a c t i v i t y i s a s s o c i a t e d w i t h p a r t i c l e s w h i c h s e d i m e n t b e t w e e n 500 and 100 ,000xg_, w h i c h i n c l u d e s t h e m i c r o s o m a l f r a c t i o n [ S t a f f o r d 1 9 6 9 ] . In b u c k w h e a t a l s o , t h e enzyme a p p e a r s on a m i c r o s o m a l p a r t i c l e [ A m r h e i n and Zenk 1 9 7 1 ] . Much i n f o r m a t i o n e x i s t s on t h e l o c a t i o n w i t h i n t h e c e l l o f p h e n o l i c compounds and t h e enzymes i n v o l v e d i n t h e i r s y n t h e s i s . L e s s i n f o r m a t i o n e x i s t s on t h e i n c r e a s e o f t h e s e enzymes a f t e r a c h a n g e i n t h e e n v i r o n m e n t . T h e p r e s e n t s t u d y work c o m b i n e s s t u d i e s o f enzyme l o c a l i z a t i o n w i t h t h o s e o f c h a n g e s i n l e v e l s o f e n z y m e s . Two a s p e c t s o f p h e n o l i c m e t a b o l i s m w e r e i n -v e s t i g a t e d . T h e f i r s t p a r t o f t h i s s t u d y i n v o l v e d t h e c h l o r o p l a s t and a g r o u p o f p h e n o l i c compounds f o u n d t h e r e , t h e p i a s t o q u i n o n e s ( F i g u r e 2 ) . L i g h t c a u s e s t h e l e v e l o f t h e s e compounds t o r i s e i n e t i o l a t e d b a r l e y [ L i c h t e n t h a l e r 1 9 6 9 ] . I t has b e e n shown t h a t 12 O H UBIQUINONE 9 PLASTOQUINONE A 9 F i g u r e 2 . F o r m a t i o n o f u b i q u i n o n e a n d p i a s t o q u i n o n e f r o m p h e n y l a l a n i n e a n d t y r o s i n e [ a f t e r W h i s t a n c e a n d T h r e l l f a l l 1 9 6 7 ] . p h e : p h e n y l a l a n i n e , t y r : t y r o s i n e , c i n n : c i n n a m i c a c i d , £ - c o u m : p _ - c o u m a r i c a c i d , p - O H b e n z : p _ - h y d r o x y b e n z o i c a c i d , p h p p : p _ - h y d r o x y p h e n y I p y r u v i c a c i d , h g : h o m o g e n t i s i c a c i d . 13 D- and L - t y r o s i n e , and i n some p l a n t s , p h e n y l a l a n i n e , a r e v e r y g o o d p r e c u r s o r s o f t h e s e c o mpounds, and t h a t t h e m e t h y l g r o u p meta t o t h e p o l y p r e n y l c h a i n i s d e r i v e d f r o m t h e |3 - c a r b o n o f t y r o s i n e [ W h i s t a n c e and T h r e l f a l l 1 9 6 8 ] , The m o s t l o g i c a l and d i r e c t r o u t e f o r t h e c o n v e r s i o n o f t y r o s i n e t o p i a s t o q u i n o n e i s v i a p - h y d r o x y p h e n y l p y r u v i c a c i d (PHPP) and h o m o g e n t i s i c a c i d (HG) [ W h i s t a n c e and T h r e l f a l l 1 9 6 7 ] . A l t h o u g h t h e enzymes f o r t h e s e c o n v e r s i o n s a r e known i n b a c t e r i a t h e y a r e n o t a l l known i n p l a n t s . F u r t h e r c r e d e n c e i s g i v e n t h i s s u g g e s t i o n , h o w e v e r , by t h e f i n d i n g s t h a t r a d i o a c t i v e PHPP [ T h r e l f a l l and W h i s t a n c e 1 9 6 8 ] and HG [ W h i s t a n c e and T h r e l f a l l 1 970] a r e w e l l i n c o r p o r a t e d i n t o p l a s t o q u i n o n e . S i m i l a r l y , t h e u b i q u i n o n e s a c c u m u l a t e i n t h e m i t o c h o n d r i a [ G r i f f i t h s e t a l _ . 1 9 6 7 ] , T h e u b i q u i n o n e s h a v e b e e n shown t o be f o r m e d f r o m a d m i n i s t e r e d p h e n y l a l a n i n e o r t y r o s i n e v i a p _ - c o u m a r i c a c i d and p _ - h y d r o x y b e n z o i c a c i d [ W h i s t a n c e and T h r e l f a l l 1 9 6 8 ] . Some o f t h e enzymes c a p a b l e o f c a r r y i n g o u t t h e s e r e a c t i o n s h a v e b e e n l o c a t e d w i t h i n t h e c e l l ( F i g u r e 1 ) . In o r d e r t o s e t t l e t h e p r o b l e m o f t h e r o l e o f e a c h o r g a n e l l e i n a s p e c i f i c a r e a o f m e t a b o l i s m , t h e enzymes i n v o l v e d must be l o c a t e d w i t h r e s p e c t t o t h e s e o r g a n e l l e s . I f , f o r e x a m p l e , a l l t h e enzymes s y n t h e s i z i n g t h e r i n g o f 14 p i a s t o q u i n o n e w e r e f o u n d t o be e x t r a c h l o r o p l a s t i d i c , t h e n t h e p r e s e n c e o f p1 a s t o q u i n o n e s i n t h e c h l o r o p l a s t a f t e r i l l u m i n a t i o n w o u l d be a r e s u l t o f t r a n s p o r t and n o t o f i n s i t u s y n t h e s i s . T h e s e c o n d p a r t o f t h e work i n v o l v e s t h e i n c r e a s e i n p h e n o l i c a c i d s i n p o t a t o a f t e r a g e i n g i n t h e l i g h t [ Z u c k e r 1 9 6 3 ] . T h e i n t r a c e l l u l a r l o c a t i o n o f enzymes i n -v o l v e d i n t h i s i n c r e a s e was i n v e s t i g a t e d i n d o r m a n t p o t a t o t i s s u e and i n d i s k s a g e d i n t h e l i g h t . I t was f e l t t h a t t h e c o m b i n a t i o n o f t h e a p p r o a c h e s o f l o c a l i z a t i o n and c h a n g e s i n enzyme l e v e l s w o u l d y i e l d a more i n t e g r a t e d p i c t u r e o f p h e n o l i c m e t a b o l i s m i n t h e t i s s u e s i n v e s t i g a t e d . MATERIALS AND METHODS Chemi c a l s M o s t b i o c h e m i c a l r e a g e n t s and m a t e r i a l s f o r t h e p r e p a r a t i o n o f m e d i a and c e l l f r a c t i o n s w e re o b t a i n e d f r o m m C a l b i o c h e ^ , La J o l l a , C a l i f . T h e s e i t e m s i n c l u d e M i r a c l o t h , t r i c i n e b u f f e r , s o r b i t o l , d i t h i o t h r e i t o ! , ATP, a l l p y r i d i n e n u c l e o t i d e s and amino a c i d s , g l u c o s e - 6 - p h o s p h a t e d e h y d r o -g e n a s e , 3 - p h o s p h o g l y c e r a l d e h y d e and 3 - p h o s p h o g l y c e r i c a c i d k i n a s e . j D - H y d r o x y p h e n y l p y r u v i c a c i d was o b t a i n e d f r o m C a l b i o c h e m b e c a u s e t h e i r p r o d u c t ( a l t h o u g h l a b e l l e d B g r a d e ) was p u r e r t h a n t h e b e s t g r a d e s f r o m o t h e r c o m p a n i e s . L-Amino a c i d o x i d a s e ( N a j a n a j a ) , s h i k i m i c a c i d , m a l i c a c i d , c a f f e i c a c i d , f e r u l i c a c i d , j p _ - h y d r o x y b e n z a l d e h y d e and m - h y d r o x y b e n z a l d e h y d e w ere o b t a i n e d f r o m K o c h - L i g h t , C o l n b r o c k , B u c k . , E n g l a n d . F i c o l l , D e x t r a n T 40, and S e p h a d e x G-200 were o b t a i n e d f r o m P h a r m a c i a ( C a n a d a ) L t d . , M o n t r e a l . 15 16 V a n i l l i n was o b t a i n e d f r o m A l d r i c h C h e m i c a l s , M i l w a u k e e , W i s e . B e n z a l d e h y d e was f r o m BDH C h e m i c a l s , T o r o n t o . R a d i o a c t i v e t y r o s i n e , p h e n y l a l a n i n e and c i n n a m i c a c i d w e r e o b t a i n e d f r o m New E n g l a n d N u c l e a r , B o s t o n , M a s s . , as were a l l r e a g e n t s u s e d i n p r e p a r a t i o n o f s a m p l e s f o r l i q u i d s c i n t i l l a t i o n c o u n t i n g . S - A d e n o s y l m e t h i o n i n e - m e t h y l - 1 ^ C was o b t a i n e d f r o m I n t e r n a t i o n a l C h e m i c a l and N u c l e a r C o r p o r a t i o n , I r v i n e , C a l i f . S i l i c a g e l G and GHR were o b t a i n e d f r o m E . M e r c k , D a r m s t a d t , Germany. MN c e l l u l o s e was p u r c h a s e d f r o m M a c h e r y , N a g e l and C o . , D u r e n , Germany. A l l o t h e r c h e m i c a l s and s o l v e n t s w e r e o f r e a g e n t g r a d e , b u t w e r e f r o m v a r i o u s s o u r c e s . B i o l o g i c a l M a t e r i a l  S p i n a c h S e e d s w e r e p l a n t e d i n f l a t s o f s o i l m a i n t a i n e d i n a g r o w t h chamber a t 2 0 ° C on 12 h o u r d a y s . P l a n t s w e r e h a r -v e s t e d a t 4-6 w e e k s . O t h e r s p i n a c h was p u r c h a s e d l o c a l l y . 17 B a r l e y S e e d s w e r e s u r f a c e s t e r i l i z e d i n 2% s o d i u m h y p o -c h l o r i t e , r i n s e d and sown i n s t e r i l e v e r m i c u l i t e i n f l a t s . F o r s t e r i l e e x p e r i m e n t s u s i n g e t i o l a t e d b a r l e y , t h e s e e d s w ere r i n s e d and sown u s i n g s t e r i l e t e c h n i q u e i n m o i s t v e r m i c u l i t e i n a l a r g e s t e e l b e a k e r c o v e r e d by f o i l . An a i r v e n t was c o v e r e d w i t h a c o t t o n p l u g . No w a t e r i n g was r e q u i r e d i f t h e s e e d l i n g s w e re u s e d b e f o r e 9 d a y s o l d . T o m a t o T o m a t o f r u i t s w e r e s u r f a c e s t e r i l i z e d i n e t h a n o l . U s i n g s t e r i l e t e c h n i q u e , s e e d s were r e m o v e d and 6 w e r e * p l a c e d on t h e s u r f a c e o f n u t r i e n t a g a r i n a 250 ml f l a s k . T h e s e e d l i n g s w e re m a i n t a i n e d a t 2 3 ° C i n 12 h o u r d a y s . T h e s e e d l i n g s w e r e u s e d i n t h e f l a s k , o r w e r e h a r v e s t e d t h u s : 30 ml o f s t e r i l e w a t e r was a d d e d t o t h e f l a s k , and t h e s e e d l i n g s w e r e c u t o f f u n d e r t h e w a t e r w i t h c u r v e d s c i s s o r s ( 5 " a r t e r i o t o m y s c i s s o r s ) . N u t r i e n t a g a r : p e r I, 1.32 gm C a ( N 0 3)2; 0.25 gm of KHaPOi*; 0.25 gm M g S O ^ T H a O ; 5 gm a g a r and t a p w a t e r . 18 T i s s u e c u l t u r e R u t a g r a v e o l e n s t i s s u e was m a i n t a i n e d f r o m s t o c k s o b t a i n e d f r o m D r . 0. Gamborg, P r a i r i e R e g i o n a l L a b o r a t o r y N a t i o n a l R e s e a r c h C o u n c i l , S a s k a t o o n . T i s s u e was m a i n t a i n e d i n s h a k e c u l t u r e on B 5 o r B 5 C 2 medium a t 2 3 ° C i n 16 h o u r d a y s . M a t e r i a l was s u b c u l t u r e d e v e r y t h r e e w e e k s . T i s s u e c u 1 1 u re medi urn ( B s C 2 ) \l NaHaPCU 'HaO 1 50 mg K N 0 3 3 . 5 gm (NrU) aSCU 1 34 mg M g S O W H z O 250 mg C a C I 2 * 2 H 2 0 1 50 mg Fe ( s e q u e s t r e n e ) * 28 mg K l 0 . 7 5 mg S u c r o s e 20 gm ** N - Z ami ne 2 gm ( o m i t f o r B 5 medium) 2 , 4 - D 1 mg v i t a m i n s o l u t i o n 10 m 1 m i c r o n u t r i e n t s o 1 . 1 ml pH 5 . 5 * G e i g y I n d u s t r i a 1 C h e m i c a l s , A r d s l e y , New Y o r k . S h e f f i e l d C h e m i c a l s , N o r w i c h , New Y o r k , 19 V i t a m i n s o l u t i o n ( i n 1 0 0 m l ) n i c o t i n i c a c i d I 0 mg t h i a m i n e I 0 0 mg p y r i d o x i n e 10 mg m y o - i n o s i t o I I gm M i c r o n u t r ? e n t s o l u t i o n ( i n 1 0 0 m l ) M n S 0 ^ * H 2 0 I gm H 3 B 0 3 3 0 0 mg Z n SO^ • 7 H 2 0 3 0 0 mg N a s M o O i j - Z H a O 25 mg CuSOij 25 mg C o C I 2 * 6 H 2 0 25 mg P o t a t o L a r g e f i r m t u b e r s o f p o t a t o v a r i e t y ' N e t t e d Gem' were w a s h e d and c y l i n d e r s were r e m o v e d w i t h a s h a r p c o r k b o r e r . D i s k s a p p r o x i m a t e l y 2 mm t h i c k were c u t f r o m t h e c y l i n d e r s and w e r e w a s h e d i n c o l d r u n n i n g w a t e r f o r 15 m i n u t e s . T h e d i s c s were e i t h e r u s e d f r e s h , o r aged 18 h o u r s u n d e r f l u o r e s c e n t l i g h t s a t 23° C i n P e t r i d i s h e s 20 (5 g m / d i s h w i t h 5 ml w a t e r ) . A f t e r 18 h o u r s , t h e d i s k s w e r e w a s h e d and c h i l l e d b e f o r e g r i n d i n g . F r a c t i o n a t i o n P r o c e d u r e s P r e p a r a t i o n o f c h l o r o p l a s t s C h i l l e d w a s h e d t i s s u e was c u t w i t h s c i s s o r s and t h e n h o m o g e n i z e d i n a b l e n d o r f o r t h r e e 2 - s e c o n d b u r s t s i n t w i c e t h e v o l u m e o f s o r b i t o l medium [ a f t e r C o c k b u r n e t a l . 1968; s o r b i t o l 0.33 M, M g C l 2 5 mM, s o d i u m i s o a s c o r b a t e 2 mM, pH 6.7 s o d i u m p y r o p h o s p h a t e b u f f e r 10 mM] o r m o d i f i e d Honda medium [ R a n a l e t t i e t aj_. 1969; F i c o l l 2.5%, D e x t r a n T - 4 0 5%, s u c r o s e 0.25 M, m e r c a p t o e t h a n o l 8 mM, t r i c i n e b u f f e r 0.1 M, M g C l 2 1 mM]. The h o m o g e n a t e was f i l t e r e d t h r o u g h M i r a c l o t h , t h e n c e n t r i f u g e d a t 2 7 0 x £ f o r 5 m i n u t e s . T h e s u p e r n a t a n t was c e n t r i f u g e d a t 6 0 0 0 x £ f o r 5 m i n u t e s . T h i s was t e r m e d t h e 1 c h l o r o p l a s t - r i c h 1 p r e p a r a t i o n . A ' m i t o -c h o n d r i o n - r i c h ' p r e p a r a t i o n was made by c e n t r i f u g i n g t h e s u p e r n a t a n t a t 2 0 , 0 0 0 x £ f o r 20 m i n u t e s . N o n - a q u e o u s c e l l f r a c t i o n a t i o n P l a n t t i s s u e was f r o z e n i n l i q u i d n i t r o g e n , p l a c e d o v e r P 2 0 5 ( a n h y d r o u s ) o r H 2S0i» i n t h e c o l d and e v a c u a t e d 21 w i t h a pump f o r two h o u r s . The m a t e r i a l was s t o r e d i n a d e s i c c a t o r i n a f r e e z e r . T i s s u e c u l t u r e lumps h a v e l i t t l e c u t i c l e and c a n be f r e e z e - d r i e d i n an o r d i n a r y l y o p h i l i z e r . A b o u t 0.2 gm o f f r e e z e - d r i e d t i s s u e was t h o r o u g h l y g r o u n d i n a V i r T i s O m n i m i x e r i n an i c e - c o l d m i x t u r e o f c h l o r o f o r m - h e x a n e w i t h s p e c i f i c g r a v i t y (<°) 1.40. T h i s was s t r a i n e d t h r o u g h c h e e s e c l o t h (8 l a y e r s ) and 5 ml o f t h e m i x t u r e was a d d e d t o a 15 ml g l a s s c e n t r i f u g e t u b e . On t o p w e r e l a y e r e d 2 ml e a c h o f m i x t u r e s o f c h l o r o f o r m and h e x a n e w i t h ^ o f 1.35, 1.30 and 1.20. The d e n s i t y g r a d i e n t s w e r e c e n t i f u g e d a t 2 0 , 0 0 0 x £ f o r 20 m i n u t e s i n a S o r v a l l RC-2B c e n t r i f u g e u s i n g t h e HB s w i n g i n g b u c k e t r o t o r . B ands w e r e c o l l e c t e d f r o m t h e t o p o f t h e g r a d i e n t w i t h a p i p e t t e . P r e p a r a t i o n s o f p o t a t o f r a c t i o n s by d i f f e r e n t i a l  c e n t r i f u g a t i on P l a n t m a t e r i a l was g r o u n d o r c h o p p e d i n an e q u a l v o l u m e o f g r i n d i n g medium l a f t e r R u i s 1 9 7 1 ; s u c r o s e 0.4 M, t r i c i n e b u f f e r 0.165 M pH 7.5, KC1 .01 M, EDTA 1 mM, M g C l 2 - 6 H 2 0 .01 M, d i t h i o t h r e i t o ! .01 MJ, t h e n f i l t e r e d t h r o u g h M i r a c l o t h t o r emove t h e c e l l c l u m p s . T h e f i l t r a t e was c e n t r i f u g e d i n a r e f r i g e r a t e d c e n t r i f u g e 5 m i n u t e s a t 2 7 0 x £ t o remove w h o l e c e l l s . T h e s u p e r n a t a n t was c e n t i f u g e d f o r 10 m i n u t e s a t 10,000xg_. T h i s p e l l e t was t e r m e d t h e 22 p a r t i c u l a t e f r a c t i o n a n d was s u s p e n d e d i n f r e s h g r i n d i n g m e d i u m . T h e s u p e r n a t a n t was c e n t r i f u g e d i n a S p i n c o M o d e l L 2 - 6 5 B p r e p a r a t i v e u l t r a c e n t r i f u g e f r o m 1 t o 4 h o u r s i n a 5 0 T i r o t o r a t 4 0 , 0 0 0 r p m . T h e s u p e r n a t a n t f r o m t h i s t r e a t -m e n t was t e r m e d t h e s o l u b l e f r a c t i o n , o r 1 0 0 , 0 0 0 x g _ s u p e r -n a t a n t a n d t h e p e l l e t , t h e m i c r o s o m a l f r a c t i o n o r 1 0 0 , 0 0 0 x g _ s u p e r n a t a n t . P r e p a r a t i o n o f c e l l f r a c t i o n s by s u c r o s e  d e n s i t y g r a d i e n t s e p a r a t i o n T h e 1 0 , 0 0 0 x £ p e l l e t f r o m t h e a b o v e p r o c e d u r e was r e s u s p e n d e d i n f r e s h m e d i u m a n d was l a y e r e d g e n t l y o n a p r e v i o u s l y p r e p a r e d g r a d i e n t . T h e d i s c o n t i n u o u s g r a d i e n t s w e r e f o r m e d b y l a y e r i n g w i t h a p i p e t t e . A t y p i c a l g r a d i e n t c o n s i s t e d o f 5 ml o f 70% w/v s u c r o s e , 10 ml o f 60% w / v s u c r o s e , 10 ml o f 51% w / v s u c r o s e a n d 8 ml o f 44% w/v s u c r o s e i n a l x 3 £ " c e l l u l o s e n i t r a t e t u b e . A l l s o l u t i o n s u s e d f o r g r a d i e n t s c o n t a i n e d s u c r o s e , t r i c i n e b u f f e r . 0 5 M , pH 7 . 5 , a n d 1 mM E D T A . On t o p o f t h i s was l a y e r e d a b o u t 6 ml o f r e s u s p e n d e d p e l l e t . T h e t u b e s w e r e c e n t r i f u g e d f r o m 4 t o 18 h o u r s i n a SW27 r o t o r i n a S p i n c o M o d e l L 2 - 6 5 B p r e p a r a t i v e u l t r a c e n t r i f u g e . 23 F r a c t i o n s w e r e c o l l e c t e d f r o m t h e g r a d i e n t w i t h a c a p i l l a r y t u b e ( g e n t l y i n s e r t e d t o t h e b o t t o m o f t h e c e n t r i f u g e t u b e ) a t t a c h e d t o a LKB V a r i o p e r p e x p u m p . T h e pump was a d j u s t e d t o a f l o w r a t e o f 1 m l / m i n , a n d 2 m i n f r a c t i o n s w e r e c o l l e c t e d w i t h an ISCO M o d e l 272 f r a c t i o n c o l l e c t o r . R e f r a c t i v e i n d i c e s w e r e m e a s u r e d w i t h a B a u s c h a n d Lomb Abbe" r e f r a c t o m e t e r . E x t r a c t i o n a n d C h r o m a t o g r a p h y P r o c e d u r e s E x t r a c t i o n a n d c h r o m a t o g r a p h y o f p h e n o l i c a c i d s P l a n t m a t e r i a l was e x t r a c t e d w i t h h o t 80% e t h a n o l . T h e e x t r a c t was d r i e d t h e n t a k e n up i n a s m a l l v o l u m e o f h o t w a t e r . T h e h o t w a t e r / e x t r a c t was f i l t e r e d t h r o u g h a p a d o f C e l i t e ( J o h n s - M a n v i 1 1 e C o m p a n y ) t o r e m o v e l i p i d s . T h e l i p i d - f r e e e x t r a c t was a c i d i f i e d t o pH 2 a n d e x t r a c t e d c o n t i n u o u s l y 4 t o 6 h o u r s i n a l i q u i d - l i q u i d e x t r a c t o r . I f t h e l i p i d c o n t e n t o f t h e e t h a n o l i c e x t r a c t w e r e l o w , t h e n t h e e t h a n o l was r e m o v e d i_n v a c u o a n d t h e a q u e o u s e x t r a c t was e x t r a c t e d w i t h e t h e r d i r e c t l y . T h e e t h e r e x t r a c t was d r i e d o v e r a n h y d r o u s Na 2 S0it t a k e n t o d r y n e s s i_n v a c u o a n d made t o a s m a l l v o l u m e f o r c h r o m a t o g r a p h y . 24 S e v e r a l s o l v e n t s y s t e m s were e m p l o y e d f o r chroma-t o g r a p h y o f compounds on MN c e l l u l o s e t h i n l a y e r p l a t e s . System A) ben z e n e r a c e t i c acid:water (10:7:3, upper phase) System B) butano I:pyridine:water (7:1.5:1.5) System C) ani s o I e : a c e t i c a c i d r w a t e r (70:29:1) ( e x c e l l e n t f o r r e s o l u t i o n of HG and t y r o s i ne) System D) 2% formic a c i d ( g e n e r a l l y used f o r the second dimension) System E) butano I : a c e t i c a c i d r w a t e r (40:10:1) System F) to Iuene:ethy I formate:formic a c i d (5:4:1) P h e n o l i c compounds were v i s u a l i z e d u n d e r UV, and by s p r a y i n g w i t h d i a z o t i z e d p_-ni t r o a n i 1 i ne r e a g e n t . ( S p r a y w i t h a f r e s h m i x t u r e o f 5 ml o f 0.3% p _ - n i t r o a n i 1 i n e , 1 ml o f 5% N a N 0 2 and 15 ml o f 20% Na a c e t a t e . O v e r s p r a y w h i l e s t i l l damp w i t h 5% NaOH.) F o r m a t i o n , e x t r a c t i o n and c h r o m a t o g r a p h y o f  2 , 4 - d i n i t r o p h e n y 1 h y d r a z o n e s (DNPH) [ a f t e r  K a t s u k i e t a l _ . 1 9 6 8 ] To 4 o r 5 ml o f m a t e r i a l ( d e p r o t e i n i z e d i f n e c e s -s a r y w i t h 1% HC10*) was a d d e d 1 ml o f DNPH r e a g e n t (1 mg 25 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e i n 1 ml 2 N H C l ) . A f t e r 30 m i n u t e s a t 3 0 ° C , t h e r e a c t i o n m i x t u r e was e x t r a c t e d w i t h e t h y l a c e t a t e , a n d t h e e t h y l a c e t a t e e x t r a c t was e v a p o r a t e d t o d r y n e s s . T h i s was s h a k e n w i t h 10 ml b e n z e n e a n d 20 ml o f 0 . 1 N s o d i u m b i c a r b o n a t e . T h e b i c a r b o n a t e l a y e r c o n -t a i n e d t h e k e t o a c i d D N P H , a n d t h e b e n z e n e l a y e r , t h e o t h e r DNPH a n d e x c e s s r e a g e n t . T h e f o l l o w i n g s o l v e n t s y s t e m s w e r e u s e d : b u t a n o l : 3% a m m o n i a ( 4 : 1 ) , a n d w a t e r - s a t u r a t e d i s o a m y l a l c o h o l : b u t a n o l ( 3 : 1 ) , b o t h on S i l i c a G e l G . T h e u n k n o w n s w e r e c o m p a r e d w i t h s t a n d a r d DNPH p r e p a r e d f r o m a u t h e n t i c c o m -p o u n d s i n t h e a b o v e m a n n e r . E x t r a c t i o n a n d c h r o m a t o g r a p h y o f l i p i d s  ( i n c l u d i n g p 1 a s t o q u i n o n e s ) [ a f t e r  G r i f f i t h s e t aj_. 1 9 6 7 ] I n d i m l i g h t f r e e z e - d r i e d t i s s u e was e x t r a c t e d w i t h s e v e r a l v o l u m e s o f e t h a n o l : e t h y l e t h e r ( 1 : 2 ) a n d t h r i c e w i t h 1 ml o f a c e t o n e u n t i l c o l o u r l e s s . T h e a c e t o n e a n d e t h a n o l w e r e r e m o v e d by w a s h i n g w i t h w a t e r . T h e w a t e r e x t r a c t was b a c k e x t r a c t e d w i t h e t h e r . T h e e t h e r e a l e x -t r a c t s w e r e d r i e d o v e r a n h y d r o u s NaaSO i* a n d t a k e n t o d r y n e s s 26 i n v a c u o . T h e e x t r a c t s w e r e made t o v o l u m e i n p e t r o l e u m e t h e r ( 3 0 ° - 6 0 ° b.p.) and s t o r e d a t - 2 0 ° C . C h r o m a t o g r a p h y was on S i l i c a G e l GHR ( M e r c k ) i n c h l o r o f o r m : h e p t a n e ( 4 : 1 ) . S t a n d a r d P I a s t o q u i n o n e A ( 4 5 ) was o b t a i n e d f r o m R o c h e , B a s e l , S w i t z e r l a n d . The P r e p a r a t i o n o f p - H y d r o x y p h e n y l p y r u v a t e From L - T y r o s i n e - 1 **C  U s i n g L-Amino A c i d O x i d a s e F i v e u C i o f L - t y r o s i n e - 1 "*C ( s p e c i f i c a c t i v i t y 5 u C i / u m o l e ) , 0.1 mmoles o f KC1, 1 mg c a t a l a s e p o w d e r , 0.1 mmoles o f t r i s b u f f e r pH 8.0, and 4 u n i t s o f L - a m i n o a c i d o x i d a s e w ere m i x e d i n a f l a s k . T h e m i x t u r e was i n c u b a t e d a t 37°C. R a d i o t r a c e r M e t h o d s L i q u i d s c i n t i l l a t i o n c o u n t i n g L i q u i d s a m p l e s w ere d i s s o l v e d i n , and s o l i d s a m p l e s s u s p e n d e d i n , t o l u e n e - b a s e d s c i n t i l l a t i o n c o u n t i n g f l u i d s (4 gm PPO, 100 mg P0P0P p e r 1 t o l u e n e ) . To t h e p o w d e r e d s a m p l e s w ere a d d e d 2 c c o f C a b o s i l t o a i d s u s p e n s i o n . S a m p l e s w ere c o u n t e d i n a N u c l e a r C h i c a g o model 720 l i q u i d s c i n t i l l a t i o n c o u n t e r . 27 E x a m i n a t i o n o f c h r o m a t o g r a m s T n i n l a y e r and p a p e r c h r o m a t o g r a m s were p l a c e d w i t h X - r a y f i l m ( K o d a k B l u e B r a n d ) and d e v e l o p e d a f t e r two t o t h r e e weeks w i t h Kodak X - r a y d e v e l o p i n g s o l u t i o n s . O n e - d i m e n s i o n a l c h r o m a t o g r a m s were s c a n n e d on a N u c l e a r C h i c a g o A c t i g r a p h I I . P r o t e i n D e t e r m i n a t i o n T h e m e t h o d o f L o w r y e_t a J L [ 1 9 5 1 ] was u s e d f o r t h e e x p e r i m e n t s w i t h c h l o r o p l a s t s . F o r p r o t e i n m e a s u r e m e n t s o f s u c r o s e d e n s i t y g r a d i e n t f r a c t i o n s , t h e E 2 8 o / E 2 6 o method was u s e d [ C h a y k i n 1 9 6 6 ] . O p t i c a l d e n s i t y a t 540 nm was u s e d as an e s t i m a t e o f t u r b i d i t y . T he method o f B r a m h a l l e t a l _ . [ 1 9 6 9 ] w h i c h was n o t s u i t a b l e f o r u s e w i t h t h e h e a v y s u c r o s e f r o m t h e g r a d i e n t f r a c t i o n s was u s e d a t a l l o t h e r t i m e s . S a m p l e s o f 10 j u l , c o n t a i n i n g l e s s t h a n 25 jugm o f p r o t e i n w ere s p o t t e d on 2x2 cm s q u a r e s m a r k e d on Whatman #50 p a p e r . The s q u a r e s w e r e d r i e d , c u t a p a r t , and t h e p r o t e i n was f i x e d i n c o l d 7.5% t r i c h l o r o a c e t i c a c i d . T h i s was h e a t e d t o 8 0 ° C f o r o n e - h a l f h o u r t o h y d r o l y z e n u c l e i c a c i d s . The p a p e r s w e r e w a s h e d i n e t h a n o l r e t h y l e t h e r ( 1 : 1 ) , d r i e d , and s t a i n e d w i t h a s o l u t i o n o f x y l e n e b r i l l i a n t c y a n i n G ( E d w a r d G u r r L t d . , 28 L o n d o n ) ( 1 0 mg/ml i n 7% a c e t i c a c i d ) f o r 15 m i n u t e s a t 5 0 ° C . I t was i m p o r t a n t t h a t t h e p a p e r s w e r e o c c a s i o n a l l y s w i r l e d , b u t n o t a l l o w e d a b o v e t h e s u r f a c e o f t h e s t a i n s o l u t i o n . T h e s t a i n c o u l d be r e - u s e d f o u r o r f i v e t i m e s . E x c e s s s t a i n was r e m o v e d i n 7% a c e t i c a c i d a t 5 0 ° C - T h e s q u a r e s w e r e b l o t t e d a n d p l a c e d i n s e p a r a t e t e s t t u b e s , a n d d e - s t a i n e d w i t h 5 ml e a c h o f m e t h a n o l : w a t e r : 3 % a m m o n i a ( 6 6 : 3 4 : 1 ) . T h e a b s o r b a n c e o f t h e s o l u t i o n s was r e a d a t 610 n m . P r e p a r a t i o n o f P o t a t o PAL T h r e e h u n d r e d gm o f p o t a t o s l i c e s i n c u b a t e d 20 h o u r s i n t h e n o r m a l m a n n e r w e r e g r o u n d i n a c h i l l e d b l e n d o r w i t h 2 0 0 m l o f 0 . 1 M t r i s b u f f e r pH 8 . 6 . T h e s l u r r y was f i l t e r e d t h r o u g h M i r a c l o t h a n d t h e f i l t r a t e c e n t r i f u g e d 20 m i n u t e s a t 1 0 , 0 0 0 x g _ . The 3 0 - 6 0 % ammonium s u l f a t e f r a c t i o n was p r e p a r e d , a n d r e d i s s o l v e d i n 20 ml o f . 0 5 M t r i s b u f f e r pH 8 . 6 . F i f t e e n ml o f t h i s w e r e p u t on a S e p h a d e x G - 2 0 0 c o l u m n ( 2 7 x 2 . 6 c m ) . PAL a c t i v i t y was m e a s u r e d s p e c t r o -p h o t o m e t r i c a l l y ( s e e A s s a y s ) a n d t h e m o s t a c t i v e f r a c t i o n s p o o l e d . P r o t e i n was p r e c i p i t a t e d i n 70% ammonium s u l f a t e a n d was t a k e n up i n 14 ml o f p o t a t o g r i n d i n g m e d i u m [ R u i s 1 9 7 1 ] a n d s t o r e d a t - 2 0 ° C o v e r n i g h t . 29 Enzyme A s s a y s A l l t h e f o l l o w i n g a s s a y s w ere c a r r i e d o u t a t 3 0 ° C . T h e s p e c t r o p h o t m e t r i c a s s a y s w ere c a r r i e d o u t on a U n i c a m SP500 w i t h SP45 a t t a c h m e n t and c o n s t a n t t e m p e r a t u r e c e l l h o l d e r . C a t a l a s e [ E . C . 1.11.1.6; a f t e r A b e r 1 9 7 0 ] . I n a 1 ml c u v e t t e w e r e m i x e d 0.9 ml 0.05 M p h o s p h a t e b u f f e r pH 7.0 and 0.1 ml enzyme. T h e r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f 0.02 ml o f 1.8% h y d r o g e n p e r o x i d e ( 6 % d i l u t i o n o f 30% r e a g e n t g r a d e ) . The r e a c t i o n was f o l l o w e d a t 240 nm. T h e m o l a r e x t i n c t i o n c o - e f f i c i e n t o f h y d r o g e n p e r o x i d e i s 4 3 , 6 0 0 . F u m a r a s e [ L - m a l a t e h y d r o l y a s e , E.C. 4.2.1.2;  f r o m t h e method o f R a c k e r 1 9 5 0 ] In a 1 ml c u v e t t e w e r e m i x e d 0.9 ml o f .05 M s o d i u m m a l a t e i n 0.1 M t r i c i n e b u f f e r pH 7.5, and 0.1 ml enzyme. The a p p e a r a n c e o f f u m a r i c a c i d was f o l l o w e d a t 240 nm. T h e m o l a r e x t i n c t i o n c o - e f f i c i e n t o f f u m a r i c a c i d was c a l c u l a t e d as 2360 f r o m t h e d a t a o f R a c k e r . 3 0 PAL [ p h e n y l a l a n i n e a m m o n i a - l y a s e , E . C . 4 . 2 . 1 . 5 ;  f r o m t h e m e t h o d o f Z u c k e r 1 9 6 5 ] I n 3 ml c u v e t t e w e r e m i x e d 1 . 5 ml o f 0 . 0 5 M t r i s b u f f e r pH 8 . 6 a t 3 0 ° C , a n d 0 . 5 ml e n z y m e . The r e a c t i o n was s t a r t e d b y t h e a d d i t i o n o f 1 . 0 ml o f 2 . 5 mM ^ - p h e n y l a l a n i n e i n t h e b u f f e r . T h e a p p e a r a n c e o f c i n n a m i c a c i d was f o l l o w e d a t 2 9 0 n m . T h e m o l a r e x t i n c t i o n c o - e f f i c i e n t o f c i n n a m i c a c i d a t 2 9 0 nm i s 1 0 , 6 0 0 . When t h e e n z y m e a c t i v i t y was v e r y l o w , t h e r e a c t i o n t i m e was e x t e n d e d t o o v e r n i g h t . Q - M e t h y l t r a n s f e r a s e a c t i n g on c a f f e i c a c i d  [ E . C . 2 . 1 . 1 . ; f r o m t h e m e t h o d o f B a s m a d j i a n  a n d P a u l 1 9 7 1 ] C a f f e i c a c i d ( 7 2 j u m o l e s / m l i n . 0 5 M t r i c i n e b u f f e r pH 7 . 5 ) , 0 . 5 juCi S - a d e n o s y I m e t h i o n i ne m e t h y l - 1 I f C ( s p e c i f i c a c t i v i t y 5 2 u C i / u m o l e ) i n b u f f e r , a n d 0 . 1 ml e n z y m e w e r e m i x e d i n a t e s t t u b e . A f t e r one h o u r i n c u b a t i o n , t h e r e a c -t i o n was s t o p p e d w i t h 0 . 2 ml o f 0 . 1 1 N_ H C l , a n d 1 ml o f w a t e r was a d d e d . T h e m i x t u r e was e x t r a c t e d t h r e e t i m e s w i t h 2 ml e t h y l a c e t a t e , a n d t h e c o m b i n e d e x t r a c t s w e r e w a s h e d w i t h . 0 5 N_ H C l a n d d r i e d o v e r a n h y d r o u s N a 2 S 0 i * . T h e e x t r a c t was t r a n s f e r r e d t o a s c i n t i l l a t i o n v i a l , d r i e d , t h e n t a k e n 31 up i n 0 . 1 ml o f e t h a n o l . T h e n o r m a l v o l u m e o f t o l u e n e s c i n t i l l a t i o n f l u i d was a d d e d f o r c o u n t i n g . T h e i d e n t i t y o f t h e r e a c t i o n p r o d u c t was e s t a b -l i s h e d b y c o - c h r o m a t o g r a p h y o f t h e e t h y l a c e t a t e e x t r a c t o f t h e r e a c t i o n p r o d u c t w i t h a u t h e n t i c f e r u l i c a c i d . C i n n a m i c a c i d 4 - h y d r o x y l a s e [ E . C . 1 . 9 9 . 1 ;  a f t e r R u s s e l l a n d C o n n 1 9 7 1 ] T h e N A D P H 2 - g e n e r a t i n g s y s t e m c o n s i s t e d o f 5 /umoles g l u c o s e - 6 - p h o s p h a t e , 2 j u m o l e s N A D P , a n d 0 . 4 u n i t s o f g l u c o s e -6 - p h o s p h a t e d e h y d r o g e n a s e i n a t o t a l o f t h r e e ml o f 0 . 1 M p h o s p h a t e b u f f e r pH 7 . 5 . T h i s was i n c u b a t e d a t 30°C f o r f i v e m i n u t e s . T h e h y d r o x y l a t i n g s y s t e m c o n s i s t e d o f 1 ml e n z y m e , 0 . 3 mg c i n n a m i c a c i d , 0 . 1 juCi c i n n a m i c a c i d lhC ( 1 . 4 j u C i / j u m o l e , 1 . 0 ml o f b u f f e r , a n d 0 . 2 4 mg m e r c a p t o e t h a n o l . T h e a d d i t i o n o f t h e N A D P H 2 g e n e r a t i n g s y s t e m s t a r t e d t h e r e a c -t i o n . I n c u b a t i o n t i m e was o n e h o u r . I t was n o t n e c e s s a r y t o s h a k e t h e r e a c t i o n m i x t u r e d u r i n g i n c u b a t i o n i f t h e r e a c t i o n v e s s e l s w e r e u n c o v e r e d . T h i s s e e m e d t o p r o v i d e e n o u g h o x y g e n t o a l l o w t h e r e a c t i o n t o p r o c e e d . T h e r e a c t i o n was s t o p p e d w i t h 0 . 1 ml o f 6 N H C 1 . T h e r e a c t i o n m i x t u r e was e x t r a c t e d t h r e e t i m e s w i t h 4 ml o f e t h y l a c e t a t e , a n d t h e e x t r a c t s p o o l e d . T h e 32 e x t r a c t was t h e n d r i e d and t h e w h o l e e x t r a c t s p o t t e d w i t h a u t h e n t i c p _ - c o u m a r i c a c i d on Whatman #1 p a p e r and c h r o m a t o -g r a p h e d i n one d i m e n s i o n i n s o l v e n t s y s t e m A. The s p o t c o r r e s p o n d i n g t o p _ - c o u m a r i c a c i d was l o c a t e d u n d e r UV l i g h t a f t e r f u m i n g w i t h ammonia v a p o u r , c u t o u t and c o u n t e d d i r e c t l y i n a s c i n t i l l a t i o n v i a l . p_-Coumaric a c i d h y d r o x y l a s e S e v e r a l p r o c e d u r e s w ere i n v e s t i g a t e d . a . The s y s t e m a s d e s c r i b e d f o r c i n n a m i c a c i d 4 - h y d r o -x y l a s e was e m p l o y e d w i t h t h e s u b s t i t u t i o n o f p _ - c o u m a r i c a c i d f o r c i n n a m i c a c i d . b . A m o d i f i c a t i o n o f m e t h o d o f S a t o H\966l was u s e d . The r e a c t i o n m i x t u r e c o n s i s t e d o f I ml o f e n z y m e , 2 ml 0 . 1 M p h o s p h a t e b u f f e r pH 6 . 7 , 0 . 1 ml o f 0 . 0 2 M p _ - c o u m a r i c a c i d and . 0 6 uCi o f p _ - c o u m a r i c a c i d - l l f C ( p r e p a r e d by B . E l l i s o f t h i s l a b o r a t o r y ) . In b o t h c a s e s a. and b . , a f t e r t h e e x t r a c t i o n o f t h e r e a c t i o n m i x t u r e s t h e m a t e r i a l was r e p e a t e d l y c h r o m a t o -g r a p h e d i n 1 d i m e n s i o n i n s o l v e n t A t o s e p a r a t e t h e c a f f e i c a c i d f r o m t h e o r i g i n . 33 c . The m e t h o d o f N a m b u d i r i [ 1 9 7 1 ] was f o l l o w e d . The t i s s u e was g r o u n d i n t h e b u f f e r e d s u c r o s e medium p r e p a r e d w i t h o u t m e r c a p t o e t h a n o I . The e n z y m e was i n c u b a t e d w i t h 2 u m o l e s o f £ _ - c o u m a r i c a c i d and 2 u m o l e s o f p h o s p h a t e b u f f e r pH 7 . 5 . The £ _ - c o u m a r i c a c i d f o r m e d was m e a s u r e d by t h e m e t h o d o f A rnow [ 1 9 3 7 ] . d . A m o d i f i c a t i o n o f t h e m e t h o d o f S t a f f o r d and D r e s l e r [ 1 9 7 2 ] was u s e d . The t i s s u e was g r o u n d i n b u f f e r e d s u c r o s e w i t h m e r c a p t o e t h a n o I , b u t was p a s s e d t h r o u g h a S e p h a d e x G - 2 5 c o l u m n i n an a t t e m p t t o remove t h e m e r c a p t o e t h a n o I . The i n c u b a t i o n m i x t u r e c o n s i s t e d o f 2 u m o l e s o f p _ - c o u m a r i c a c i d - 1 I f C , 0 . 2 ml e n z y m e , . 4 u m o l e s o f a s c o r b i c a c i d , and 150 u m o l e s o f p h o s p h a t e b u f f e r pH 6 . 0 . A f t e r s h o u r a t 30°C t h e r e a c t i o n was s t o p p e d w i t h 0 . 1 ml 2N_ H C l , and t h e m i x t u r e was e x t r a c t e d w i t h e t h y l e t h e r . The e x t r a c t was c h r o m a t o g r a p h e d w i t h a u t h e n t i c c a f f e i c a c i d i n s o l v e n t s A and E on c e l l u l o s e TLC p l a t e s . C a f f e i c a c i d s p o t s w e r e s c r a p e d and c o u n t e d i n s c i n t i l l a t i o n v i a l s . S h i k i m i c a c i d : N A D P o x i d o r e d u c t a s e [ E . C . 1 . 1 . 1 . 2 5 ;  a f t e r B a l i n s k y a n d D a v i e s 1 9 6 1 ] N i n e y m o l e s o f s h i k i m i c a c i d , 1 u m o l e o f N A D P , a n d 160 u m o l e s o f g l y c i n e :NaOH b u f f e r , pH 9 . 0 w e r e m i x e d i n a 1 ml c u v e t t e . Up t o 0 . 4 ml o f e n z y m e was a d d e d . T h e r e a c t i o n was f o l l o w e d a t 340 nm. 34 P r e p h e m ' c a c i d : N A D P o x i d o r e d u c t a s e [ E . C . 1.1.1; a f t e r t h e method o f Gamborg 1 9 6 6 ] P r e p a r a t i o n o f p r e p h e n i c a c i d f r o m t h e b a r i u m  s a 1 t — [ A f t e r t h e met h o d o f Gamborg and S i m p s o n 1 9 6 4 ] . F i f t y mg o f p r e p h e n i c a c i d b a r i u m s a l t ( o b t a i n e d f r o m D r . 0. Gamborg) were d i s s o l v e d i n 10 ml o f .05 M t r i s . A s l i g h t e x c e s s ( 1 . 4 8 mg) o f p o t a s s i u m s u l f a t e was a d d e d . T he m i x -t u r e was s t i r r e d f o r 15 m i n u t e s , t h e n f i l t e r e d t h r o u g h g l a s s f i b e r p a p e r . E s t i m a t i o n o f p r e p h e n i c a c i d c o n c e n t r a t i o n — To 50 jul o f t h e a b o v e s o l u t i o n w e r e a d d e d 50 ju 1 o f w a t e r a n d 0.1 ml IH HC1 t o c o n v e r t t h e s o l u t i o n t o PHPP. A f t e r 15 m i n u t e s i n c u b a t i o n a t 3 0 ° C , 2.3 ml o f 0.5 N_ NaOH w e r e a d d e d . The m o l a r e x t i n c t i o n c o e f f i c i e n t a t 320 nm o f t h e r e s u l t i n g p _ - h y d r o x y b e n z o i c a c i d i s 16 ,000, and t h e o p t i c a l d e n s i t y o f t h e s o l u t i o n a t hand was 0.8. T h i s i n d i c a t e s a p r e p h e n i c a c i d c o n c e n t r a t i o n i n t h e o r i g i n a l s o l u t i o n o f 2.5 u m o l e s / m l . T h e pH o f t h e s o l u t i o n o f p r e p h e n i c a c i d was l o w e r e d t o 8.0 f o r u s e i n t h e enzyme a s s a y . In a 1 ml c u v e t t e w e r e p l a c e d 50 jumoles o f p y r o p h o s p h a t e b u f f e r pH 7.5, 0.5 jumoles o f NADP, and 1 jumole o f p r e p h e n i c a c i d . Up t o 0.4 ml o f enzyme were u s e d . T he r e a c t i o n was f o l l o w e d a t 340 nm. 35 A l c o h o l d e h y d r o g e n a s e s [ a f t e r D a v i e s e t a l . 1 9 7 2 ] A l k y l a l c o h o l d e h y d r o g e n a s e — F i v e j j m o l e s o f a c e t a l d e h y d e , . 2 5 y m o l e s o f N A D H 2 , 50 y m o l e s o f p h o s p h a t e b u f f e r pH 7 . 5 , a n d p o t a t o e n z y m e t o a t o t a l o f 1 ml w e r e m i x e d i n a 1 ml c u v e t t e . T h e r e a c t i o n was f o l l o w e d a t 3 4 0 n m . N A D P H 2 was a l s o t r i e d i n p l a c e o f N A D H 2 . A r o m a t i c a l c o h o l d e h y d r o g e n a s e — T h e a b o v e p r o -p o r t i o n s w e r e u s e d , w i t h v a n i l l i n , b e n z a l d e h y d e , m - h y d r o x y -b e n z a l d e h y d e , a n d £ - h y d r o x y b e n z a l d e h y d e as t h e s u b s t r a t e s . B o t h N A D P H 2 a n d N A D H 2 w e r e u s e d . D - G l y c e r a l d e h y d e - 3 - p h o s p h a t e : N A D P o x i d o r e d u c t a s e  ( p h o s p h o r y l a t i n g ) ( E . C . 1 . 2 . 1 . 9 ) T h e r e a c t i o n m i x t u r e c o n s i s t e d o f 3 - p h o s p h o g l y -c e r a l d e h y d e , 2 j u m o l e s , 100 jumoles o f p h o s p h a t e b u f f e r , pH 7 . 0 , 10 j j m o l e s o f M g C 1 2 , 5 j u m o l e s o f r e d u c e d g l u t a t h i o n e , 5 j u m o l e s o f A T P , 0 . 2 u n i t s o f 3 - p h o s p h o g l y c e r i c a c i d k i n a s e . T h i s m i x t u r e was i n c u b a t e d o n e m i n u t e a t 3 0 ° C . E n z y m e a n d 0 . 2 y m o l e s N A D P H 2 w e r e a d d e d t o a t o t a l v o l u m e o f 1 m l . T h e r e a c t i o n was f o l l o w e d a t 3 4 0 n m . PART I METABOLISM OF TYROSINE BY ISOLATED CHLOROPLASTS RESULTS I n m o s t o f t h e f o l l o w i n g e x p e r i m e n t s , l a b e l l e d s u b s t r a t e o f l o w s p e c i f i c a c i t i v i t y was u s e d . W i t h h i g h s p e c i f i c a c t i v i t y s u b s t r a t e , e v e n a s m a l l a m o u n t o f b a c -t e r i a l c o n t a m i n a t i o n c a n r e s u l t i n c o n v e r s i o n s w h i c h a r e m i s l e a d i n g [Gnanam e_t aj_. 1 9 6 9 ] . The p r e s e n t s t u d y was an i n v e s t i g a t i o n o f t h e p o s s i b l e m e t a b o l i s m o f t y r o s i n e t o p _ - h y d r o x y p h e n y 1 p y r u v i c a c i d ( P H P P ) a n d h o m o g e n t i s i c a c i d ( H G ) . S i n c e b o t h t h e s e c o m p o u n d s a r e p r o d u c e d b y t h e b a c -t e r i a l b r e a k d o w n o f t y r o s i n e , i t i s e s p e c i a l l y i m p o r t a n t t o u s e l o w s p e c i f i c a c t i v i t y s u b s t r a t e s . E x p e r i m e n t s w i t h S p i n a c h C h l o r o p l a s t s ( i ) I n o r d e r t o f o r m HG f r o m s u p p l i e d t y r o s i n e v i a t h e e x p e c t e d p a t h w a y t o p i a s t o q u i n o n e [ W h i s t a n c e a n d T h r e l f a l l , 1 9 6 7 , 1 9 6 8 , 1 9 7 0 ] , t h e c h l o r o p l a s t s w o u l d f i r s t h a v e t o c o n v e r t t h e t y r o s i n e t o PHPP a n d t h e n t o H G . T h e 36 37 f o l l o w i n g e x p e r i m e n t s w e r e an a t t e m p t t o f i n d t h e s e c o n d s t e p o f t h i s p a t h w a y ; t h e c o n v e r s i o n o f PHPP t o H G . T h e a m i n o a c i d o x i d a s e s y s t e m ( s e e M e t h o d s ) was u s e d t o g e n e r a t e PHPP f r o m t y r o s i n e - U L - 1 ( s p e c i f i c a c t i v i t y 10 u C i / u m o l e ) . A f t e r f i v e m i n u t e s p r e - i n c u b a t i o n o f t h e o x i d a s e s y s t e m , s p i n a c h c h l o r o p l a s t s f r o m 20 gm o f s p i n a c h ( i s o l a t e d i n H o n d a m e d i u m ) w e r e a d d e d . A f t e r 30 m i n u t e s i n c u b a t i o n , t h e m a t e r i a l was e x t r a c t e d a n d c h r o m a t o g r a p h e d as f o r p i a s t o q u i n o n e d e t e r m i n a t i o n . C h r o m a t o g r a p h y i n h e p t a n e : c h l o r o f o r m a n d i n s y s t e m s A a n d D ( 2 d i m e n s i o n s ) s h o w e d no r a d i o a c t i v e c o m p o u n d s o t h e r t h a n t h e PHPP g e n e r a t e d b y t h e o x i d a s e . E i t h e r t h e r e h a d b e e n no f u r t h e r m e t a b o l i s m , t h e t u r n o v e r o f t h e HG was v e r y f a s t , o r t h e HG h a d b e e n d e g r a d e d i n t h e e x t r a c t i o n p r o c e d u r e . ( i i ) T o p r e v e n t c h e m i c a l b r e a k d o w n d u r i n g i s o l a t i o n a n d c h r o m a t o g r a p h y , t h e f o l l o w i n g e x p e r i m e n t was d e v i s e d . I n c u -b a t i o n m i x t u r e s w e r e s e t up as b e f o r e , w i t h t y r o s i n e - U L - 1 ^ C . A f t e r f i v e m i n u t e s p r e - i n c u b a t i o n , f r a c t i o n s f r o m a n o n -a q u e o u s p r e p a r a t i o n o f s p i n a c h w e r e a d d e d . A t t h e e n d o f 30 m i n u t e s i n c u b a t i o n , 9 . 5 mg o f HG w e r e a d d e d t o e a c h f r a c t i o n . A f t e r e x t r a c t i o n a n d c h r o m a t o g r a p h y , i t a p p e a r e d t h a t f r o m t h e e n t i r e e x t r a c t , e q u i v a l e n t t o 3 0 0 mg o f f r e e z e -d r i e d s p i n a c h , o n l y 9 2 0 cpm w e r e r e c o v e r e d i n H G . 38 T h u s , t h e r e a p p e a r s t o be o n l y a low l e v e l o f m e t a b o l i s m o f t y r o s i n e i n t o p h e n o l i c compounds. T he s p i n a c h p l a n t s u s e d a b o v e were y o u n g ( n o t o v e r one month o l d ) , and wer e q u i t e g r e e n . L e a f l i p i d s a r e f o r m e d r a p i d l y on e x p o s u r e o f t h e l e a f t o l i g h t [ L i c h t e n t h a l e r 1 9 6 9 ] , and g r e e n i n g t i s s u e m i g h t be t h e b e s t s o u r c e o f enzymes m e t a b o l i z i n g t y r o s i n e and PHPP. E x p e r i m e n t s w i t h G r e e n i n g B a r l e y ( i ) T h e f o l l o w i n g e x p e r i m e n t was d e s i g n e d t o v e r i f y t h a t p i a s t o q u i n o n e s w ere b e i n g f o r m e d f r o m t y r o s i n e . S h o o t s ( 1 0 0 ) o f e t i o l a t e d 9 d a y o l d b a r l e y grown u n d e r s t e r i l e c o n d i t i o n s w ere p l a c e d i n 10 ml o f w a t e r w i t h 7.5 j j C i D L - t y r o s i n e - U L - 1 "*C ( s p e c i f i c a c t i v i t y 50 j u C i / u m o l e ) . A f t e r t h e p l a n t s had b e e n a l l o w e d t o g r e e n f o r 29 h o u r s , t h e y w ere e x t r a c t e d i n dim l i g h t w i t h e t h e r and a c e t o n e . W h i l e c h r o m a t o g r a p h y o f t h e p h e n o l i c a c i d f r a c t i o n showed no l a b e l i n HG, c h r o m a t o g r a p h y o f t h e l i p i d s on S i l i c a G e l GHR showed many r a d i o a c t i v e f l u o r e s c e n t b a n d s i n c l u d i n g one c o r r e s p o n d i n g t o a u t h e n t i c p i a s t o q u i n o n e A 45 ( F i g u r e 3 ) . Th e UV s p e c t r a i n m e t h a n o l o f compounds 2, 3 and 4, b u t n o t 5, showed s h i f t s w i t h NaBHt, c h a r a c t e r i s t i c o f q u i n o n e s . FRONT p carotene d p m / 1 0 0 s h o o t s 39 Q D O P O A 34% 404B 1720 4 3 2 C 2 3 3556 C D 3882 ORIGIN ^/v/V/* chlorophylls F i g u r e 3. C h r o m a t o g r a m o f l i p i d e x t r a c t f r o m b a r l e y s h o o t s a d m i n i s t e r e d t y r o s i n e - U L - 1 ^ C . coat 1ng s o l v e n t PQ A s i I i ca gel GHR ch I oroform:heptane 4:1 a u t h e n t i c p I astoquinone A 45 40 T h i s e x p e r i m e n t s u g g e s t s t h a t t u r n o v e r o f HG i n t o p i a s t o q u i n o n e s i s v e r y r a p i d , s i n c e t h e r e i s no a c c u m u l a -t i o n o f l a b e l l e d m a t e r i a l , a l t h o u g h t h e e n d p r o d u c t i s l a b e l l e d . ( i i ) S i n c e i t i s known t h a t PHPP i s a n u n s t a b l e c o m -p o u n d , an a t t e m p t was made t o p r e v e n t b r e a k d o w n b y t h e f o r m a -t i o n o f t h e 2 , 4 - d i n i t r o p h e n y 1 h y d r a z i n e d e r i v a t i v e ( D N P H ) . C h l o r o p l a s t s w e r e p r e p a r e d i n H o n d a m e d i u m f r o m 9 . 5 g-iih o f y o u n g b a r l e y . T h e c h l o r o p l a s t s w e r e r e s u s p e n d e d i n 4 ml o f b u f f e r a n d t h e s u p e r n a t a n t was s a v e d . A b r e i was p r e p a r e d f r o m a s e c o n d b a t c h o f b a r l e y ( 1 0 . 5 g m ) . T y r o s i n e - U L - 1 ^ C , 1 u C i / u m o l e , 1 u C i / r e a c t i o n ) was a d d e d . T h e r e a c t i o n was s t o p p e d a f t e r o n e h o u r b y t h e a d d i t i o n o f 2 , 4 - D N P r e a g e n t . E x t r a c t i o n , s e p a r a t i o n a n d c h r o m a t o g r a p h y w e r e c a r r i e d o u t as i n M e t h o d s . T h e l o w c o u n t s o f PHPP ( T a b l e 2) i n d i c a t e t h a t t h i s c o m p o u n d d o e s n o t a c c u m u l a t e i n t h e p l a n t , i f i n d e e d i t i s f o r m e d a t a l l . ( i i i ) I n an a t t e m p t t o s e p a r a t e c h l o r o p l a s t s w i t h a c t i v e e n z y m e s f r o m t h e r e s t o f t h e c e l l , a n o n - a q u e o u s f r a c t i o n a -t i o n was c a r r i e d o u t w i t h 7 - d a y o l d b a r l e y . T h e 41 T a b I e 2 R a d i o a c t i v i t y i n 2 , 4 - d i n i t r o p h e n y h y d r a z i n e d e r i v a t i v e s o f c o m p o u n d s f r o m t y r o s i n e - 1 "*C f e d t o a l l f r a c t i o n s o f y o u n g b a r l e y Ba r 1 e y F r a c t i o n V o l u m e R e a c t i on Mi x t u r e ( m l ) CPM i n K e t o A c i d DNPH CPM i n PHPP DNPH CPM i n O t h e r DNPH c h 1 o r o p l a s t ! s u p e r n a t a n t j 4 1 6 2 2 4 8 181 22 1 4 0 7 1410 b r e i 20 1 7 0 0 66 4 8 6 c h l o r o p l a s t - r i c h f r a c t i o n was o f s p e c i f i c g r a v i t y 1 . 3 0 - 1 . 3 5 as j u d g e d b y t h e c h l o r o p h y l 1 - p r o t e i n r a t i o a n d t h e p r e s e n c e o f 3 - p h o s p h o g l y c e r a l d e h y d e d e h y d r o g e n a s e ( N A D P ) . To t h e f r a c t i o n s w e r e a d d e d 1 . 0 ml 0 . 1 M t r i s b u f f e r pH 7 . 4 , a n d 1 u C i t y r o s i n e - 3 - 1 " C (8.8 u C i / u m o l e ) . A f t e r 30 m i n u t e s a t 3 7 ° , t h e r e a c t i o n was s t o p p e d b y f r e e z i n g . C h r o m a t o g r a p h y o f t h e e t h e r e x t r a c t s h o w e d o n e r a d i o a c t i v e c o m p o u n d w h i c h a g r e e d i n v a l u e i n two s o l v e n t s y s t e m a n d i n c o l o u r r e a c t i o n ( d i a z o t i z e d £_-ni t r o a n i 1 i n e ) w i t h a u t h e n t i c p _ - h y d r o x y -b e n z a l d e h y d e . 42 Up t o t h i s p o i n t , t y r o s i n e o f low s p e c i f i c a c t i v i t y had b e e n u s e d i n an e f f o r t t o m i n i m i z e t h e e f f e c t o f b a c t e r i a l c o n t a m i n a t i o n . H o w e v e r , t h i s meant t h a t t h e s p e c i f i c a c t i v i t y o f t h e p r o d u c t was a l s o l o w , and i f t h e r e were o n l y a few m o l e c u l e s o f t h e p r o d u c t p r e s e n t , d e t e c t i o n and a n a l y s i s became d i f f i c u l t . T h e u s e o f s t e r i l e m a t e r i a l and s u b s t r a t e was t h o u g h t t o o v e r c o m e t h i s p r o b l e m and p e r m i t t h e u s e o f t y r o s i n e o f h i g h e r s p e c i f i c a c t i v i t y . E x p e r i m e n t s w i t h T o m a t o S e e d l i n g s ( i ) T h e i n h i b i t o r . <*, <*. 1 d i p y r i d y l p r e v e n t s t h e e n z y m i c o x i d a t i o n o f HG i n mammalian s y s t e m s [ L a Du and Z a n n o n i 1 9 5 5 ] , and was u s e d i n an a t t e m p t t o p r e v e n t f u r t h e r m e t a b o l i s m o f any HG t h a t m i g h t be f o r m e d . T e n ml o f a s t e r i l e s o l u t i o n o f .02 M o c " , <*'di-p y r i d y l i n .06 M p h o s p h a t e b u f f e r pH 6 were p o u r e d i n t o two f l a s k s o f s i x 4-week o l d p l a n t s . C o n t r o l p l a n t s r e c e i v e d b u f f e r a l o n e . A f t e r 12 h o u r s ( b y w h i c h t i m e t h e i n h i b i t o r had t u r n e d t h e a g a r i n t h o s e f l a s k s p i n k ) 1.25 juCi o f s t e r i l e t y r o s i n e - U L - 1 ^ C (50 u C i / n m o l e ) were a d d e d . A f t e r an a d d i -t i o n a l 60 h o u r s , when t h e e x p e r i m e n t a l p l a n t s were b e c o m i n g n e c r o t i c , t h e p l a n t m a t e r i a l was r e m o v e d , r i n s e d w e l l and e x t r a c t e d w i t h b o i l i n g 8 0% e t h a n o l . 43 F i v e m a i n c o m p o u n d s w e r e l a b e l l e d , a n d t h e i r c h a r -a c t e r i s t i c s a r e s h o w n i n T a b l e 3. T a b l e 3 R a d i o a c t i v e c o m p o u n d s a f t e r a d m i n i s t e r i n g t y r o s i n e - 1 t o t o m a t o s e e d l i n g s S p o t R f S y s t e m Co 1 o u r , pNA S p e c t r u m Re 1 a t i ve L a b e 1 B D i nh i b i t o r c o n t r o 1 1 . 7 8 - . 8 4 . 5 7 - . 6 1 ( p - h y d r o x y -b e n z o a t e ) ++++ +++ 2 . 7 8 - . 8 4 . 7 7 - . 8 0 p e a k 3 8 2 ++++ + 3 . 6 7 - . 7 3 . 8 7 (HG) ++++ + 4 . 3 9 - . 4 0 . 8 3 g r e e n 3 3 2 , s h 3 0 0 ( c a f f e a t e ) + +++ 5 . 2 2 - . 3 0 . 9 0 - . 9 3 r e d d i s h 3 7 8 , s h 3 8 6 ++++ ++++ pNA - d i a z o t i z e d p_-n i t r o a n i I i ne r e a g e n t . Compound 3 c o - c h r o m a t o g r a p h s w i t h H G . T h i s e x p e r i -m e n t i n d i c a t e s t h a t t h e m e t a b o l i s m o f t o m a t o i s a f f e c t e d b y t h e i n h i b i t o r , a n d t h a t a b u i l d u p o f HG s e e m s t o o c c u r . 44 B e c a u s e t h e p r e s e n c e o f t h e i n h i b i t o r c a u s e d t h e p l a n t s t o b e c o m e n e c r o t i c , i t was i m p o s s i b l e t o p r e p a r e c h l o r o p l a s t s f r o m t h e m , t o f i n d w h e r e t h e r a d i o a c t i v e c o m -p o u n d s w e r e l o c a l i z e d . T h e r e f o r e , t h e u s e o f t h e i n h i b i t o r was d i s c o n t i n u e d . ( i i i ) I n t h e a b o v e e x p e r i m e n t , t h e e n t r a n c e o f s u b s t r a t e d e p e n d e d u p o n t r a n s p o r t a c r o s s t h e m e m b r a n e s o f t h e r o o t h a i r s a n d s t e m . I n an a t t e m p t t o g e t t h e r a d i o a c t i v e m a t e r i a l t o e n t e r t h e p l a n t s as q u i c k l y as p o s s i b l e , s t e r i l e t y r o s i n e was f e d t h r o u g h c u t s t e m s . S t e r i l e t e c h n i q u e s w e r e u s e d t h r o u g h o u t . T e n s t e m s o f t o m a t o s e e d l i n g s w e r e p l a c e d i n each, o f t h r e e v i a l s i n a l a r g e s e a l e d c h a m b e r . T h e s t e r i l e f e e d -i n g s o l u t i o n ( 3 m l ) was 5 , u C i e a c h o f L - t y r o s i n e - U L - 1 ( 5 0 j u C i / u m o l e ) i n 0 . 1 M p h o s p h a t e b u f f e r pH 6 . 3 . A t 1 , 2 , a n d 4 h o u r s , o n e v i a l was r e m o v e d a n d t h e p l a n t s u s e d f o r c h l o r o -p l a s t a n d m i t o c h o n d r i o n p r e p a r a t i o n s . C h i o r o p l as t - r i c h , m i t o c h o n d r i o n - r i c h a n d s u p e r n a t a n t f r a c t i o n s w e r e e x t r a c t e d a n d c h r o m a t o g r a p h e d . T y r o s i n e was f o u n d t o be t a k e n up i n b o t h c h l o r o -p l a s t i d i c a n d n o n - c h l o r o p l a s t i d i c p r o t e i n ( F i g u r e 4 ) , a n d i n t o t h e e t h e r - e x t r a c t a b l e m a t e r i a l ( F i g u r e 5 ) . 45 4000.. F i g u r e 4. I n c r e a s e w i t h t i m e o f r a d i o a c t i v i t y i n t h e p r o t e i n o f s u b c e l l u l a r f r a c t i o n s o f t o m a t o p l a n t s a f t e r t h e a d m i n i s t r a t i o n o f t y r o s i n e - U L - 1 . c h l o r o : c h I o r o p I a s t - r i c h f r a c t i o n A * m i t o : m i t o c h o n d r i a - r i c h f r a c t i o n & a s u p e r : s u p e r n a t a n t f r a c t i o n o o 2000.. c p m 1000 hr F i g u r e 5. I n c r e a s e w i t h t i m e o f r a d i o a c t i v i t y i n t h e e t h e r e x t r a c t o f s u b c e l l u l a r f r a c t i o n o f t o m a t o p l a n t s a f t e r t h e a d m i n i s t r a t i o n o f t y r o s i n e - U L - 1 . c h I o r o c h I o r o p I a s t - r i c h f r a c t i o n s u p e r o o : c e l l s u p e r n a t a n t 47 H o w e v e r , c h r o m a t o g r a p h y , f o l l o w e d by l i q u i d s c i n -t i l l a t i o n c o u n t i n g o f t h e a p p r o p r i a t e s p o t s on t h e c h r o m a t o -g r a m s o f t h e e t h e r e x t r a c t s s h o w e d no r a d i o a c t i v i t y i n HG o r a n y o f t h e o t h e r e x p e c t e d p h e n o l i c a c i d s i n e i t h e r s u p e r -n a t a n t o f c h l o r o p l a s t f r a c t i o n s . E x p e r i m e n t s w i t h Ruta T i s s u e C u l t u r e -The d u a l r e q u i r e m e n t s o f l a r g e a m o u n t s o f m a t e r i a l f o r c h l o r o p l a s t s e x t r a c t i o n , a n d o f s t e r i l i t y f o r l o n g t e r m f e e d i n g s u g g e s t e d t h e u s e o f t i s s u e c u l t u r e . T h e s t r a i n o f R u t a g r a v e o l e n s u s e d d o e s n o t f o r m e t i o l a t e d t i s s u e t h a t g r e e n s r e a d i l y i n t h e l i g h t . ( i ) U s i n g s t e r i l e t e c h n i q u e s t h r o u g h o u t , t i s s u e a b o u t o n e m o n t h o l d was c h o p p e d a n d r i n s e d i n 1% s u c r o s e . A l i q u o t s o f a b o u t 0 . 5 gm w e r e r e s u s p e n d e d i n 8 f l a s k s ; 4 i n n o r m a l B 5 C 2 m e d i u m a n d 4 i n B5 m e d i u m w i t h o u t a d d e d a m i n o a c i d s . I n t o e a c h f l a s k w e r e i n t r o d u c e d 2 juCi t y r o s i n e - U L - 1 ''C o f s p e c i f i c a c t i v i t y 3 5 0 u C i / u m o l e . T h e f l a s k s w e r e i n c u b a t e d a t 20°C w i t h s h a k i n g , i n r o o m l i g h t up t o s e v e n d a y s . P a i r s o f f l a s k s w e r e r e m o v e d a t v a r i o u s t i m e s a n d c h l o r o p l a s t s w e r e p r e p a r e d f r o m t h e c e l l s . F i g u r e 6 s h o w s t h e u p t a k e w i t h t i m e o f t y r o s i n e i n t o p r o t e i n . T h e t i s s u e s a c t i v e l y 48 days F i g u r e 6 I n c r e a s e U L - ^ C w i t h t i m e o f r a d i o a c t i v i t y f r o m t y r o s i n e i n t h e p r o t e i n o f s u b c e l l u l a r f r a c t i o n s o f Ru t a c u l t u r e s , g r o w i n g on B 5 o r B 5 C 2 medium. R u t a g r o w n on B5C2 g r o w n on B5 c h l o r o p l a s t p r o t e i n • - -c e l l s u p e r n a t a n t p r o t e i n x - -c h l o r o p l a s t p r o t e i n o o c e l l s u p e r n a t a n t p r o t e i n * + + v a l u e s shown a r e 1/10 a c t u a l v a l u e s 49 m e t a b o l i z e d t h e s u p p l i e d m a t e r i a l . F i g u r e 7 s h o w s t h a t r a d i o a c t i v i t y r e c o v e r e d i n t h e s p o t s c o r r e s p o n d i n g t o HG i n c r e a s e d w i t h t i m e , i n t h e e t h e r e x t r a c t o f t h e c h l o r o p l a s t -p o o r s u p e r n a t a n t . T h e R u t a t i s s u e g r o w n on t h e m e d i u m w i t h o u t a d d e d a m i n o a c i d s p r o d u c e d m o r e HG t h a n d i d t h e R u t a g r o w n on B 5 C 2 m e d i u m . C h r o m a t o g r a p h y o f t h e c h l o r o p l a s t f r a c t i o n s f o l l o w e d b y l i q u i d s c i n t i l l a t i o n c o u n t i n g s h o w e d no r a d i o a c t i v i t y a t a l l i n t h e s p o t s c o r r e s p o n d i n g t o H G . ( i i ) C o m p o u n d s o t h e r t h a n HG a r e f o r m e d b y t h e R u t a c u l t u r e s , h o w e v e r . A f e e d i n g s i m i l a r t o t h e l a s t was s e t u p , b u t i n B 5 C 2 m e d i u m f o r b e t t e r g r o w t h . Up t o t w o d a y s , HG p r o d u c t i o n a p p e a r s t h e same i n c e l l s g r o w n i n b o t h m e d i a ( F i g u r e 7) . C e l l s w e r e r e m o v e d a t t i m e s t o two d a y s a n d e x -t r a c t e d w i t h b o i l i n g 80% e t h a n o l . R a d i o a c t i v i t y i n a t l e a s t two c o m p o u n d s i n c r e a s e d w i t h t i m e . I n s o l v e n t C t h e s e c o m -p o u n d s h a v e R.p v a l u e s o f 0 . 2 4 ( y e l l o w f l u o r e s c e n t u n d e r UV) a n d 0 . 5 2 ( b l u e f l u o r e s c e n t u n d e r U V ) . T h e y e l l o w s p o t c o r -r e s p o n d s t o HG i n R f a n d f l u o r e s c e n c e . 50 8 0 0 -600 400 £ Q. u B5 200-B5C2 •5 day 4 s 6 F i g u r e 7. C h a n g e w i t h t i m e o f r a d i o a c t i v i t y i n h o m o g e n t i s i c a c i d f r o m R u t a c u l t u r e s a d m i n i s t e r e d t y r o s i n e ~ U L 1 ' 'C ( s e e t e x t f o r d e t a i l s ) . g r o w n on B 5 C 2 medium o o g r o w n on B5 med ium © e> 51 ( i i i ) The two e x p e r i m e n t s a b o v e c o n s i s t e d o f a r a d i o -a c t i v e f e e d i n g s , f o l l o w e d by e x t r a c t i o n o f c h l o r o p l a s t s , i n an a t t e m p t s t o l o c a l i z e t h e p r o d u c t o f a r e a c t i o n . T h e f o l l o w i n g i s an a t t e m p t t o f i n d t h e s i t e o f t h e r e a c t i o n i t s e l f . A n o n - a q u e o u s c e l l f r a c t i o n a t i o n was made o f 4-week o l d R u t a t i s s u e . T he f r a c t i o n s w e r e s u p p l i e d w i t h 0.5 u C i t y r o s i n - U L - 1 o f 1 u C i / u m o l e i n 0.5 ml o f 0.1 M t r i s b u f f e r , pH 7.5. A f t e r one h o u r a t 30° t h e r e a c t i o n was s t o p p e d w i t h o . l ml o f 1 M H C l , and t h e r e a c t i o n m i x t u r e was e x t r a c t e d w i t h e t h e r . C h r o m a t o g r a p h y f o l l o w e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g showed t h a t HG was s c a r c e l y r a d i o a c t i v e b u t a b l u e f l u o r e s c e n t s p o t o f R f 0.15 i n s y s t e m C showed some r a d i o a c t i v i t y . M o s t c o n v e r s i o n had t a k e n p l a c e i n t h e f r a c t i o n o f p i . 2 0 , a l t h o u g h t h e c h l o r o p l a s t f r a c t i o n i s p 1 . 3 0 - 1 . 3 5 . DISCUSSION T h e r e a r e s e v e r a l g e n e r a l c o n c l u s i o n s t o be drawn f r o m t h i s s e r i e s o f e x p e r i m e n t s . F i r s t l y i t a p p e a r s t h a t HG i s f o r m e d f r o m t y r o s i n e by s t e r i l e p l a n t m a t e r i a l . T h i s c o n f i r m s t h e work o f E l l i s [ 1 9 7 0 ] . T h e f o r m a t i o n o f PHPP i n t h i s m a t e r i a l i s n o t d e f i n i t e l y known, a l t h o u g h i t i s a l m o s t c e r t a i n l y t h e i n t e r m e d i a t e i n t h e f o r m a t i o n o f HG f r o m t y r o s i n e . S e c o n d l y , HG i s n o t s t o r e d i n t h e c h l o r o p l a s t , as shown by t h e e x p e r i m e n t s i n w h i c h c h l o r o p l a s t s w e re p r e -p a r e d f r o m p l a n t s f e d r a d i o a c t i v e s u b s t r a t e . T h i r d l y , e i t h e r t h e s e c o n v e r s i o n s do n o t t a k e p l a c e i n t h e c h l o r o p l a s t s , o r t h e c h l o r o p l a s t i d i c enzymes h a v e s u c h a h i g h t u r n o v e r number t h a t t h e i n t e r m e d i a t e s a r e n e v e r d e t e c t e d . T h e s i m p l e s t e x p l a n a t i o n w o u l d be t h a t t h e r i n g o f t h e p i a s t o q u i n o n e n u c l e u s i s f o r m e d o u t s i d e t h e c h l o r o p l a s t and t h e n t r a n s p o r t e d i n t o i t . Some s u p p o r t f o r t h i s i d e a comes f r o m t h e d i s c o v e r y o f L - a m i n o a c i d o x i d a s e i n g l y o x y -somes [ B e e v e r s 1 9 7 0 ] . T h i s enzyme w o u l d f o r m PHPP f r o m 52 53 t y r o s i n e . F u r t h e r , i n R i c i n u s g l y o x y s o m e s , t y r o s i n e i s c o n v e r t e d t o H G , a l t h o u g h t h i s was n o t a s t e r i l e p r e p a r a t i o n [ K i n d l a n d R u i s 1 9 7 1 ] . T h e s t a g e a t w h i c h t h e p _ - d i h y d r i c r i n g i s o x i d i z e d t o t h e q u i n o n e i s u n k n o w n , as i s t h e s i t e o f t h i s o x i d a t i o n . W h i l e p h e n o l a s e s w i t h £ - d i p h e n o l o x i d a s e a c t i v i t y a r e p r e s e n t i n c h l o r o p l a s t s , m i t o c h o n d r i a [ P a r i s h 1 9 7 2 ] , m i c r o s o m e s , m i c r o b o d i e s a n d s o l u b l e f r a c t i o n s [ R u i s 1 9 7 2 ] , t h e l o c a t i o n o f p _ - d i p h e n o l o x i d a s e i s u n k n o w n . I t a p p e a r s m o s t p r o b a b l e t h a t t h e a t t a c h m e n t o f t h e p i a s t o q u i n o n e i s o p r e n o i d t a i l o c c u r s i n t h e c h l o r o p l a s t , s i n c e l i p i d m e t a b o l i s m i s v e r y a c t i v e i n t h e s e o r g a n e l l e s [ R o g e r s e t a l _ . 1 9 6 8 ] . W i t h i n f o r m a t i o n l i k e t h i s , a p i c t u r e o f i n t r a -c e l l u l a r t r a n s p o r t c a n g r a d u a l l y be b u i l t u p . I t i s s t i l l n o t c e r t a i n t h a t t h i s i s t h e way t h a t p i a s t o q u i n o n e s a r e f o r m e d ; i t i s m e r e l y a p l a u s i b l e r o u t e . PART I I INTRACELLULAR L O C A L I Z A T I O N OF ENZYMES OF PHENYLPROPANOID METABOLISM IN POTATO TUBER RESULTS G e n e r a l O b s e r v a t i o n s N a t u r e o f t h e b a n d s o b s e r v e d a f t e r d e n s i t y  g r a d i e n t c e n t r i f u g a t i o n P o t a t o — In m o s t p r e p a r a t i o n s , a b o u t f o u r m a i n b a n d s w ere n o t e d . T h e s e c o r r e s p o n d i n F i g u r e s 8 t o 12, t o p e a k s o f p r o t e i n c o n t e n t , o r o f a b s o r b a n c e a t 540 nm. Band 1 a l w a y s c o n t a i n e d c a t a l a s e ( a n enzyme w h i c h i s c h a r a c t e r i s t i c o f t h e m i c r o b o d y ) i n h i g h s p e c i f i c a c t i v i t y . T h e b u o y a n t d e n s i t y o f t h i s b a n d was 1.25 t o 1.26. Band 3 c o n t a i n e d f u m a r a s e , a m i t o c h o n d r i a l enzyme, and had a b u o y a n t d e n s i t y o f 1.18 t o 1.19. Bands 1 and 3 c o r r e s p o n d e d t o t h e m i c r o -b o d y and m i t o c h o n d r i o n b a n d s i d e n t i f i e d i n p o t a t o by Huang and B e e v e r s [ 1 9 7 1 ] . A b a n d o f d e n s i t y 1.22 was n o t d e s c r i b e d by t h e s e w o r k e r s , a l t h o u g h a low p e a k o f enzyme a c t i v i t y a p p e a r e d h e r e i n t h e i r g r a p h s o f g r a d i e n t p r o f i l e s . T h i s band a p p e a r e d i n t h e g r a d i e n t p r o f i l e s o f o t h e r w o r k e r s as w e l l [ R u i s 1 9 7 1 ] . 54 55 F i g u r e 8, G r a d i e n t p r o f i l p e l l e t f r o m m n t h e l i g h t . e o f e n z y m e s f r o m t h e 1 0 , 0 0 0 x g _ 100 gm o f p o t a t o d i s k s a g e d i n p r o t e i n : p r o • A , c a t a l a s e : c a t P A L : + »- , c i n n a m i c h y d r o x y l a s e : s p e c i f i c g r a v i t y : p c o ' 9 0 0 56 F i g u r e 9. G r a d i e n t p r o f i l e o f enzymes f r o m t h e 10,000xg_ p e l l e t f r o m 100 gm o f d o r m a n t p o t a t o . P r o t e i n : p r o k A , c a t a l a s e : c a t e 1, PAL: + +, cinnamic h y d r o x y l a s e : CO 0 o, s p e c i f i c g r a v i t y : p 57 F i g u r e 1 0 . G r a d i e n t p r o f i l e o f e n z y m e s f r o m t h e 1 0 , 0 0 0 x £ p e l l e t f r o m 70 gm o f p o t a t o d i s k s a g e d i n t h e l i g h t . O p t i c a l d e n s i t y at 540 nm: A A , c a t a l a s e : c a t • • , fumarase: fum + • • • + , PAL: + +, s p e c i f i c g r a v i t y : p ••••• 58 4 0 . . 1 6 0 • • • • • o E 2 0 . . 8 0 A \ pro I o 6 a. < \ s fum 4 126 A, o L. a. E . .40,000 2 3 C C . 2QCO0g E a F i g u r e 1 1 . G r a d i e n t p r o f i l e o f e n z y m e s f r o m t h e 1 0 , 0 0 0 x £ p e l l e t f r o m 45 gm o f d o r m a n t p o t a t o . p r o t e i n : p r o A A , c a t a l a s e : c a t • fumarase: fum +•••+, O-methyl t r a n s f e r a s e : O - m t A A, s p e c i f i c g r a v i t y : ••••• 59 F i g u r e 1 2 . G r a d i e n t p r o f i l e o f e n z y m e s f r o m t h e 1 0 , 0 0 0 x g _ p e l l e t f r o m 80 gm o f p o t a t o d i s k s a g e d i n s t r e p t o m y c i n i n t h e l i g h t . p r o t e i n : p r o k c a t a l a s e : c a t • • , fumarase: fum +•••+, PAL: + +, O-methyl t r a n s f e r a s e : 0 - m t A» #*A, s p e c i f i c g r a v i t y : p ••••• 60 I n many p l a n t s e . g . s p i n a c h , t h e m i t o c h o n d r i a a r e o f t h i s b u o y a n t d e n s i t y [ H u a n g a n d B e e v e r s 1 9 7 0 ] , b u t i n p o t a t o b a n d 3 was a l w a y s f o u n d t o c o n t a i n m o r e f u m a r a s e t h a n b a n d 2 . S i n c e t h e s i z e a n d e n z y m e a c t i v i t i e s o f t h i s b a n d w e r e e x t r e m e l y v a r i a b l e , i t w e l l m i g h t be t h a t i t r e p r e s e n t e d b r o k e n a n d a g g r e g a t e d m i c r o b o d y a n d m i t o c h o n d r i o n p a r t i c l e s . I t i s k n o w n t h a t d a m a g e d c a s t o r b e a n g l y o x y s o m e s s e d i m e n t w i t h t h e m i t o c h o n d r i a [ G e r h a r d t a n d B e e v e r s 1 9 7 0 ] . F u r t h e r e v i d e n c e f o r t h i s i n t e r p r e t a t i o n comes f r o m F i g u r e 1 0 , w h i c h r e p r e s e n t s an e x p e r i m e n t i n w h i c h o n l y o n e - h a l f t h e u s u a l v o l u m e o f g r i n d i n g m e d i u m was u s e d . D u r i n g d i s r u p t i o n , t h e p l a n t m a t e r i a l b r o w n e d s l i g h t l y . T h i s , t h e n , was o b v i o u s l y a d a m a g e d p r e p a r a t i o n a n d i t i s o n l y i n t h i s p r e p a r a t i o n t h a t PAL was l o c a t e d i n b a n d 1 , a n d f u m a r a s e i n b a n d 2 , as w e l l as i n t h e u s u a l b a n d s . T h e f o u r t h b a n d , o f b u o y a n t d e n s i t y 1 . 1 5 t o 1 . 1 7 , c o r r e s p o n d e d t o t h e h e t e r o g e n o u s b a n d i n v e s t i g a t e d b y R u i s [ 1 9 7 1 ] . P a r t i c l e s w h i c h s e d i m e n t f r o m t h e a p p l i e d l a y e r , b u t w h i c h do n o t p e n e t r a t e t h e g r a d i e n t , c o n t a i n e d some o f t h e £ - d i p h e n o l a s e o f t h e p o t a t o t u b e r . I t was l a t e r s h o w n t h a t t h i s e n z y m e i s p r e s e n t i n t h e m i c r o s o m e s o f p o t a t o [ R u i s 1 9 7 2 ] . s o p e r h a p s t h e r e was some m i c r o s o m a l c o n t a m i n a -t i o n i n b a n d 4 . F u m a r a s e , t o o , was a l s o f o u n d i n b a n d 4 ; 61 p e r h a p s t h i s r e p r e s e n t e d t h e ' l i g h t m i t o c h o n d r i a 1 f o u n d i n p r e p a r a t i o n s f r o m s l i c e d p o t a t o [ V e r l e u r et_ aj_« 1 9 7 0 ] . Sp i n a c h — S p i n a c h p r e p a r a t i o n s a l s o d i s p l a y e d 4 m a i n b a n d s ( F i g u r e 1 3 ) . B a n d 1 c o n t a i n e d o n l y c a t a l a s e , a n d i s p r o b a b l y p e r o x i s o m a l i n n a t u r e . B a n d 2 c o n t a i n e d a v e r y h i g h l e v e l o f c a t a l a s e a l s o , b u t s i n c e t h i s b a n d was a l s o r i c h i n f u m a r a s e , i t p r o b a b l y r e p r e s e n t e d t h e m i t o c h o n d r i o n - r i c h f r a c t i o n . T h i s a l s o m i g h t be a c a s e o f d a m a g e d m i c r o b o d i e s s e d i m e n t i n g w i t h t h e m i t o c h o n d r i a [ G e r h a r d t a n d B e e v e r s 1 9 7 0 ] . B a n d 3 c o n t a i n e d a l o w l e v e l o f f u m a r a s e , a h i g h c a t a l a s e l e v e l , a n d a v e r y h i g h l e v e l o f p r o t e i n . A l m o s t a l l t h e c h l o r o p h y l l i n t h e p r e p a r a t i o n was l o c a t e d i n t h i s b a n d ; i t p r o b a b l y r e p r e s e n t e d t h e c h l o r o p l a s t s . B a n d 4 s e e m e d t o be v e r y h e t e r o g e n o u s , a n d i t s n a t u r e i s u n k n o w n . E f f e c t o f a n t i b i o t i c s on t h e p r e s e n c e o f e n z y m e s M o s t e x p e r i m e n t s w e r e p e r f o r m e d on p o t a t o t i s s u e t h a t h a d b e e n i n c u b a t e d f o r 18 h o u r s i n d i s t i l l e d w a t e r . H o w e v e r , t h e r e i s a s i g n i f i c a n t b a c t e r i a l f l o r a i n s i d e t h e 62 F i g u r e 13. G r a d i e n t p r o f i l e o f enzymes f r o m t h e 10 ,000xg_ p e l l e t f r o m 50 gm o f s p i n a c h . p r o t e i n : p r o • A , c a t a l a s e : c a t t f u m a r a s e : fum +•••+, s p e c i f i c g r a v i t y : p 0 , , iO, c i n n a m i c h y d r o x y l a s e : CO 0 0, 0 - m e t h y l t r a n s f e r a s e : 0-mt A » » » A , c h l o r o p h y l l : c h l X X, PAL: + + 63 p o t a t o t u b e r [R. B a n d o n i , p e r s . comm.] and t h i s , as w e l l as t h o s e c o n t a m i n a n t s i n t r o d u c e d d u r i n g n o n - s t e r i l e h a n d l i n g c o u l d r e s u l t i n a m a s s i v e g r o w t h o f b a c t e r i a d u r i n g t h e i n c u b a t i o n p e r i o d . To s e e i f b a c t e r i a made any d i f f e r e n c e t o t h e a c t i v i t y o f some o f t h e enzymes i n q u e s t i o n , p o t a t o e s w e re i n c u b a t e d i n t h e p r e s e n c e o f s t r e p t o m y c i n s u l f a t e (1 gm/1). F i g u r e 12 shows t h e g r a d i e n t p r o f i l e o f t h i s t i s s u e T h e r e d i d n o t seem t o be any s i g n i f i c a n t e f f e c t s o f t h e a n t i b i o t i c . T i s s u e i n c u b a t e d i n n e o m y c i n s u l f a t e was u s e d f o r t h e p r e p a r a t i o n o f May 16 ( T a b l e 4 ) , and a g a i n , t h e r e d i d n o t a p p e a r t o be any s i g n i f i c a n t d i f f e r e n c e s i n PAL p r o d u c -t i o n o r d i s t r i b u t i o n . I t seems t h a t t h e p r e s e n c e o r a b s e n c e o f b a c t e r i a d i d n o t m a r k e d l y a f f e c t t h e p r o d u c t i o n and d i s t r i b u t i o n o f s e v e r a l o f t h e enzymes e x a m i n e d i n t h i s s t u d y . P h e n y l a l a n i n e a m m o n i a - l y a s e ( E . C . 4.3.1.5) P o t a t o t i s s u e , e s p e c i a l l y a f t e r s l i c i n g and a g e i n g , c o n t a i n s many a r o m a t i c compounds [ H a n s o n and Z u c k e r 1 9 6 3 ] . E l e c t r o n m i c r o g r a p h s o f t h e I 0 , 0 0 0 x £ p e l l e t s h o w e d m i c r o b o d i e s and m i t o c h o n d r i a , b u t no b a c t e r i a l c e l l s t h a t c o u be p o s i t i v e l y i d e n t i f i e d . T a b l e 4 D i s t r i b u t i o n o f PAL i n s u b c e l l u l a r f r a c t i o n s o f p o t a t o a g e d In t h e l i g h t Ce11 F r a c t i on E x p e r i m e n t 1 E x p e r i m e n t 2 E x p e r i m e n t 3 I 0 , 0 0 0 x g p e l l e t 1 0 0 , 0 0 0 x £ p e l 1 e t (mi c r o s o m e s ) 1 0 0 , 0 0 0 x £ s u p e r n a t a n t S . A . % >v S . A . % S . A . % ( m i c r o b o d i e s 0% b a n d 2 . 1 3 , mi t o c h o n d r i a . 2 4 " b a n d 4 and^ s o l u b l e - 2 . 0 2 e n z y m e s 2 . 4 $ 1 3 . 9 8 . 7 5 1 6 4 . 0 2 . 4 4 . 8 9 2 . 8 7 . 14 3 . 0 3 156 4 . 3 1 . 8 9 4 . 0 5 . 4 2 0 . 4 1 8 0 . 1 2 . 6 9 . 9 8 7 . 6 1 8 6 . 6 1 0 0 . 0 1 6 6 . 2 1 0 0 . 1 2 0 6 . 9 1 0 0 . I S p e c i f i c A c t i v i t y i n j j m o l e s c i n n a m i c a c i d f o r m e d h r gm f r e s h w e i g h t 4* 65 I t i s t h e r e f o r e a d a n g e r t h a t t h e m e a s u r e m e n t o f PAL by c h a n g e i n o p t i c a l d e n s i t y a t 290 nm m i g h t be c o n f o u n d e d by t h e r e l e a s e d u r i n g i n c u b a t i o n o f p r e v i o u s l y b o u n d a r o m a t i c c o m -p o u n d s . On t h e o t h e r h a n d , t h e d e s t r u c t i o n o f p a r t i c l e s on l o n g i n c u b a t i o n r e d u c e s t h e a p p a r e n t o p t i c a l d e n s i t y i n t h o s e f r a c t i o n s . T o e v a l u a t e t h e s e f a c t o r s , t h e c h a n g e i n o p t i c a l d e n s i t y m e a s u r e d i n a l i q u o t s o f m a t e r i a l i n c u b a t e d o v e r n i g h t i n b u f f e r w i t h o u t a d d e d p h e n y l a l a n i n e was c o m p a r e d t o t h e c h a n g e s o b t a i n e d d u r i n g t h e u s u a l PAL a s s a y . F i g u r e 14 i n d i c a t e s t h a t t h e e r r o r s i n t r o d u c e d b y o v e r n i g h t i n c u b a t i o n w i t h u n d i a l y s e d p a r t i c u l a t e f r a c t i o n s w e r e r e l a t i v e l y m i n o r . T h e 1 0 , 0 0 0 x £ p e l l e t , 1 0 0 , 0 0 0 x £ p e l l e t a n d t h e 1 0 0 , 0 0 0 x £ s u p e r n a t a n t f r o m a g e d p o t a t o w e r e e x a m i n e d f o r t h e p r e s e n c e o f PAL ( T a b l e 4 ) . W h i l e 90% o f t h e e n z y m e a p p e a r e d t o be s o l u b l e , t h e r e was c o n s i d e r a b l e a c t i v i t y i n b o t h t h e o r g a n e l l e a n d m i c r o s o m a l f r a c t i o n s ( 1 0 , 0 0 0 X J J a n d 1 0 0 , 0 0 0 x j j p e l l e t s , r e s p e c t i v e l y ) . The l a t t e r f r a c t i o n c o n t a i n s a b o u t 10% o f t h e t o t a l a c t i v i t y o f t h e c e l l ; i t i s u n l i k e l y t h a t 10% o f t h e s o l u b l e m a t e r i a l w o u l d be c a r r i e d by t h e u n w a s h e d p e l l e t . T h u s i t a p p e a r s t h a t some o f t h e e n z y m e i s s o l u b l e a n d some s e d i m e n t s w i t h t h e o r g a n e l l e s . T h e n a t u r e o f t h e 2-3% o f t h e t o t a l e n z y m e t h a t i s l o c a t e d i n t h e 10 , 0 0 0 x g _ p e l l e t was i n v e s t i g a t e d w i t h a bottom '10 20 ml. 30 top F i g u r e 14. A s s e s s m e n t o f e r r o r s i n t h e PAL a s s a y due t o o v e r n i g h t i n c u b a t i o n . u s u a l a s s a y : x x c o n t r o l ( w i t h o u t p h e n y l a l a n i n e ) : t • a c t u a l c h a n g e i n n m o I e s / m I / h r : 0***0 67 d i s c o n t i n u o u s g r a d i e n t . F i g u r e s 8 and 12 a r e t y p i c a l g r a -d i e n t p r o f i l e s . PAL a p p e a r e d i n t h e s o l u b l e f r a c t i o n s o f a l l p r e p a r a t i o n s . PAL a l s o a p p e a r e d c o n s i s t e n t l y i n t h e b a n d s 2, 3 and 4. T h e r e i s t h e p o s s i b i l i t y t h a t t h e enzyme m o l e c u l e s ( o r p a r t i c l e s ) w e re a d s o r b e d o n t o , o r a d h e r e d t o t h e s u r -f a c e s o f t h e l a r g e r o r g a n e l l e s . To t e s t t h i s p o s s i b i l i t y , a p a r t i a l l y p u r i f i e d p r e p a r a t i o n o f PAL f r o m 1 8 - h o u r o l d p o t a t o d i s k s was p u t t h r o u g h a S e p h a d e x G-200 c o l u m n and t h i s p r e p a r a t i o n was i n c u b a t e d w i t h t h e p a r t i c u l a t e f r a c t i o n f r o m f r e s h p o t a t o e s , w h i c h had l i t t l e o r no PAL ( s e e F i g u r e 9 ) . I f t h e r e had b e e n a d h e r e n c e o f PAL t o t h e o r g a n e l l e s f r o m t h e f r e s h p o t a t o , t h e n t h i s s h o u l d h a v e b e e n d i s t i n g u i s h a b l e by t h e d i s t r i b u t i o n o f t h e enzyme on a s u c r o s e d e n s i t y g r a d i e n t . F i g u r e 15 shows t h e r e s u l t s o f s u c h a g r a d i e n t s e p a r a t i o n , and shows t h a t , w i t h i n e x p e r i m e n t a l e r r o r , t h e r e was no ' s m e a r i n g ' o f t h e s o l u b l e PAL on t h e g r a d i e n t . C i n n a m i c A c i d 4 - H y d r o x y 1 a s e ( E . C . 1.99.1) The e f f e c t s o f a g e i n g i n t h e l i g h t on t h e p r o d u c t i o n o f t h i s enzyme were e x a m i n e d i n p o t a t o d i s c s . T a b l e 5 shows t h a t s p e c i f i c a c t i v i t y i n c r e a s e d a f t e r a g e i n g when e x p r e s s e d e i t h e r as u n i t s p e r mg p r o t e i n o r p e r gm f r e s h w e i g h t . 68 F i g u r e 1 5 . G r a d i e n t p r o f i l e o f p a r t i a l l y p u r i f i e d PAL m i x e d w i t h o r g a n e l l e s f r o m d o r m a n t p o t a t o . PAL a l o n e : o o o r g a n e l l e s a l o n e : • • PAL p l u s o r g a n e l l e s : + + s p e c i f i c g r a v i t y : 69 T a b l e 5 a l s o s h o w s t h a t m o s t o f t h e e n z y m e a p p e a r e d i n t h e 0 - 3 0 % ammonium s u l f a t e f r a c t i o n i n c o n t r a s t t o P A L , w h i c h was f o u n d i n t h e 2 7 - 4 0 % f r a c t i o n [ H a v i r a n d H a n s o n 1968]. T a b I e 5 The e f f e c t o f a g e i n g i n t h e l i g h t on t h e a c t i v i t y o f c i n n a m i c a c i d 4 - h y d r o x y I a s e i n p o t a t o AGE Dorma n t Aged 18 H o u r s * E x p e r i m e n t 1 * * E x p e r i m e n t 2 0 - 3 0 ? arnmon i urn s u 1 f a t e 3 0 - 7 0 ? ammon i urn s u1 f a t e 641±13? dpm/gm f r wt 6 5 2 cpm/mg p r o t e i n 142 cpm/mg p r o t e i n I 4 4 0 ± 4 $ dpm/grn f r wt 2 0 , 8 0 0 cpm/mg p r o t e i n 1730 cpm/mg p r o t e i n E x p e r i m e n t I: m a t e r i a l c h o p p e d t o p r e s e r v e o r g a n e l l e s . E x p e r i m e n t 2 : m a t e r i a l g r o u n d f o r c o m p l e t e e x t r a c t i o n . W h i l e t h e r e was r e l a t i v e l y l i t t l e a c t i v i t y i n t h e 1 0 , 0 0 0 x g _ p e l l e t i n t h e f r e s h m a t e r i a l , t h e r e was up t o 20% o f t h e t o t a l a c t i v i t y i n t h i s f r a c t i o n i n a g e d m a t e r i a l ( T a b l e 6). Much l e s s o f t h e t o t a l a c t i v i t y o f c i n n a m a t e T a b I e 6 S u b c e l l u l a r d i s t r i b u t i o n o f c i n n a m i c a c i d 4 - h y d r o x y I a s e In d o r m a n t p o t a t o t u b e r and t i s s u e aged i n t h e l i g h t Ce11 F r a c t I on D o r m a n t T i s s u e Aged T i s s u e a c t i v i t y % o r g a n e 1 1 e s a c 11 v i t y % o r g a n e 1 1 e s 1 0 , 0 0 0 x £ . 165 2 . 4 ± . 2 3 . 1 6 2 0 . 4 ± 3 . 1 0? p e l l e t I n c l u d i n g : m1c r o b o d I e s . 0 6 ? 0? band 2 . 0 6 ? 1 . 9 ? m I t o c h o n d r I a . 17? 6 . 3 band 4 2 . 1 ? 1 2 . 2 1 0 0 , 0 0 0 x £ p e l l e t . 9 0 0 1 3 . 7 ±4 . 6 9 . 7 6 6 7 . 3 ± 4 . 0 (mi c r o s o m a 1 ) I 0 0 , 0 0 0 x £ s u p e r n a t a n t 5 . 5 2 8 4 . 0 ± 2 0 1 . 1 7 1 2 . 2 1 . 7 2 ( s o l u b l e ) 6 . 59 1 4 . 7 A c t i v i t y i n n m o l e s / h r / g m f r e s h w e i g h t . D e t e r m i n a t i o n s done i n d u p l i c a t e . 71 h y d r o x y l a s e was f o u n d i n t h e s o l u b l e f r a c t i o n i n a g e d t h a n i n f r e s h p o t a t o ; i t a p p e a r s t h a t t h e e n z y m e b e c o m e s p a r t i c u -l a t e as t h e p o t a t o a g e s . O f t h e m a t e r i a l w h i c h i s f o u n d i n t h e 10 ,000xg_ p e l l e t , t h e r e a p p e a r s t o be t h e same d i s t r i b u t i o n o f e n z y m e as was s h o w n by P A L ; c i n n a m a t e h y d r o x y l a s e o c c u r r e d i n a l l o r g a n e l l e s e x c e p t t h e m i c r o b o d i e s . I n s p i n a c h ( T a b l e 7, F i g u r e 1 4 ) , some c i n n a m a t e h y d r o x y l a s e d i d a p p e a r t o be a s s o c i a t e d w i t h t h e p e r o x i s o m e s . T a b l e 7 D i s t r i b u t i o n o f c i n n a m i c a c i d 4 - h y d r o x y I a s e i n t h e I 0 ,000xg_ pe I I e t o f s p i n a c h p e r o x i s o m e s 6.8% m i t o c h o n d r i a \ \ .7% c h 1 o r o p 1 a s t s 22.7% b a n d 4 58.8% O - M e t h y l T r a n s f e r a s e M e t h y l a t i n g C a f f e i c A c i d ( E . C . 2 . 1 . 1 ) A g e i n g i n l i g h t c a u s e d a 2 - f o l d i n c r e a s e i n t h e a m o u n t o f t h i s e n z y m e ( T a b l e 8 ) , b u t t h e r e d i d n o t a p p e a r t o T a b l e 8 S u b c e l l u l a r d i s t r i b u t i o n o f 0 - m e t h y l t r a n s f e r a s e i n d o r m a n t p o t a t o t u b e r and t i s s u e a g e d i n t h e l i g h t Ce1 1 F r a c t i on D o r m a n t T i s s u e Aged T i s s u e *t a c t i v i t y 9 ac 1 1 v i t y 9 I 0 , 0 0 0 x £ pe 1 l e t . 0 0 9 4 . 7 ± . 98 . 0 1 5 2 . 9 ± . 84 1 0 0 , 0 0 0 x g _ pe 1 l e t (mi c r o s o m a 1 ) . 0 0 4 2 . 1 ± . 47 . 0 0 8 1 . 4 ± . 21 1 0 0 , 0 0 0 x £ s u p e r n a t a n t ( s o l u b l e ) . 1 85 9 3 . 2 ± l 6 . 4 5 3 9 5 . 6 ± l 5 . 3 . 198 . 4 7 6 n m o I e s / h r / g m f r e s h w e i g h t , d e t e r m i n a t i o n s done i n d u p l i c a t e . 73 be a n y c h a n g e i n t h e d i s t r i b u t i o n . I n b o t h f r e s h a n d a g e d t i s s u e , a b o u t 95% o f t h e t o t a l a c t i v i t y was s o l u b l e . I n two d i f f e r e n t e x p e r i m e n t s ( F i g u r e s 11 a n d 1 2 ) , t h e 10 ,000xg_ p e l l e t was e x a m i n e d on a s u c r o s e g r a d i e n t . I n b o t h c a s e s , t h e r e a p p e a r e d e n z y m e a c t i v i t y w i t h t h e v a r i o u s b a n d s , b u t o n l y a b o u t 5-10% o f t h e a p p l i e d a c t i v i t y was r e c o v e r e d i n t h e s e b a n d s , a n d t h e r e s t o f t h e a c t i v i t y r e -m a i n e d i n t h e s u p e r n a t a n t a b o v e t h e s u c r o s e g r a d i e n t . T h e g r a d i e n t d i s t r i b u t i o n i n s p i n a c h i s v e r y s i m i l a r ( F i g u r e 1 3 ) . S h i k i m i c A c i d r N A D P O x i d o r e d u c t a s e ( E . C . 1 . 1 . 1 . 2 5 ) ( S O R ) ,  P r e p h e n i c A c i d r N A D P O x i d o r e d u c t a s e ( E . C . 1 . 1 . 1 ) ( P O R ) T h e s e t w o e n z y m e s b e h a v e d i n a s i m i l a r m a n n e r . B o t h i n c r e a s e d i n a c t i v i t y on a g e i n g , b u t t h e e x t e n t o f i n -c r e a s e i n u n k n o w n , f o r i n one e x p e r i m e n t ( T a b l e 9 ) , n e i t h e r e n z y m e c o u l d be l o c a t e d i n f r e s h p o t a t o , w h i l e i n a n o t h e r e x p e r i m e n t , c o n s i d e r a b l e a c t i v i t y was p r e s e n t b e f o r e a g e i n g . I n a n y e v e n t , t h e r e was a t l e a s t a t w o - f o l d i n c r e a s e on a g e i n g . T h e s e e n z y m e s s h o w e d v e r y s i m i l a r d i s t r i b u t i o n p a t t e r n s ; b o t h w e r e e x c l u s i v e l y s o l u b l e . W h i l e SOR i s q u i t e s t a b l e d u r i n g t h e p e r i o d o f c e n t r i f u g a t i o n , 40% o f t h e o r i g i n a l a c t i v i t y i n POR was l o s t d u r i n g f r a c t i o n a t i o n . T a b l e 9 S u b c e l l u l a r d i s t r i b u t i o n o f s h i k i m i c a c i d o x i d o r e d u c t a s e (SOR) and p r e p h e n i c a c i d o x i d o r e d u c t a s e (POR) i n d o r m a n t p o t a t o t u b e r and t i s s u e a g e d i n t h e l i g h t E x p e r i ment Enzyme T i s s u e F r a c t i o n A c t i v i t y u m o l e s / h r / g m f . w . ? T o t a l 1 SOR d o r m a n t a g e d 1 0 ,000xg_ p e l 1 e t m I c r o s o m a 1 s o l u b l e 1 0 , 0 0 0 x £ pe1 l e t m I c r o s o m a 1 s o l u b l e 0 0 0 . 5 2 4 0 0 1 . 1 7 1 0 0 ? POR d o r m a n t , . a g e d I 0 , 0 0 0 x g _ pe 1 l e t m i c r o s o m a 1 s o l u b l e 1 0 ,000xg_ p e l 1 e t m i c r o s o m a 1 s o l u b l e 0 0 0 . 2 5 0 0 0 0 . 6 2 6 1 0 0 ? 2 SOR dorma n t a g e d 1 0 ,000xg_ p e l 1 e t m i c r o s o m a 1 s o l u b l e I 0 , 0 0 0 x £ p e l l e t m i c r o s o m a 1 s o l u b l e 0 0 0 0 0 1 . 5 6 1 0 0 ? POR d o r m a n t a g e d 1 0 , 0 0 0 x £ p e l 1et m i c r o s o m a 1 s o l u b l e 1 0 , 0 0 0 x £ pe 1 l e t m i c r o s o m a 1 s o l u b l e 0 0 0 0 0 0 . 7 8 6 1 0 0 ? 75 I n s p i n a c h , POR was p r e s e n t i n t h e 10 ,000xg_ s u p e r -n a t a n t i n t r a c e a m o u n t s . I t was n o t e s t a b l i s h e d w h e t h e r t h e e n z y m e was s o l u b l e o r p a r t i c u l a t e . H i g h e r a c t i v i t i e s o f SOR w e r e p r e s e n t , a n d t h e a c t i v i t y was a l l s o l u b l e . p _ - C o u m a r i c A c i d H y d r o x y l a s e No h y d r o x y 1 a t i on o f p _ - c o u m a r i c a c i d t o c a f f e i c a c i d by e x t r a c t s o f a g e d p o t a t o d i s c s c o u l d be d e t e c t e d b y a n y o f t h e m e t h o d s t r i e d ( s e e M e t h o d s ) . A l c o h o l D e h y d r o g e n a s e s T h r e e a l c o h o l d e h y d r o g e n a s e s s e p a r a b l e on S e p h a d e x G - 2 0 0 , a n d w i t h d i f f e r e n t s p e c i f i c i t i e s h a v e b e e n f o u n d i n f r e s h p o t a t o [ D a v i e s e t a l _ . 1 9 7 2 ] . One e n z y m e r e q u i r e s NAD a n d i s a c t i v e w i t h a l k y l a l c o h o l s ; t h e s e c o n d i s an i s o p r e -n o i d a l c o h o l e n z y m e u s i n g NADP o r N A D , a n d t h e t h i r d e n z y m e o x i d i z e s a r o m a t i c a l c o h o l s w i t h N A D P . I n c r u d e e n z y m e p r e p a r a t i o n s f r o m ' N e t t e d Gem 1 s l i c e s a g e d i n t h e l i g h t a n d p r e p a r a t i o n s f r o m w h i c h l o w m o l e c u l a r w e i g h t c o m p o u n d s h a d b e e n r e m o v e d , t h e a r o m a t i c a l c o h o l d e h y d r o g e n a s e ( N A D P ) as d e s c r i b e d b y D a v i e s e t a l . was n o t p r e s e n t . T a b l e 10 c o m p a r e s t h e a c t i v i t i e s f o u n d i n 76 ' N e t t e d Gem' w i t h t h o s e w h i c h w e r e f o u n d i n t h e p o t a t o e s e x a m i n e d b y D a v i e s e_t a l . Table 10 Potato a l c o h o l dehydrogenases as d e s c r i b e d by Davies et a I., and r e d u c t i o n of aldehydes by e x t r a c t s of aged 'Netted Gem' s l i c e s S u b s t r a t e Davies et a 1 . Re 1a t T V e A c t i v i t y * 'Netted Gem' AOD 3 i, o /m i n/m 1 NADH NADPH NADH.7, NADPH-aceta1dehyde 1 0 .066 . 0 26 benza1dehyde 0.0 12 1 , 0.14 .008 v a n i l l i n 0 8 0 0 p_-hyd roxy benza 1 dehyde 0 4.9 0 0 m-hydroxybenzaldehyde 0 1 .3 0 0 R e l a t i v e to the r a t e with acetaldehyde f o r the a l k y l (.NIADH?) enzyme and to the r a t e with benzaldehyde f o r the a r y l (NADPH2) enzyme. 77 B i o s y n t h e t i c C a p a b i l i t i e s o f S u b c e l l u l a r F r a c t i o n s  S u p p l i e d w i t h R a d i o a c t i v e S u b s t r a t e P o t a t o d i s c s a g e d 18 h o u r s i n t h e l i g h t i n n e o m y c i n s u l f a t e w e r e s e p a r a t e d i n t o a p a r t i c u l a t e f r a c t i o n ( 1 0 , 0 0 0 x g _ f r a c t i o n ) c o n t a i n i n g m i c r o b o d i e s a n d m i t o c h o n d r i a , a m i c r o -s o m a l p e l l e t ( 1 0 0 ,000xg_ p e l l e t ) a n d a s o l u b l e f r a c t i o n ( 1 0 0 ,000xg_ s u p e r n a t a n t ) . T y r o s i n e - 2 - 1 "C ( 5 0 j u C i / u m o l e , 0 . 5 j j C i / f e e d i n g ) a n d p h e n y l a l a n i n e l - 1 I f C ( 1 6 . 5 j u C i / u m o l e , 0 . 5 j u C i / f e e d i n g ) w e r e f e d t o a l i q u o t s o f e a c h f r a c t i o n . A f t e r o n e h o u r o f i n c u b a t i o n , t h e f r a c t i o n s w e r e e x t r a c t e d a n d c h r o m a t o g r a p h e d i n t h e u s u a l m a n n e r . C o m p o u n d s c o r r e s p o n d i n g t o f o g g e d a r e a s on r a d i o a u t o g r a m s w e r e e l u t e d a n d r e - c h r o m a t o -g r a p h e d w i t h v a r i o u s a u t h e n t i c c o m p o u n d s i n s o l v e n t s A , D a n d F. P h e n y l a l a n i n e f e e d i n g : I n a l l c a s e s , o n l y c i n n a m i c a c i d was l a b e l l e d . T h i s s u g g e s t s t h a t t h e e n z y m e c a t a l y z i n g c h a i n s h o r t e n i n g i s v e r y a c t i v e . No p _ c o u m a r i c a c i d b e c a m e l a b e l l e d , b u t t h i s i s t o be e x p e c t e d as t h e c i n n a m i c a c i d 4 - h y d r o x y l a s e r e a c t i o n r e q u i r e s t h e p r e s e n c e o f N A D P H 2 . T y r o s i n e f e e d i n g : I n a g e d p o t a t o t h e r e was no t r a c e o f T A L a c t i v i t y . C o m p o u n d s w h i c h c o - c h r o m a t o g r a p h e d i n t h r e e s o l v e n t s y s t e m s w i t h p _ - h y d r o x y p h e n y 1 a c e t i c a c i d ( P H P A ) a n d p _ - h y d r o x y p h e n y 1 p y r u v i c a c i d ( P H P P ) w e r e f o r m e d f r o m t h e 78 a d m i n i s t e r e d t y r o s i n e . I t w o u l d a p p e a r t h a t t h e f a t e o f t y r o s i n e i n p o t a t o i s t r a n s a m i n a t i o n f o l l o w e d by c h a i n s h o r t e n i n g . T h e m i c r o s o m a l f r a c t i o n f o r m e d v e r y l i t t l e PHPA b u t i t d i d f o r m P H P P . T h e e x t r a c t o f t h e p a r t i c u l a t e f r a c t i c o n t a i n e d b o t h c o m p o u n d s . I t i s c o n c l u d e d t h a t t h e f i r s t r e a c t i o n t o o k p l a c e i n b o t h f r a c t i o n s , w h i l e t h e s e c o n d r e a c t i o n t o o k p l a c e m a i n l y i n t h e l a r g e r o r g a n e l l e s . DISCUSSION D i s t r i b u t i o n o f PAL i n S u b c e l l u l a r F r a c t i o n s f r o m  Aged P o t a t o T u b e r In a g e d p o t a t o , PAL was f o u n d i n e v e r y s u b c e l l u l a r f r a c t i o n e x c e p t t h e m i c r o b o d y f r a c t i o n ( T a b l e 4 ) . R u i s [ 1 9 7 1 ] c o u l d f i n d no t r a c e o f PAL i n t h e s e o r g a n e l l e s . S i n c e t h e s e f r a c t i o n s w e re a l l u n w a s h e d , i t w o u l d be a m i s t a k e t o c l a i m t h a t an enzyme i s l o c a l i z e d i n an o r g a n e l l e b e c a u s e i t i s f o u n d i n a f r a c t i o n r i c h i n t h a t o r g a n e l l e . S u c h a d i s t r i b u t i o n c o u l d a r i s e i n any o f s e v e r a l w a y s . F i r s t l y t h e r e c o u l d be s e v e r a l i s o z y m e s f o r m e d , and e a c h m i g h t be a s s o c i a t e d w i t h a s e p a r a t e o r g a n e l l e . A n o t h e r e x p l a n a t i o n c o u l d be t h e a d s o r p t i o n o f PAL m o l e c u l e s t o t h e s u r f a c e s o f o r g a n e l l e s a f t e r c e l l d i s r u p t i o n . O r , b i t s o f c y t o p l a s m c o n t a i n i n g PAL c o u l d be a t t a c h e d t o t h e o r g a n e l l e s . Y e t a n o t h e r e x p l a n a t i o n i s t h a t a g g r e g a t e s o f PAL m o l e c u l e s c o u l d s e d i m e n t w i t h t h e c e l l f r a c t i o n s , d e p e n d i n g o f c o u r s e , on s i z e and d e n s i t y . 79 80 T h e f i r s t e x p l a n a t i o n i s u n l i k e l y , s i n c e i n ?. d e -t a i l e d i n v e s t i g a t i o n o n l y t w o f o r m s o f PAL w e r e f o u n d in a g e d p o t a t o [ H a v i r a n d H a n s o n 1 9 6 9 ] , A b s o r p t i o n o f PA~ m o l e c u l e s i s a l s o u n l i k e l y : c e r t a i n l y PAL m o l e c u l e s d J n o t a d s o r b o n t o o r g a n e l l e s f r o m f r e s h t i s s u e ( F i g u r e 1 5 ) . T h e s u g g e s t i o n t h a t c y t o p l a s m i c c o n t a m i n a t i o n m i g h t a c c o u n t f o r t h e d i s t r i b u t i o n i s m o r e r e a s o n a b l e . A p h o t o g r a p h y o f a m i c r o b o d y - r i c h f r a c t i o n f r o m c u c u m b e r c o t y l e d o n s [ T r e l e a s e e_t a l_. 1 9 7 1 ] s h o w s p o r t i o n s o f c y t o p l a s m i n c l u d e d w i t h i n m i c r o b o d i e s . I f s u c h i n c l u s i o n s w e r e a s s o -c i a t e d w i t h o t h e r o r g a n e l l e s , d i s t r i b u t i o n o f PAL m i g h t be f o u n d . T h e r e a r e a r g u m e n t s a g a i n s t t h i s s u g g e s t i o n . F i r s t , why w o u l d c y t o p l a s m i c c o n t a m i n a t i o n o c c u r i n a l l f r a c -t i o n s b u t t h e m i c r o b o d i e s ? S e c o n d l y , i f PAL w e r e a s o l u b l e c y t o p l a s m i c e n z y m e , t h e n t h e r a t i o o f s p e c i f i c a c i t i v i t i e s o f PAL a n d t h e m a i n l y c y t o p l a s m i c e n z y m e 0 - m e t h y l t r a n s f e r a s e s h o u l d be t h e same i n e a c h f r a c t i o n . S i n c e i t i s n o t ( T a b l e 1 1 ) , t h e d i s t r i b u t i o n o f t h e e n z y m e s c a n n o t be t h e s a m e , a n d t h e p r e s e n c e o f PAL c a n n o t be d u e t o c y t o p l a s m i c c o n t a m i n a -t i o n . T h e f o u r t h s u g g e s t i o n d e a l s w i t h t h e p r e s e n c e o f PAL i n a g g r e g a t e s . I n b u c k w h e a t , PAL has b e e n l o c a t e d i n p a r t i c l e s t h a t s e d i m e n t a t 100 ,000xg_ b u t a r e n o t r i b o s o m e s 81 T a b l e II R a t i o o f PAL and 0 - m e t h y I t r a n s f e r a s e a c t i v i t i e s i n s u b c e l l u l a r f r a c t i o n s o f a g e d p o t a t o F r a c t i on P A L nm m 1 _ 1 h r - 1 0 - m e t h y l t r a n s f e r a s e dpm m 1 - 1 h r - 1 R a t i o m i c r o b o d i e s 0 3 8 1 5 -b a n d 2 7 . 5 4 2 2 0 564 mi t o c h o n d r i a 1 5 . 0 1 6 0 5 0 1 0 6 5 b a n d 4 a n d s o l u b l e 1 1 5 . 9 1 8 0 5 0 0 1 560 [ A m r h e i n a n d Z e n k 1 9 7 1 ] . S u c h p a r t i c l e s c o u l d w e l l be t r a p p e d i n t h e p a c k e d p e l l e t a f t e r c e n t r i f u g a t i o n a t 1 0 , 0 0 0 x g _ . S c h o p f e r [ 1 9 7 1 ] s u g g e s t t h a t s u c h p a r t i c l e s m i g h t be a g g r e g a -t i o n p r o d u c t s , s i n c e m u s t a r d PAL i s o l a t e d i n t r i s b u f f e r o f l o w c o n c e n t r a t i o n f o r m e d a g g r e g a t e s o f m o l e c u l a r w e i g h t > 4 x 1 0 6 , w h i c h m i g h t s e d i m e n t w i t h t h e r i b o s o m e s . B u t i n t h e p r e s e n t s t u d y , t h e e n z y m e was n e v e r i n b u f f e r o f c o n c e n -t r a t i o n l e s s t h a n 0 . 0 5 M , a n d t h i s i s a l e s s s e v e r e c o n d i t i o n t h a n u s e d b y S c h o p f e r t o o b t a i n a g g r e g a t e s . F u r t h e r , p a r -t i a l l y p u r i f i e d PAL p r e p a r e d i n 0 . 0 5 M t r i s b u f f e r i s r e t a r d e d i n a S e p h a d e x G - 2 0 0 c o l u m n , w h i c h i m p l i e s a m o l e c u l a r w e i g h t 82 < 8 0 0 , 0 0 0 f o r a g l o b u l a r p r o t e i n ( P h a r m a c i a t e c h n i c a l b u l l e t i n 1 9 6 6 ) . On t h e o t h e r h a n d , PAL f r o m p o t a t o e s g r o u n d i n t o o l i t t l e b u f f e r d i d a p p e a r a t g r e a t e r b u o y a n t d e n s i t i e s t h a n u s u a l . T h u s , t h e PAL m o l e c u l e s f r o m p o t a t o may f o r m a g g r e g a t e s u n d e r c o n d i t i o n s o t h e r t h a n t h o s e m e n t i o n e d b y S c h o p f e r . The m o s t r e a s o n a b l e e x p l a n a t i o n f o r t h e s e d i m e n -t a t i o n b e h a v i o u r o f PAL i s t h a t t h e e n z y m e was m o s t l y s o l u b l e b u t a l s o o c c u r s i n a p a r t i c u l a t e f o r m . T h i s p a r t i c l e was l a r g e r t h a n a r i b o s o m e , b u t t o o s m a l l t o s e d i m e n t w i t h t h e l a r g e r o r g a n e l l e s . T h e p r e s e n c e o f PAL i n t h e s e l a r g e r o r g a n e l l e s c o u l d h a v e b e e n d u e t o c o n t a m i n a t i o n b y , o r f u r t h e r a g g r e g a t i o n o f , t h e s e p a r t i c l e s . S u c h p a r t i c l e s w o u l d be d i f f i c u l t t o i d e n t i f y w i t h e l e c t r o n m i c r o s c o p y . I n a n y e v e n t , m o r e w o r k i s n e e d e d w i t h p u r i f i e d o r g a n e l l e f r a c t i o n s b e f o r e t h e e n d o g e n o u s p r e s e n c e o f PAL c a n be u n e q u i v o c a l l y d e m o n s t r a t e d . PAL f r o m f r e s h p o t a t o ( p r e s e n t a t v e r y l o w l e v e l s ) d i d n o t a p p e a r w i t h a n y o f t h e l a r g e r o r g a n e l l e s , a n d may be m o s t l y s o l u b l e . T h i s f i n d i n g a g r e e s w i t h e a r l i e r w o r k on p o t a t o [ R u i s 1 9 7 1 ] . 83 D i s t r i b u t i o n o f C i n n a m i c A c i d 4 - H y d r o x y l a s e M o s t o f t h e c i n n a m a t e h y d r o x y l a s e i n a g e d p o t a t o seems t o be a s s o c i a t e d w i t h p a r t i c u l a t e f r a c t i o n s ( T a b l e 6 ) . S u m m i n g t h e p e r c e n t a g e s f o u n d i n t h e 1 0 , 0 0 0 x £ a n d 100 ,000xg_ p e l l e t s , 88% o f t h e t o t a l a c t i v i t y was n o t s o l u b l e . T h i s a g r e e s w e l l w i t h S t a f f o r d ' s f i n d i n g ( 1 9 6 9 ) t h a t a l l t h e c i n n a m a t e h y d r o x y l a s e i n S o r g h u m was p a r t i c u l a t e . As i n t h e c a s e o f P A L , no 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 m i c r o b o d i e s , a n d a l a r g e a m o u n t o f a c t i v i t y was a s s o c i a t e d w i t h b a n d 4 . I t i s i n t e r e s t i n g t h a t i n s p i n a c h , t h e e n z y m e a p p e a r e d i n t h e p e r o x i s o m a l as w e l l as t h e o t h e r f r a c t i o n s . A m r h e i n a n d Z e n k s h o w e d by g r a d i e n t c e n t r i f u g a t i o n o f t h e 1 0 0 , 0 0 0 x £ p e l l e t f r o m b u c k w h e a t t h a t t h e m i c r o s o m a l p a r t i c l e s c o n t a i n i n g PAL w e r e q u i t e d i f f e r e n t f r o m t h o s e c o n t a i n i n g c i n n a m a t e h y d r o x y l a s e . T h e s i t u a t i o n n e e d s t o be d e t e r m i n e d i n p o t a t o a l s o . T h e E f f e c t o f A g e i n g on E n z y m e s o f P h e n y l p r o p a n o i d  M e t a b o l i s m o f P o t a t o T h e m o s t o b v i o u s e f f e c t o f a g e i n g on t h e e n z y m e s o f T a b l e 12 i s a s t r o n g i n c r e a s e i n a c t i v i t y . T h e i n c r e a s e o f SOR on a g e i n g was n o t e d b y S a c h e r e_t a j k [ 1 9 7 2 J . T h e y f o u n d 84 T a b l e 12 The e f f e c t o f a g e i n g i n t h e l i g h t on some e n z y m e s o f p h e n o l i c m e t a b o l i s m i n d i s k s o f p o t a t o t u b e r En zyme E x p e r i m e n t I n c r e a s e i n A g e i n g (?) SOR exp t . 1 ( n o a c t i v i t y i n f r e s h t i s s u e ) e x p t . 3 2 2 0 POR exp t . 2 ( n o a c t i v i t y i n f r e s h t i s s u e ) e x p t . 3 250 PAL e x p t . 3 ( n o a c t i v i t y i n f r e s h t i s s u e ) c i n n a m a t e hyd r o x y 1 a s e e x p t . 1 220 e x p t . 3 3 5 0 0 0 - m e t h y 1 t r a n s f e r a s e e x p t . 4 2 4 0 a 5 0 0 - 6 0 0 % i n c r e a s e on a g e i n g a n d t h i s m i g h t be a r e f l e c t i o n o f d i f f e r e n c e s b e t w e e n p o t a t o v a r i e t i e s o r c o n d i t i o n s o f a g e i n g . G a m b o r g [ 1 9 6 6 ] was n o t a b l e t o d e t e c t POR i n t i s s u e c u l t u r e f r o m p o t a t o t u b e r , a l t h o u g h SOR was p r e s e n t . P e r h a p s 85 h i g h l e v e l s o f t h e f i r s t e n z y m e a r e i n v o l v e d i n r e s p o n s e s t o w o u n d i n g . PAL i s t h e o n l y e n z y m e o f t h o s e s t u d i e d p r e s e n t c o n s i s t e n t l y i n o n l y t r a c e a m o u n t s i n f r e s h p o t a t o . I t w e l l m i g h t be t h e e n z y m e l i m i t i n g p h e n o l i c b i o s y n t h e s i s i n d o r m a n t t i s s u e . C i n n a m i c h y d r o x y l a s e a n d 0 - m e t h y l t r a n s f e r a s e a r e known t o i n c r e a s e i n t h e l i g h t i n p a r s l e y c e l l c u l t u r e s [ H a l h b r o c k e_t a l_ . 1 9 7 1 ] . C i n n a m a t e h y d r o x y l a s e b e h a v e s l i k e P A L , t h e l e v e l s p e a k i n g 18 h o u r s a f t e r t h e s t a r t o f i l l u m i -n a t i o n , w h i l e 0 - m e t h y l t r a n s f e r a s e l e v e l s c o n t i n u e t o i n c r e a s e i n a c t i v i t y e v e n a f t e r 24 h o u r s . A l t h o u g h no h y d r o x y l a t i on o f p _ - c o u m a r i c a c i d t o c a f f e i c a c i d c o u l d be d e t e c t e d i n e x t r a c t s f r o m a g e d d i s k s , t h i s r e a c t i o n was c a r r i e d o u t b y e x t r a c t s f r o m t h e p e e l o f ' K e n n e b e c ' p o t a t o e s [ P a t i l a n d Z u c k e r 1 9 6 5 ] . T h e p o t a t o d i s k s u s e d i n t h e p r e s e n t s t u d y w e r e f r e e o f p e e l . P e r h a p s t h e m o n o p h e n o l o x i d a s e a c t i v i t y i s r e s t r i c t e d t o t h e o u t e r l a y e r s o f t h e t u b e r . C h l o r o g e n i c a c i d i s t h e q u i n i c a c i d e s t e r o f c a f f e i c a c i d , a n d t h i s c o m p o u n d a c c u m u l a t e s e v e n i n t h e s t e l a r r e g i o n o f p o t a t o u p o n a g e i n g o f s l i c e s [ L e v y a n d Z u c k e r I 9 6 0 ] . T h e m a j o r r o u t e o f f o r m a t i o n o f t h i s c o m p o u n d i s v i a t h e q u i n i c a c i d e s t e r s o f c i n n a m i c a c i d a n d p _ - c o u m a r i c a c i d . I t 86 w e l l may be t h a t t h e h y d r o x y 1 a t i o n o c c u r s o n l y when t h e p _ - c o u m a r i c a c i d i s p r e s e n t as t h e q u i n i c a c i d e s t e r . A l t h o u g h no m o n o p h e n o l o x i d a s e was d e t e c t a b l e i n t h e p o t a t o d i s c s , e a r l i e r w o r k h a s s h o w n t h a t t h e o - d i p h e n o l o x i d a s e s o f p o t a t o w e r e p r e s e n t a n d i n c r e a s e d 3 - f o l d on a g e i n g [ R u i s 1 9 7 2 ; S a c h e r e t a l _ . 1 9 7 2 ] . T h e d i s t r i b u t i o n o f t h e s o l u b l e , m i c r o s o m a l and m i c r o b o d y f o r m s o f t h e e n z y m e d i d n o t c h a n g e on a g e i n g o f t h e t i s s u e [ R u i s 1 9 7 2 ] . T h e d i s t r i b u t i o n o f c i n n a m a t e h y d r o x y l a s e d o e s u n d e r g o a c h a n g e u p o n a g e i n g o f p o t a t o i n t h e l i g h t . I n d o r m a n t t i s s u e , t h e e n z y m e i s l a r g e l y s o l u b l e ; i n a g e d t i s s u e i t i s l a r g e l y p a r t i c u l a t e . T h e same p h e n o m e n o n m i g h t o c c u r w i t h P A L , f o r i n d i c a t i o n s a r e t h a t t h e e n z y m e i s s o l u b l e i n d o r m a n t t i s s u e a n d a t l e a s t p a r t l y p a r t i c u l a t e i n a g e d t i s s u e . H o w e v e r , t h e r e i s s o l i t t l e e n z y m e i n t h e f r e s h t i s s u e t h a t i t i s d i f f i c u l t t o t e l l . S u c h c h a n g e s o f f o r m a r e n o t u n k n o w n i n o t h e r t i s s u e s . I n r h e s u s m o n k e y l i v e r , a m i t o c h o n d r i a l t y r o s i n e t r a n s a m i n a s e was d e t e c t a b l e i n e a r l y f e t a l l i f e , w h i l e a s o l u b l e f o r m r e a c h e d s i g n i f i c a n t a c t i v i t y j u s t b e f o r e b i r t h . A c t i v i t y o f b o t h f o r m s i n c r e a s e d a t b i r t h [ K o l e r e_t ^1_. 1 9 6 9 ] , I n w h e a t l e a v e s , t h e t y p i c a l c a t a l a s e - c o n t a i n i n g m i c r o b o d i e s a r e n o t f o r m e d u n t i l t h e f o u r t h d a y a f t e r g e r m i n a t i o n [ F e i e r a b e n d a n d B e e v e r s 1 9 7 2 ] . B e f o r e t h i s t i m e , t h e e n z y m e 87 i s a s s o c i a t e d w i t h p a r t i c l e s o f l o w s p e c i f i c g r a v i t y . T h e s e a r e b e l i e v e d t o be c o m p o n e n t s o f t h e m i c r o b o d i e s w h i c h d e v e l o p l a t e r . W h i l e i n d o r m a n t p o t a t o c a t a l a s e i s f o u n d i n a f r a c t i o n w i t h t h e s p e c i f i c g r a v i t y t y p i c a l o f m i c r o b o d i e s , a s e r i e s o f c h a n g e s a n a l o g o u s t o t h o s e t a k i n g p l a c e i n g e r -m i n a t i o n m i g h t t a k e p l a c e on a g e i n g o f p o t a t o . T h e s o l u b l e c i n n a m a t e h y d r o x y l a s e may be c h a r a c t e r i s t i c o f d o r m a n t t i s s u e ; t h e p a r t i c u l a t e f o r m m i g h t be c h a r a c t e r i s t i c o f t h e a c t i v e l y m e t a b o l i z i n g t i s s u e . F o r m a t i o n o f new c i n n a m a t e h y d r o x y l a s e c o u l d t a k e p l a c e i n e i t h e r o f two w a y s . T h e p r o d u c t i o n o f b o t h f o r m s c o u l d i n c r e a s e , a l t h o u g h t o a h i g h e r e x t e n t i n t h e c a s e o f t h e p a r t i c u l a t e f o r m . T h i s w o u l d be s i m i l a r t o t h e c a s e i n m o n k e y t i s s u e . A l t e r n a t i v e l y , t h e s o l u b l e f o r m m i g h t b e c o m e p a r t i c u l a t e as c h a n g e s i n t h e i n t r a c e l l u l a r o r g a n i z a t i o n o c c u r , i n a m a n n e r s i m i l a r t o t h e b e h a v i o u r o f c a t a l a s e i n w h e a t l e a v e s . I t i s known t h a t g e n e r a l m e m b r a n e s t r u c t u r e c h a n g e s as p o t a t o s l i c e s a g e [ C a s t e l f r a n c o e_t aJU 1 9 7 0 ] , T h e s e c h a n g e s m i g h t i n c l u d e a g g r e g a t i o n o r o r g a n i z a t i o n o f c i n n a m a t e h y d r o x y l a s e m o l e c u l e s i n t o p a r t i c l e s . 88 T y r o s i n e M e t a b o l i s m b y A g e d P o t a t o T i s s u e I n R i c i n u s , t h e p r o p l a s t i d s a n d g l y o x y s o m e s d e a m i -n a t e t y r o s i n e t o p _ - h y d r o x y p h e n y 1 p y r u v i c a c i d ( P H P P ) a n d t h e n c e t o D _ - h y d r o x y p h e n y 1 a c e t i c a c i d ( P H P A ) [ R u i s a n d K i n d l 1 9 7 1 ] . I n p o t a t o , t h e f i r s t r e a c t i o n seems t o be m a i n l y m i c r o s o m a l , a l t h o u g h some c o n v e r s i o n t a k e s p l a c e i n t h e 1 0 , 0 0 0 x £ p e l l e t a l s o . T h i s p e l l e t i n c l u d e s t h e p o t a t o m i c r o -b o d y , as w e l l as o t h e r o r g a n e l l e s , a n d i n t h i s f r a c t i o n t h e PHPP l o s e s a c a r b o n a t o m t o b e c o m e P H P A . S i n c e t h i s r e a c t i o n i s g l y o x y s o m a l i n R i c i n u s , i t w o u l d seem l i k e l y t h a t i n p o t a t o i t w o u l d o c c u r i n t h e m i c r o b o d i e s . T h i s i s n o t c e r t a i n , h o w e v e r , f o r e x t r a p o l a t i o n s f r o m o n e p l a n t t o a n o t h e r a r e n o t a l w a y s v a l i d . F o r e x a m p l e , c i n n a m a t e h y d r o -x y l a s e a n d PAL h a v e b e e n s h o w n i n t h e p r e s e n t s t u d y t o be p r e s e n t i n t h e p e r o x i s o m a l f r a c t i o n o f s p i n a c h b u t n o t i n t h e m i c r o b o d y f r a c t i o n o f p o t a t o . G e n e r a l C o n c l u s i o n s To t h i s t i m e , w o r k h a s b e e n d o n e on t h e i n c r e a s e i n p h e n y l p r o p a n o i d d e r i v a t i v e s i n p o t a t o on a g e i n g , b u t t h e p r e s e n t s t u d y p r e s e n t s t h e m o s t c o h e r e n t p i c t u r e t o d a t e o f t h e i n t r a c e l l u l a r c h a n g e s i n v o l v e d i n t h i s i n c r e a s e . F i g u r e s 16 a n d 17 s u m m a r i z e t h e s e c h a n g e s . DORMANT POTATO 89 5 -DEHYDROSHIK IMIC A C I D Shi OR j l o w leve ls SHIK IMIC A C I D P R E P H E N I C A C I D p r e OR l o w leve ls p - H Y D R O X Y P H E N Y L -P Y R U V I C A C I D i P H E N Y L A L A N I N E P A L t r a c e s o l u b l e C I N N A M I C A C I D C O l o w l e v e l s m i c r o s o m e so lub le p - C O U M A R I C ACID y C A F F E I C A C I D / o-diphenolase low levels s o l u b l e and p a r t i c u la te f o r m s ( R u i s 1972) / s o l u b l e O - m t low l e v e l s F E R U L I C A C I D F i g u r e 1 6 . L o c a l i z a t i o n o f p h e n y l p r o p a n o i d r e a c t i o n s i n d o r m a n t p o t a t o . 90 AGED POTATO 5 -DEHYDROSHIK IMIC ACID shi OR I so lub le p-hydroxy phenyl acet ic acid SHIKIM1C ACID soluble particle* \ J/przOR sol P R E P H E N I C A C I D p-hy d r o x y phenyl-p y r u v i c a c i d t \ Dluble m i c r V ,/ I t y r o s i n e | m i c r o s o m e s q u i n o n e s m i c r o b o d i e s , m i c r o s o m e s o-dipheno'ase s o l u b l e [ P H E N Y L A L A N I N E par t i c le P A L s o l u b l e V U C I N N A M I C A C I D ft \ m i c r o s o m e s C O so lub le J p - C O U M A R I C A C I D C A F F E I C A C I D 'not 'detectable O - m t so lub le F E R U L I C ACID F i g u r e 17. L o c a l i z a t i o n o f p h e n y l p r o p a n o i d r e a c t i o n s i n p o t a t o a g e d i n t h e l i g h t . 91 T h e i n c r e a s e i n SOR n o t e d by o t h e r s [ S a c h e r e t a l . 1 9 7 2 ] h a s b e e n v e r i f i e d . T h i s i m p l i e s t h a t an i n c r e a s e i n c a r b o n f l o w i s p e r m i t t e d t h r o u g h t h e w h o l e s h i k i m i c a c i d p a t h w a y . A h i g h e r f l o w o f c a r b o n t o t y r o s i n e i s a l s o p e r -m i t t e d t h r o u g h t h e i n c r e a s e i n POR d i s c o v e r e d i n t h i s s t u d y . T h e r e h a v e n o t b e e n a n y r e p o r t s o f o r g a n i s m s i n w h i c h t h e s e t w o e n z y m e s a r e u n d e r c o - o r d i n a t e c o n t r o l , s o i t seems l i k e l y t h a t i n p o t a t o a l s o t h i s w o u l d p r o v i d e a m e c h a n i s m f o r c h a n n e l i n g o f c a r b o n i n t o p h e n y l a l a n i n e o r t y r o s i n e . W h i l e p h e n y l a l a n i n e i s d e a m i n a t e d b y PAL t o c i n -n a m i c a c i d i n p o t a t o , t y r o s i n e i s n o t c o n v e r t e d t o £ - c o u m a r i c a c i d b y T A L . I n s t e a d , t y r o s i n e i s c o n v e r t e d t o p _ - h y d r o x y -p h e n y l p y r u v i c a c i d a n d p _ - h y d r o x y p h e n y 1 a c e t i c a c i d . E v e n b e f o r e a g e i n g , t h e r e i s a s i g n i f i c a n t l e v e l o f c i n n a m a t e h y d r o x y l a s e a c t i v i t y , a n d a f t e r a g e i n g , t h e r e i s p r o b a b l y a h i g h e n o u g h l e v e l t o s u p p l y a n y £ - c o u m a r i c a c i d n e e d e d b y t h e p l a n t . T h e s i g n i f i c a n c e o f t h e p a r t i c u l a t e a n d s o l u b l e f o r m s o f PAL a n d c i n n a m a t e h y d r o x y l a s e i s u n k n o w n . T h e r e a r e two i s o e n z y m e s o f PAL i n o a k [ B o u d e t e_t aj_. 1 9 7 1 ] , o n e o f w h i c h i s s u b j e c t t o i n h i b i t i o n b y p h e n y l p r o p a n o i d d e r i v a -t i v e s , t h e o t h e r b y b e n z o i c a c i d d e r i v a t i v e s . The t w o t y r o s i n e t r a n s a m i n a s e s , s o l u b l e a n d m i t o c h o n d r i a l , i n m o n k e y t i s s u e a r e a l s o s u b j e c t t o d i f f e r e n t c o n t r o l m e c h a n i s m s 92 [ K o l e r e_t a_l_. 1 9 6 9 ] . A s i m i l a r s i t u a t i o n m i g h t be i n o p e r a -t i o n i n t h e c a s e o f p o t a t o . I n a n y e v e n t , t h e n a t u r e o f t h e d i f f e r e n t e n z y m i c f o r m s o f PAL a n d c i n n a m a t e h y d r o x y l a s e , w h e t h e r a g g r e g a t e s o r i s o e n z y m e s , w h e t h e r p r e s e n t i n o r g a n e l l e s o r m e r e l y c o n t a m i n a t i o n , r e m a i n s t o be s e e n . T h e a b s e n c e o f a p _ - c o u m a r a t e h y d r o x y l a s e b u t t h e p r e s e n c e o f an a c t i v e 0 - m e t h y l t r a n s f e r a s e o f c a f f e i c a c i d , i s a p u z z l e . As d i s c u s s e d b e f o r e , t h e h y d r o x y l a t i on o f p_-c o u m a r y 1 - q u i n a t e a p p a r e n t l y d o e s o c c u r i n p o t a t o t i s s u e . An i n c r e a s e i n t h e e n z y m e m e t h y l a t i n g c a f f e i c a c i d d o e s n o t n e c e s s a r i l y i m p l y an i n c r e a s e i n t h a t s u b s t r a t e . T h i s ( o r t h e s e ) e n z y m e s ( s ) m i g h t h a v e v e r y b r o a d s p e c i f i t i e s , a n d an e s t e r o f c a f f e i c a c i d m i g h t be t h e s u b s t r a t e f o r t h e e n z y m e 1_n v i v o . T h e p o t a t o i s an e x c e l l e n t t i s s u e f o r t h e i n v e s -t i g a t i o n o f i n t r a c e l l u l a r c o n t r o l o f p h e n y l p r o p a n o i d m e t a b o l i s m . T h e r e l a t i v e l y d r a m a t i c s t e p - u p i n p r o d u c t i o n o f p h e n y l p r o p a n o i d d e r i v a t i v e s u n d e r r e p r o d u c i b l e c o n d i t i o n s means t h a t m o d e l s o f c a r b o n f l o w a n d b e h a v i o u r c a n be a s s e m b l e d . The p r e s e n t s t u d y i s a f i r s t s t e p i n t h a t d i r e c t i o n . REFERENCES A b e r , H . 1 9 7 0 . K a t a l a s e i_n M e t h o d e n d e r e n z y m a t i s c h e A n a l y s e . E d . H . V . B e r g m e y e r , V e r l a g C h e m i e . A m r h e i n , N . a n d M . H . Z e n k . 1 9 7 0 . C o n c o m i t a n t i n d u c t i o n o f p h e n y l a l a n i n e a m m o n i a - l y a s e a n d c i n n a m i c a c i d 4 - h y d r o x y l a s e d u r i n g i l l u m i n a t i o n o f e x c i s e d b u c k w h e a t h y p o c o l y t s . N a t u r w i s s . , 6^ : 3 1 2 - 3 1 3 . . 1 9 7 1 . U n t e r s u c h u n g e n z u r R o l l e du P h e n y l a l a n i n A m m o n i u m - L y a s e ( P A L ) b e i d e r R e g u l a t i o n d e r F I a v o n o i d s y n t h e s e i n B u c h w e i z e n ( F a g o p y r u m e s c u l e n t u m M o e n c h ) . Z . P f 1 a n z e n p h y s i o 1 . , 6 4 : 1 4 5 - 1 6 8 . A r n o w , L . E . 1 9 3 7 . C o l o r i m e t r i c d e t e r m i n a t i o n o f t h e c o m -p o n e n t s o f 3 , 4 - d i h y d r o p h e n y l a l a n i n e - t y r o s i n e m i s t u r e s . J . B i o l . C h e m . , 1 1 8 : 5 3 1 - 5 3 7 . B a l i n s k y , K. a n d D . D . D a v i e s . 1 9 6 1 . A r o m a t i c b i o s y n t h e s i s i n h i g h e r p l a n t s . 1 . P r e p a r a t i o n a n d p r o p e r t i e s o f d e h y d r o s h i k i m i c r e d u c t a s e . B i o c h e m . J . , 8 0 : 2 9 2 - 2 9 6 . B a s m a d j i a n , G . P . a n d A . G . P a u l . 1 9 7 1 . T h e i s o l a t i o n o f an 0 - m e t h y l t r a n s f e r a s e f r o m p e y o t e a n d i t s r o l e i n t h e b i o s y n t h e s i s o f m e s c a l i n e . L l o y d i a , 3 4 : 9 1 - 9 3 . B e e v e r s , H . 1 9 6 9 . G l y o x y s o m e s o f c a s t o r b e a n e n d o s p e r m a n d t h e i r r e l a t i o n t o g l u c o n e o g e n e s i s . A n n . N . Y . A c a d . S c i . , 1 6 8 : 3 1 3 - 3 2 4 . 1 9 7 0 . P e r o x i s o m e s a n d r e l a t e d o r g a n e l l e s . 8 t h I n t . C o n g r . B i o c h e m . A b s t r . , 2 7 5 - 2 7 6 . 9 3 94 B o u d e t , A . , R. R a n j e v a a n d P . G a d a l . 1 9 7 1 . P r o p r i e t y a l 1 o s t e > i g u e s s p £ c i f i q u e s d e s d e u x i s o e n z y m e s de l a p h e n y l a l a n i n e a m m o n i a q u e l y a s e c h e z Q u e r c u s  p e d u n c u 1 a t a . P h y t o c h e m i s t r y , 10 : 9 9 7 - 1 0 0 6 . B r a m h a l l , S . , N . N o a c k , M . Wu a n d J . R. L o e w e n b e r g . 1 9 6 9 . A s i m p l e c o l o r i m e t r i c m e t h o d f o r d e t e r m i n a t i o n o f p r o t e i n . A n a l y t i c a l B i o c h e m i s t r y , 3 1 : 1 4 6 - 1 4 8 . B r e i d e n b a c h , R . W . , A . K a h n , a n d H . B e e v e r s . 1 9 6 8 . C h a r a c -t e r i z a t i o n o f g l y o x y s o m e s f r o m c a s t o r b e a n e n d o -s p e r m . P l a n t P h y s i o l . , 4 3 : 7 0 5 - 7 1 3 . C a s t e ! f r a n c o , P . A . , W . - J . T a n g , a n d M . L . B o l a r . 1 9 7 1 . M e m b r a n e t r a n s f o r m a t i o n s i n a g e i n g p o t a t o t u b e r s l i c e s . P l a n t P h y s i o l . , 4 8 : 7 9 5 - 8 0 0 . C h a y k i n , S . 1 9 6 6 . B i o c h e m i s t r y l a b o r a t o r y t e c h n i q u e s . J o h n W i 1 e y a n d S o n s . C o c k b u r n . W. , D . A . W a l k e r a n d C . W . B a l d r y . 1 9 6 8 . T h e i s o -l a t i o n o f s p i n a c h c h l o r o p l a s t s i n p y r o p h o s p h a t e m e d i a . P l a n t P h y s i o l . , 43_: 141 5 - 1 4 1 8 . D a v i e s , D . D . , E . U g o c h u k w u , K . P a t i l a n d G . H . N . T o w e r s . 1 9 7 2 . I n p r e p a r a t i o n . E b e l , J . , K . H a h l b r o c k a n d H . G r i s e b a c h . 1 9 7 2 . P u r i f i c a t i o n a n d p r o p e r t i e s o f an o ^ - d i h y d r i c p h e n o l m e t a - o -m e t h y l t r a n s f e r a s e f r o m c e l l s u s p e n s i o n c u l t u r e s o f p a r s l e y a n d i t s r e l a t i o n t o f l a v o n o i d b i o s y n -t h e s i s . B i o c h i m . B i o p h y s . A c t a , 269 : 3 1 3 - 3 2 6 . E l l i s , B . E . 1 9 6 9 . P h e n o l i c m e t a b o l i s m i n h i g h e r p l a n t s . P h . D . T h e s i s , 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 . E n g e l s m a , G . 1 9 6 7 . P h o t o i n d u c t i o n o f p h e n y l a l a n i n e d e a m i n a s e i n g h e r k i n s e e d l i n g s . I . E f f e c t o f b l u e l i g h t . P l a n t a , 7 ^ : 2 0 7 - 2 1 9 . . 1 9 6 7 a . P h o t o i n d u c t i o n o f p h e n y l a l a n i n e d e a m i n a s e i n g h e r k i n s e e d l i n g s . I I . E f f e c t o f r e d a n d f a r - r e d l i g h t . P l a n t a , 7 7 : 4 9 - 5 7 . 95 E n g e l s m a , G . 1 9 6 8 . P h o t o i n d u c t i o n o f p h e n y l a l a n i n e d e a m i n a s e i n g h e r k i n s e e d l i n g s . I I I . E f f e c t s o f e x c i s i o n a n d i r r a d i a t i o n on e n z y m e d e v e l o p m e n t i n h y p o c o t y l s e g m e n t s . P l a n t a , 8 2 : 3 5 5 - 3 6 8 . —. 1 9 6 9 . L o w - t e m p e r a t u r e d e p e n d e n t d e v e l o p m e n t o f p h e n y l a l a n i n e a m m o n i a - l y a s e i n g h e r k i n h y p o c o t y l s . N a t u r w i s s . , 5 6 : 5 6 3 . F e i e r a b e n d , J . a n d H . B e e v e r s . 1 9 7 2 . D e v e l o p m e n t a l s t u d i e s on m i c r o b o d i e s i n w h e a t l e a v e s . I I . O n t o g e n y o f p a r t i c u l a t e e n z y m e a s s o c i a t i o n s . P l a n t P h y s i o l . , 4 9 : 3 3 - 3 9 . F r e d e r i c k , S . E . a n d E . H . N e w c o m b . 1 9 6 9 . A m i c r o b o d y - 1 i k e o r g a n e l l e i n l e a f c e l l s . S c i e n c e , 1 6 3 : 1 3 5 3 - 1 3 5 5 . G a m b o r g , 0 . 1 9 6 6 . A r o m a t i c m e t a b o l i s m i n p l a n t s . I . A s t u d y o f t h e p r e p h e n a t e d e h y d r o g e n a s e f r o m b e a n p l a n t s . B i o c h i m . B i o p h y s . A c t a , 1 1 5 : 6 5 - 7 2 . . 1 9 6 6 a . A r o m a t i c m e t a b o l i s m i n p l a n t s . I I . E n z y m e s o f t h e s h i k i m a t e p a t h w a y i n s u s p e n s i o n c u l t u r e s o f p l a n t c e l l s . C a n . J . B i o c h e m . , 4 4 : 7 9 1 - 7 9 9 . G a m b o r g , 0 . a n d F . J . S i m p s o n . 1 9 6 4 . P r e p a r a t i o n o f p r e p h e n i c a c i d a n d i t s c o n v e r s i o n t o p h e n y l a l a n i n e a n d t y r o s i n e by p l a n t e n z y m e s . C a n . J . B i o c h e m . , 4 2 : 5 8 3 - 5 9 1 . G e r h a r d t , B . P . a n d H . B e e v e r s . 1 9 7 0 . D e v e l o p m e n t a l s t u d i e s on g l y o x y s o m e s i n R i c i nus e n d o s p e r m . J . C e l l B i o l . , 4 4 : 9 4 - 1 0 2 . G n a n a m , A . , A . T . J a g e n d o r f a n d M . - L . R a n a l l e t t i . 1 9 6 9 . C h l o r o p l a s t s a n d b a c t e r i a l a m i n o a c i d i n c o r p o r a -t i o n : a f u r t h e r c o m m e n t . B i o c h i m . B i o p h y s . A c t a , 186 : 2 0 5 - 2 1 3 . G r i f f i t h s , W . T . , D . R . T h r e l f a l l a n d T . W . G o o d w i n . 1 9 6 7 . N a t u r e , i n t r a c e l l u l a r d i s t r i b u t i o n a n d f o r m a t i o n o f t e r p e n o i d q u i n o n e s i n m a i z e a n d b a r l e y s h o o t s . B i o c h e m . 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A n d e r u n g e n v o n E n z y m a k t i v i t a t e n w a h r e n d d e s W a c h s t u m s v o n Z e l l s u s p e n s i o n s k u l t u r e n v o n G l y c i n e m a x : P h e n y l a -l a n i n e Ammoni u m - L y a s e und p _ - C o u m a r a t : C o A L i g a s e . P l a n t a , 9 9 : 3 1 1 - 3 1 8 . H a n s o n , K . R . a n d M. Z u c k e r . 1 9 6 3 . T h e b i o s y n t h e s i s o f c h l o r o g e n i c a c i d a n d r e l a t e d c o n j u g a t e s o f t h e h y d r o x y c i n n a m i c a c i d s . C h r o m a t o g r a p h i c s e p a r a t i o n a n d c h a r a c t e r i z a t i o n . J . B i o l . C h e m . , 2 3 8 : 1 1 0 5 -1 1 1 5 . H a t t o r i , S . a n d M. S a t o . 1 9 6 3 . T h e o x i d a t i o n o f a r b u t i n b y i s o l a t e d c h l o r o p l a s t s o f a r b u t i n - c o n t a i n i n g p l a n t s . P h y t o c h e m i s t r y , 2 ^ : 3 8 5 - 3 9 5 . H a v i r , E . A . a n d K . R . H a n s o n . 1 9 6 8 . L - P h e n y l a l a n i n e a m m o n i a -l y a s e . I . P u r i f i c a t i o n a n d m o l e c u l a r s i z e o f t h e e n z y m e f r o m p o t a t o t u b e r s . B i o c h e m i s t r y , 7 : 1 8 9 6 - 1 9 0 3 . H e b e r , U . , N . G . P o n a n d M. H e b e r . 1 9 6 3 . L o c a l i z a t i o n o f c a r b o x y d i s m u t a s e a n d t r i o s p h o s p h a t e d e h y d r o g e n a s e s i n c h l o r o p l a s t s . P l a n t P h y s i o l . , 3 8 ; 3 5 5 - 3 6 0 . H u a u n g , A . H . C . a n d H . B e e v e r s . 1 9 7 1 . I s o l a t i o n o f m i c r o -b o d i e s f r o m p l a n t t i s s u e s . P l a n t P h y s i o l . , 4 8 : 6 3 7 - 6 4 1 . 97 I m a s e k i , H . , M . U c h i y a m a a n d I . U r i t a n i . 1 9 6 8 . E f f e c t o f e t h y l e n e on t h e i n d u c t i v e i n c r e a s e i n m e t a b o l i c a c t i v i t i e s i n s l i c e d s w e e t p o t a t o r o o t s . A g r . B i o l . C h e m . , 3 2 : 3 8 7 - 3 8 7 . 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H o p p e - S e y l e r ' s Z . P h y s i o l . C h e m . , 3 5 2 : 1 3 5 - 3 1 6 . P a r i s h , R . W . 1 9 7 1 . T h e i s o l a t i o n o f p e r o x i s o m e s , m i t o c h o n -d r i a a n d c h l o r o p l a s t s f r o m l e a v e s o f s p i n a c h b e e t ( B e t a v u l g a r i s L . s s p . v u l g a r i s ) . E u r . J . B i o c h e m 2 2 ^ : 4 2 3 - 4 2 9 . . 1 9 7 2 . T h e i n t r a c e l l u l a r l o c a t i o n o f p h e n o l o x i d a s e s a n d p e r o x i d a s e i n s t e m s o f s p i n a c h b e e t ( B e t a v u l g a r i s L . ) . Z . P f 1 a z e n p h y s i o l . , 6 6 : 1 7 6 -1 8 8 . P a t i l , S . S . a n d Z u c k e r , M . 1 9 6 5 . P o t a t o p h e n o l a s e s . P u r -i f i c a t i o n a n d p r o p e r t i e s . J . B i o l . C h e m . , 2 4 0 : 3 9 3 8 . P h a r m a c i a F i n e C h e m i c a l s . 1 9 6 6 . S e p h a d e x - g e l f i l t r a t i o n i n t h e o r y a n d p r a c t i c e . P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a , S w e d e n . 99 R a c k e r , E . 1 9 5 0 . S p e c t r o p h o t o m e t r i c m e a s u r e m e n t s o f t h e e n z y m a t i c f o r m a t i o n o f f u m a r i c a n d c i s ~ a c o n i t i c a c i d s . B i o c h i m . B i o p h y s . A c t a , 4 _ : 2 1 1 - 2 1 4 . R a n a l l e t t i , M . , A . Gnanam a n d A . T . J a g e n d o r f . 1 9 6 9 . A m i n o a c i d i n c o r p o r a t i o n b y i s o l a t e d c h l o r o p l a s t s . B i o c h i m . B i o p h y s . A c t a , 1 8 6 : 1 9 2 - 2 0 4 . R o g e r s , L . J . , S . P . J . S h a h a n d T . W . G o o d w i n . 1 9 6 8 . C o m p a r t -m e n t a t i o n o f b i o s y n t h e s i s o f t e r p e n o i d s i n g r e e n p l a n t s . P h o t o s y n t h e t i c a , 2 ^ : 1 8 4 - 2 0 7 . R u i s , H . 1 9 7 1 . I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f p e r o x i -somes f r o m p o t a t o t u b e r s . H o p p e - S e y l e r ' s Z . P h y s i o l . C h e m . , 3 5 2 : 1 1 0 5 - 1 1 1 2 . . 1 9 7 2 . P a r t i c u l a t e a n d s o l u b l e f o r m s o f £ - d i p h e n o l o x i d a s e f r o m p o t a t o t u b e r s . P h y t o c h e m i s t r y , 1 1 : 5 3 - 5 8 . R u i s , H . a n d H . K i n d l . 1 9 7 0 . D i s t r i b u t i o n o f a m m o n i a - l y a s e s i n o r g a n e l l e s o f c a s t o r b e a n e n d o s p e r m . H o p e -S e y l e r ' s Z . P h y s i o l . C h e m . , 3 5 1 : 1 4 2 5 - 1 4 2 7 . . 1 9 7 1 . F o r m a t i o n o f a . ^ - u n s a t u r a t e d c a r b o x y l i c a c i d s f r o m a m i n o a c i d s i n p l a n t p e r o x i s o m e s . P h y t o c h e m i s t r y , 1 0 : 2 6 2 7 - 2 6 3 1 . R u s s e l , D . W . 1 9 7 1 . T h e m e t a b o l i s m o f a r o m a t i c c o m p o u n d s i n h i g h e r p l a n t s X . P r o p e r t i e s o f t h e c i n n a m i c a c i d 4 - h y d r o x y l a s e a n d some a s p e c t s o f i t s m e t a -b o l i c a n d d e v e l o p m e n t a l c o n t r o l . J . B i o l . C h e m . , 2 4 6 : 3 8 7 0 . R u s s e l , D . W . a n d E . E . C o n n . 1 9 6 7 . T h e c i n n a m i c a c i d 4 -h y d r o x y l a s e o f p e a s e e d l i n g s . A r c h . B i o c h e m . B i o p h y s . , 1 2 2 : 2 5 6 - 2 5 8 . S a c h e r , J . A . , G . H . N . T o w e r s , a n d D . D . D a v i e s . 1 9 7 2 . E f f e c t o f l i g h t a n d a g e i n g on e n z y m e s , p a r t i c u l a r l y p h e n y l a l a n i n e a m m o n i a l y a s e , i n d i s c s o f s t o r a g e t i s s u e . P h y t o c h e m i s t r y , l j _ : i n p r e s s . 100 Sat6, M . 1 9 6 6 . M e t a b o l i s m o f p h e n o l i c s u b s t a n c e s by t h e c h l o r o p l a s t s . I I . C o n v e r s i o n by t h e i s o l a t e d c h l o r o p l a s t s o f £ - c o u m a r i c a c i d t o c a f f e i c a c i d . P h y t o c h e m i s t r y , 5 _ : 3 8 5 - 3 8 9 . 1 9 6 7 . M e t a b o l i s m o f p h e n o l i c s u b s t a n c e s by t h e c h l o r o p l a s t s . I I I . P h e n o l a s e as an e n z y m e c o n c e r n -i n g t h e f o r m a t i o n o f e s c u l e t i n . P h y t o c h e m i s t r y , 6 : 1 3 6 3 - 1 3 7 3 . S c h e r f , H . a n d M . H . Z e n k . 1 9 6 7 . D e r E i n f l u 3 d e s L i c h t e s a u f d i e F I a v o n o i d s y n t h e s e u n d d i e E n z y m i n d u k t i o n b e i F a g o p y r u m e s c u l e n t u m M o e n c h . Z . P f 1 a n z e n p h y s i o l . , 5 7 : 4 0 1 - 4 1 8 . S c h o p f e r , P . 1 9 7 1 . D i e P h e n y l a l a n i n a m m o n i u m l y a s e ( P A L , E . C . 4 . 3 . 1 . 5 ) des S e n f k e i m l i n g s (S i n a p i s a l b a ) , e i n e l e k r o p h o r e t i s c h e e i n h e i t l i c h e s E n z y m . P l a n t a , 1 0 1 : 3 3 9 - 3 4 6 . S t a f f o r d , H . A . 1 9 6 9 . C h a n g e s - i n p h e n o l i c c o m p o u n d s a n d r e l a t e d e n z y m e s i n y o u n g p l a n t s o f S o r g h u m . P h y t o c h e m i s t r y , 8 : 7 4 3 - 7 5 2 . S t a f f o r d , H . A . a n d S . D r e s l e r . 1 9 7 2 . 4 - H y d r o x y c i n n a m i c a c i d h y d r o x y l a s e a n d p o l y p h e n o l a s e a c t i v i t i e s i n S o r g h u m  v u l g a r e . P l a n t P h y s i o l o g y , 4 9 _ : 5 9 0 - 5 9 5 . T h r e l f a l l , D . R . a n d G . R . W h i s t a n c e . 1 9 6 8 . U t i l i z a t i o n o f D - t y r o s i n e a n d £ h y d r o x y p h e n y l p y r u v a t e f o r t h e b i o s y n t h e s i s o f p i a s t o q u i n o n e a n d r e l a t e d c o m -p o u n d s by m a i z e s h o o t s . B i o c h e m . J . , 1 0 8 : 2 4 - 2 5 . T o l b e r t , N . E . a n d R . K . Y a m a z a k i . 1 9 6 9 . L e a f p e r o x i s o m e s a n d t h e i r r e l a t i o n t o p h o t o r e s p i r a t i on a n d p h o t o -s y n t h e s i s . A n n . N . Y . A c a d . S c i . , 1 6 8 : 3 2 5 - 3 4 1 . T o l b e r t , N . E . , A . O e s e r , T , K i s a k i , R . H . Hageman a n d R . K . Y a m a z a k i . 1 9 6 8 . P e r o x i s o m e s f r o m s p i n a c h l e a v e s c o n t a i n i n g e n z y m e s r e l a t e d t o a l y c o l a t e m e t a b o l i s m . J . B i o l . C h e m . , 243 ; 5 1 7 9 - 5 1 8 4 / T r e l e a s e , R . N . , W. B e c k e r , P . G r u b e r a n d E . H . N e w c o m b . 1 9 7 1 . M i c r o b o d i e s ( g l y o x y s o m e s a n d p e r o x i s o m e s ) i n c u c u m b e r c o t y l e d o n s . C o r r e l a t i v e b i o c h e m i c a l a n d u l t r a s t r u c t u r a l s t u d y i n l i g h t - a n d d a r k - g r o w n s e e d l i n g s . P l a n t P h y s i o l . , 4 8 : 4 6 1 - 4 7 5 . 1 0 1 V e r l e u r , J . D . , H . V a n d e r W i l d e a n d T . S m i n i a . 1 9 7 0 . The h e t e r o g e n e i t y o f t h e m i t o c h o n d r i a l f r a c t i o n i n r e l a t i o n t o t h e i n c r e a s e o f m i t o c h o n d r i a l c o n t e n t i n w o u n d e d a n d d i s e a s e d p o t a t o t u b e r t i s s u e . Z . P f l a n z e n . , 6 2 : 3 5 2 - 3 6 0 . V i g i l , E . L . 1 9 6 9 . I n t r a c e l l u l a r l o c a l i z a t i o n o f c a t a l a s e ( p e r o x i d a t i c ) a c t i v i t y i n p l a n t m i c r o b o d i e s . J . H i s t o c h e m . C y t o c h e m . , 1 7 : 4 2 5 - 4 2 8 . W h i s t a n c e , G . R . a n d D . R . T h r e l f a l l . 1 9 6 7 . B i o s y n t h e s i s o f p h y t o q u i n o n e s : an o u t l i n e o f t h e b i o s y n t h e t i c s e q u e n c e s i n v o l v e d i n t e r p e n o i d q u i n o n e a n d c h r o m a n o l f o r m a t i o n i n h i g h e r p l a n t s . B i o c h e m . B i o p h y s . R e s . C o m m . , 28^: 2 9 5 - 3 0 1 „ . 1 9 6 8 . B i o s y n t h e s i s o f p h y t o q u i n o n e s . B i o s y n t h e t i c o r i g i n s o f t h e n u c l e i a n d s a t e l l i t e m e t h y l g r o u p s o f p 1 a s t o q u i n o n e , t o c o p h e r o l s a n d t o c o p h e r o l q u i n o n e s i n m a i z e s h o o t s , b e a n s h o o t s a n d i v y l e a v e s . B i o c h e m . J . , 1 0 9 : 5 7 7 - 5 9 5 . . 1 9 7 0 . B i o s y n t h e s i s o f p h y t o q u i n o n e s . H o m o g e n t i s i c a c i d : a p r e c u r s o r o f p i a s t o q u i n o n e s , t o c o p h e r o l s a n d a - t o c o p h e r o l -q u i n o n e i n h i g h e r p l a n t s , g r e e n a l g a e a n d b l u e -g r e e n a l g a e . B i o c h e m . J . , 1 1 9 : 5 9 3 - 6 0 0 . Y o s h i d a , S . 1 9 6 9 . B i o s y n t h e s i s a n d c o n v e r s i o n o f a r o m a t i c a m i n o a c i d s i n p l a n t s . A n n . R e v . P l a n t P h y s i o l . , 2 0 : 4 1 - 6 2 . Z u c k e r , M . 1 9 6 3 . T h e i n f l u e n c e o f l i g h t on s y n t h e s i s o f p r o t e i n a n d o f c h l o r o g e n i c a c i d i n p o t a t o t u b e r t i s s u e . P l a n t P h y s i o l . , 3 8 : 3 7 5 - 3 8 0 . . 1 9 6 5 . I n d u c t i o n o f p h e n y l a l a n i n e d e a m i n a s e b y l i g h t a n d i t s r e l a t i o n t o c h l o r o g e n i c a c i d s y n t h e s i s i n p o t a t o t u b e r t i s s u e . P l a n t P h y s i o l o g y , 4 0 : 7 7 9 - 7 8 4 . 

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