UBC Theses and Dissertations

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UBC Theses and Dissertations

Beta carboxylation pathway of photosynthesis Berry, Joseph Andrew 1970

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THE /3-CARBOXYLATION PATHWAY OP PHOTOSYNTHESIS by Joseph Andrew Berry B.Sc, University of California, Davis, 1963 M.Sc, University of California, Davis, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and 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 by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . It 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 j s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Bo t a l l y The U n i v e r s i t y o f B r i t i s h Co lumbia V a n c o u v e r 8, Canada Date -April 14. 1970 l i A b s t r a c t The o c c u r r e n c e o f the ] B - c a r b o x y l a t l o n pathway o f p h o t o -s y n t h e s i s Is c o r r e l a t e d w i t h the p r e s e n c e o f two c h l o r o p h y -l l o u s t i s s u e s , w i t h i n a l e a f , which f u n c t i o n c o - o p e r a t i v e l y . I t i s p o s t u l a t e d t h a t : (a) Carbon d i o x i d e f i x a t i o n by 1 3 -carboxy la t ion i s c o n f i n e d t o the l e a f m e s o p h y l l t i s s u e . (b) The c a r b o x y l t r a n s f e r and the subsequent r e a c t i o n s l e a d i n g t o the s y n t h e s i s o f s t a r c h a r e c o n f i n e d t o the bund le s h e a t h t i s s u e . (c ) The c a r b o x y l t r a n s f e r r e a c t i o n proceeds by d e c a r -b o x y l a t i o n o f a C j^ -ac id c o u p l e d w i t h RuDP c a r b o x y -l a s e c a t a l y z e d s y n t h e s i s o f 3 -PGA. (d) C o o p e r a t i o n between the two c h l o r o p h y l l o u s t i s s u e s i s made p o s s i b l e by the t r a n s p o r t o f m e t a b o l i t e s between the t i s s u e s . Two p l a n t s Gomphrena g l o b o s a and Zea mays were used t o t e s t t h e s e p o s t u l a t e s . These p l a n t s were found t o form l a b e l l e d a s -p a r t a t e and mala te as e a r l y p r o d u c t s o f p h o t o s y n t h e s i s . S t u d i e s Ik o f t h e r e d i s t r i b u t i o n o f C i n p u l s e and chase type f e e d i n g s o f 1^C02 and 1 2 C 0 2 i n d i c a t e t h a t n e a r l y a l l l a b e l l e d p h o s p h o r y -l a t e d compounds may be formed from the C ^ - a c l d s by a c a r b o x y l t r a n s f e r r e a c t i o n . The l o c a t i o n o f some p h o t o s y n t h e t i c enzymes was s t u d i e d by d i f f e r e n t i a l removal o f enzymes f rom the two t i s s u e s . P - e n o l p y r u v a t e c a r b o x y l a s e which i s p r o b a b l y r e s p o n -s i b l e f o r COg f i x a t i o n was found t o o c c u r p r e d o m i n a n t l y i n the m e s o p h y l l c e l l s . " M a l i c " enzyme which w i l l d e c a r b o x y l a t e m a l i c i i i a c i d and RuDP carboxylase were found predominantly i n the c e l l s of the bundle sheath. Microradioautographs of t h i n s e c t i o n s of a Gomphrena globosa l e a f i n d i c a t e t h a t "^C l a b e l l e d C^-acids r a p i d l y appear i n the c e l l s of the bundle sheath. T h i s appar-e n t l y precedes the c a r b o x y l t r a n s f e r s t e p . S t u d i e s of the l o -c a t i o n of l a b e l l e d compounds w i t h i n l e a f c e l l s by nonaqueous f r a c t i o n a t i o n i n d i c a t e t h a t a p o r t i o n of the C^-acids a re p r e s -ent i n the cytoplasm and co u l d be t r a n s p o r t e d by cyto p l a s m i c b r i d g e s . P-enolpyruvate c a r b o x y l a s e , "malic*' enzyme, and RuDP carboxylase a r e present i n e x t r a c t s of these l e a v e s i n a c t i v i t i e s which approach o r exceed the i n v i v o photosyntheti.c r a t e . These data suggest t h a t the r e a c t i o n s of /3 - c a r b o x y l a t i o n p h o t o s y n t h e s i s proceed as p o s t u l a t e d . T h i s mechanism i n v o l v e s COg as a f r e e i n t e r m e d i a t e as w e l l as a s u b s t r a t e . An unusual C0£ e v o l v i n g process was d e t e c t e d a t the onset of ph o t o s y n t h e s i s i n Zea l e a v e s a f t e r a short dark 1^ p e r i o d . Feedings of CO^ smggfcst t h a t the C0 2 evolved i n t h i s b u r s t may be the r e s u l t of a l i g h t s t i m u l a t e d d e c a r b o x y l a t i o n of a C ^ - a c i d . S t u d i e s of the k i n e t i c p r o p e r t i e s of " m a l i c " en-zyme i n d i c a t e t h a t i t would not be a c t i v a t e d by l i g h t induced 2+ changes i n the pH and Mg c o n c e n t r a t i o n s , or i n the adenylate p o o l . i v TABLE OF CONTENTS Page PROLOGUE. • • ... ; x LITERATURE CITED x i x CHAPTER I The p h o t o s y n t h e t i c c a r b o n metabo l ism o f Zea mays and Gomphrena g l o b o s a ; the l o c a t i o n o f CO? f i x a t i o n and the c a r b o x y l t r a n s f e r r e a c t i o n s . . . INTRODUCTION 1 MATERIALS AND METHODS 3 RESULTS AND DISCUSSION 10 SUMMARY 25 REFERENCES 28 CHAPTER II Assay p r o c e d u r e s , k i n e t i c p r o p e r t i e s , and i s o l a t i o n o f "mal ic"enzyme f rom Gomphrena  g l o b o s a and Zea mays. TEXT 32 LITERATURE CITED CHAPTER I I I L o c a t i o n o f P - e n o l p y r u v a t e c a r b o x y l a s e . TEXT 42 LITERATURE CITED 45 APPENDIX I E x t r a c t i o n and a s s a y o f P - e n o l p y r u v a t e c a r b o x y l a s e RADIO CHEMICAL ASSAY 46 SPECTROPHOTOMETRIC ASSAY 47 LITERATURE CITED 49 APPENDIX II Chromatography TEXT 50 LITERATURE CITED 53 V TABLE OP CONTENTS, c o n t ' d Page APPENDIX I I I Nonaqueous 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 TEXT 5k LITERATURE CITED 69 APPENDIX IV B i o c h e m i c a l s , a b b r e v i a t i o n s and s o u r c e s o f s u p p l y 70 EPILOGUE TEXT 72 LITERATURE CITED . . 83 v i L IST OP TABLES Page C h a p t e r I T a b l e I. A c t i v i t i e s o f enzymes i n e x t r a c t s o f m e s o p h y l l and bund le shea th t i s s u e s o f Zea mays and Gomphrena g l o b o s a  T a b l e I I . Compar ison o f the i n v i v o p h o t o s y n -t h e s i s r a t e t o the e x t r a c t i b l e a c t i v i t i e s o f some p h o t o s y n t h e t i c enzymes T a b l e I I I . F r a c t i o n (y) o f l a b e l l e d m a t e r i a l c o n t a i n e d i n the c h l o r o p l a s t s Chapte r II T a b l e I. Summary o f the i s o l a t i o n o f " m a l i c " enzyme C h a p t e r I I I T a b l e I. P - e n o l p y r u v a t e c a r b o x y l a s e a c t i v i t y i n f r a c t i o n s i s o l a t e d by nonaqueous 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 Appendix I I T a b l e I. R p v a l u e s f o r some compounds s e p a r a -t e d by one d i m e n s i o n a l paper chroma-tography Appendix I I I T a b l e I. Data f rom nonaqueous f r a c t i o n a t i o n o f l e a v e s o f wheat and s p i n a c h f e d C0 2 f o r 6 sec T a b l e I I . Data f rom nonaqueous d e n s i t y g r a d i e n t f r a c t i o n a t i o n s o f l e a v e s f e d i n the manner i n d i c a t e d T a b l e I I I . C a l c u l a t i o n o f t h e f r a c t i o n o f l e a f p r o t e i n a s s o c i a t e d w i t h t h e c h l o r o -p l a s t s T a b l e IV. Sample c a l c u l a t i o n o f the f r a c t i o n o f a m e t a b o l i t e l o c a t e d i n the c h l o r o p l a s t s 17 18 21 35 51 56 59 67 68 LIST OP TABLES, c o n t ' d E p i l o g u e The l o c a t i o n o f some p h o t o s y n -t h e t i c enzymes i n l e a v e s o f p l a n t s w i t h the p - c a r b o x y l a t i o n pathway o f p h o t o s y n t h e s i s V l l l LIST OP FIGURES Page P r o l o g u e F i g u r e 1 . The pathway o f c a r b o n metabo l ism i n / 3 - c a r b o x y l a t i o n p h o t o s y n t h e s i s x i C h a p t e r I 14 F i g u r e 1 . The r e d i s t r i b u t i o n o f C between l a b e l l e d C ^ - a c i d s and phosphate e s t e r s - i n l e a v e s d u r i n g p u l s e and chase C 0 2 f e e d i n g s 11 F i g u r e 2 . M i o r o r a d i o a u t o g r a p h o f a t h i n s e c t i o n o f a Gomphrena g l o b o s a l e a f 14 F i g u r e 3» A whole l e a f r a d i © a u t o g r a p h o f a l e a f o f Gomphrena g l o b o s a . 14 C h a p t e r I I F i g u r e 1. The e f f e c t ofpH and mala te c o n c e n -t r a t i o n on the r a t e o f " m a l i c " enzyme c a t a l y z e d r e d u c t i o n o f NADP 37 . 14 F i g u r e 2 . M a l a t e - 4 - C r e m a i n i n g a f t e r i n c u b a t i o n w i t h • m a l i c " enzyme 38 Appendix I I I F i g u r e 1. Nonaqueous 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 o f f r e e z e d r i e d l e a v e s , ^ Gomphrena g l o b o s a . • 65 F i g u r e 2 . Nonaqueous 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 o f f r e e z e d r i e d l e a v e s , Zea mays • 66 E p i l o g u e F i g u r e 1. The d i v i s i o n o f r e a c t i o n s between bundle s h e a t h and m e s o p h y l l c h l o r o p l a s t s 76 ix A c knowledgement s I f e e l a deep g r a t i t u d e t o the many people who have helped with my s t u d i e s and wit h t h i s work. I would e s p e c i a l l y l i k e t o thank Dr. Bruce Tregunna f o r many h e l p f u l and s t i m u l a t i n g d i s c u s s i o n s which l e d t o and helped i n the development of t h i s study, f o r generous f i n a n c i a l support f o r t h i s r e s e a r c h p r o j -e c t , and f o r h e l p i n many stages of i t s execution, e s p e c i a l l y i n p r e p a r a t i o n of the manuscript. I would a l s o l i k e t o thank Dr. G.HvN. Towers, Dr. Bruce Bohm, and Dr. Thana B i s a l p u t r a who pro v i d e d t e c h n i c a l a d v i c e , l e n t me equipment and chemicals, and a l s o helped i n p r e p a r a t i o n of t h i s manuscript. I am g r a t e f u l f o r the a s s i s t a n c e of Dr. John Downton who helped i n the prep-a r a t i o n of microradioautographs, i n measurement of the C0£ b u r s t , and p a r t i c i p a t e d i n many hours of d i s c u s s i o n which were i n v a l u a b l e t o t h i s p r o j e c t . P r e l i m i n a r y communications of data and d i s c u s s i o n s with Drs. OILe B jorkman, R.G. Jensen and D.N. Moss were very h e l p f u l , and a r e g r a t e f u l l y acta|owledged. The author was a r e c i p i e n t of a U n i v e r s i t y of B r i t i s h Columbia 3cb.olarsb .3p In the p e r i o d I 9 6 6 - 6 ? , and of N a t i o n a l Re-search C o u n c i l of Canada s c h o l a r s h i p s i n the p e r i o d s 1 9 6 7 ^ 6 8 , 1 9 6 8 ^ -6 9 and 1 9 6 9 - 7 0 . X P r o l o g u e A new pathway o f p h o t o s y n t h e t i c CO,, f i x a t i o n d i f f e r e n t f rom the normal C a l v i n c y c l e was I n d i c a t e d by the p i o n e e r i n g work o f K o r t s c h a k e t a l . (8) w i t h s u g a r c a n e . Subsequent s t u d -i e s by o t h e r s have shown t h a t many p l a n t s i n a d d i t i o n t o s u g a r -cane have t h i s new pathway. These s t u d i e s a l s o i n d i c a t e t h a t the new pathway c o r r e l a t e s w i t h s e v e r a l o t h e r c h a r a c t e r i s t i c s o f the p l a n t s wh ich p o s s e s s I t . These i n c l u d e : the absence o f a p p a r e n t p h o t o r e s p i r a t i o n ; h i g h e r n e t p r o d u c t i v i t y ; and s e v e r a l o t h e r r e l a t e d p h y s i o l o g i c a l d i f f e r e n c e s . These plant : a l s o have a d i f f e r e n t l e a f anatomy, d i f f e r e n t c h l o r o p l a s t u l t r a s t r u c t u r e , and some have been r e l a t e d t o one a n o t h e r by proposed p h y l o -g e n e t i c r e l a t i o n s h i p s (3.6). C l e a r l y t h i s pathway i s no t s i m p l y a b i o c h e m i c a l c u r i o s i t y . I t forms the c o r e o f what appears t o be an impor tan t v a r i a t i o n on the theme o f p h o t o s y n t h e s i s . 1 W h i l e the b a s i c c a r b o n metabo-l i s m r e p r e s e n t e d by t h i s pathway, termed the J B - c a r b o x y l a t i o n pathway, i s w e l l u n d e r s t o o d , the r e a s o n f o r i t s r e l a t i o n s h i p t o l e a f anatomy, n e t p r o d u c t i v i t y , and the absence o f p h o t o -r e s p i r a t i o n i s n o t . F u r t h e r s t u d i e s o f t h e | 3 - c a r b o x y l a t i o n pathway s h o u l d be v a l u a b l e i n a t t e m p t i n g t o understand%these r e l a t i o n s h i p s . A t the t ime t h i s p r o j e c t began the b a s i c c a r b o n metabo l ism and enzymology o f the pathway had been l a r g e l y ^ w o r k e d out by M.D. Ha tch and C.vR. S l a c k i n A u s t r a l i a . A d e t a i l e d a c c o u n t o f t h e i r work w i l l appear i n C h a p t e r I. However, a t t h i s t ime i t i s impor tan t t o n o t e t h a t t h i s pathway d i f f e r s f rom the normal C a l v i n c y c l e i n t h a t the f i r s t l a b e l l e d p r o d u c t s o f p h o t o s y n -x l P r o l o g u e F i g u r e 1. The pathway of c a r b o n metabo l ism i n B - c a r b o x y l a t i o n p h o t o s y n t h e s i s . P - e n o l p y r u v a t e o x a l o a c e t a t e py ruva te Hatch & S l a c k c y c l e a s p a r t a t e malate 3 - p h o s p h o g l y c e r a t e r ibu lose-1,5-d i p h o s p h a t e C C a l v i n c y c l e ) f r u c t o s e-1,6 d i p h o s p h a t e Y s t a r c h x i i t h e s i s a r e the C ^ - d i c a r b o x y l i c a c i d s ( m a l i c a c i d , a s p a r t i c a c i d , and o x a l o a c e t i c a c i d ) . 3 - p h o s p h o g l y c e r i c a c i d and s u b s e q u e n t l y o t h e r phosphate - fes ters t y p i c a l o f the C a l v i n c y c l e become l a b e l -l e d , but r a d i o c h e m i c a l exper iments i n d i c a t e t h a t t h e s e a r e formed f rom c a r b o n f i r s t f i x e d i n t o the C ^ - a c l d s . The exhange o f c a r b o n f rom the C ^ - a c i d s t o 3 - p h o s p h o g l y c e r i c a c i d r e s u l t s f rom the meshing o f two c y c l i c a l p r o c e s s e s , which o p e r a t e i n sequence , as shown i n F i g u r e 1. One s t e p i n t h i s sequence i s s t i l l not a d e q u a t e l y u n d e r s t o o d . T h i s i s the c a r b o x y l t r a n s f e r which r e -s u l t s i n the meshing o f the two c y c l e s . T h i s c a r b o x y l t r a n s f e r i s the p r i n c i p a l t o p i c t o be d i s * c u s s e d i n t h i s t h e s i s . W h i l e t h i s appears t o be a p u r e l y b i o -c h e m i c a l p r o b l e m , gas exchange p h y s i o l o g y , and l e a f anatomy a r e a l s o impor tan t components o f the r e a s o n i n g which l e a d t o the exper iments p e r f o r m e d . The r e s u l t s o b t a i n e d i n d i c a t e a p o s s i b l e mechanism £ o r the c a r b o x y l t r a n s f e r , and p r o v i d e some i n s i g h t i n t o the r e l a t i o n s h i p o f t h i s pathway t o l e a f anatomy and gas exchange p h y s i o l o g y . The b i o c h e m i c a l r e q u i r e m e n t s o f t h i s c a r b o x y l t r a n s f e r r e -a c t i o n were d e f i n e d i n r a d i o c h e m i c a l exper iments by Ha tch and S l a c k (5)« T h i s i n v o l v e s the t r a n s f e r o f the C ^ - c a r b o x y l group o f a C ^ - a c i d t o the C^ p o s i t i o n o f $ - P G A . There a r e two known b i o c h e m i c a l mechanisms which c o u l d a c c o u n t f o r t h i s t r a n s f e r . (1) The C ^ - a c i d donor may be d e c a r b o x y l a t e d by one enzyme (perhaps " m a l i c " enzyme) and the l i b e r a t e d CO2 used by a n o t h e r enzyme t o c a r b o x y l a t e RuDP f o r m i n g 3 -PGA. Carbon d i o x i d e would p a r t i c i p a t e as a f r e e i n t e r m e d i a t e i n t h i s type o f r e a c t i o n . $2) The d e c a r b o x y l a t i o n o f the c a r b o x y l donor and c a r b o x y l a -x i i i t l o n o f the c a r b o x y l a c c e p t o r might o c c u r on a s i n g l e enzyme. T r a n s c a r b o x y l a s e enzymes o f t h i s t y p e a r e known (7. 14,':) and have been d e t e c t e d i n p l a n t t i s s u e (4). The i n t e r m e d i a t e i n t h i s type o f r e a c t i o n i s a p p a r e n t l y b i o t i n - b o u n d r a t h e r than f r e e CC" . E x p e r i m e n t a l e v i d e n c e (5) a rgues a g a i n s t the p a r t i c i p a -t i o n o f f r e e CO^ as an i n t e r m e d i a t e i n t h i s s tepi , C a r b o n-l4 once f i x e d i n t o the C ^ - a c l d s does no t exchange w i t h CO^ o f the a tmosphere . Thus a t r a n s c a r b o x y l a s e mechanism o f the second t y p e which does not i n v o l v e GO^ as a f r e e i n t e r m e d i a t e seems more l i k e l y . However, e f f o r t s t o f i n d the t r a n s c a r b o x y l a s e have so f a r been u n s u c c e s s f u l . A t r a n s f e r o f c a r b o n f rom a C ^ - a c i d ( m a l i c a c i d ) t o 3-PGA by a mechanism s i m i l a r t o the f i r s t type o c c u r s i n c r a s s u l a c e a n a c i d m e t a b o l i s m . T h i s p r o c e s s , which i s p e c u l i a r t o some s u c -c u l e n t p l a n t s , r e s u l t s i n a d i u r n a l f l u c t u a t i o n i n the a c i d c o n t e n t o f the p h o t o s y n t h e t i c t i s s u e . M a l i c a c i d a p p a r e n t l y accumula tes a t n i g h t as the r e s u l t o f GO^ f i x a t i o n . Under some c o n d i t i o n s n e a r l y a l l o f the CO^ f i x e d by the p l a n t e n t e r s a t n i g h t by t h i s mechanism. D u r i n g the day m a l i c a c i d i s consumed i n p h o t o s y n t h e s i s . T h i s d e a c i d i f i c a t i o n i s p r o b a b l y mediated by " m a l i c " enzyme. The C0 2 produced i n t h i s d e c a r b o x y l a t i o n i s then r e f i x e d by RuDP c a r b o x y l a s e f o r m i n g 3-PGAi, The stomates ot'•,ythese p l a n t s c l o s e d u r i n g t h i s d e a c i d i f i c a t i o n p r o c e s s hence the CO^ accumula ted a t n i g h t does no t e s c a p e . I f the c a r b o x y l t r a n s f e r i n t h e / 3 - c a r b o x y l a t i o n pathway o f p h o t o s y n t h e s i s a l s o o c c u r s i n an i s o l a t e d environment f r e e CO^ might no t be d e t e c t e d a s a n i n t e r m e d i a t e . x i v Work by Downton and Tregunna (. 3 ) showed t h a t p l a n t s which have the ^ - c a r b o x y l a t l o n pathway have a v e r y low C0 2 compensa-t i o n p o i n t . T h i s i n d i c a t e s t h a t v e r y l i t t l e CO2 f rom any s o u r c e i s l o s t f rom the l e a f i n the l i g h t . O ther p l a n t s have a h i g h e r C0 2 compensat ion p o i n t , and a p p a r e n t l y l o s e a g r e a t d e a l of C0 2 d u r i n g p h o t o s y n t h e s i s by a p r o c e s s termed p h o t o r e s p i r a t i o n (1.2). The p l a n t s w i t h /5 - c a r b o x y l a t i o n may l a c k the m e t a b o l i c r e a c t i o n s r e s p o n s i b l e f o r p h o t o r e s p i r a t i o n , o r they may p o s s e s s a mecha-n ism (as r e c y c l i n g ) which masks i t . Such a mechanism c o u l d a l s o Ik preven t exchange of C0 2 between the C ^ - a c i d s and C0 2. The e x -i s t e n c e of such a mechanism c o u l d e x p l a i n bo th the low C0 2 com-p e n s a t i o n p o i n t and p r o v i d e a f e a s i b l e mechanism f o r the t r a n s -f e r of the c a r b o x y l w i thout l o s s of c a r b o n . The C0 2 - m e d i a t e d mechanism c o u l d p r o c e e d by s t e p s f o r which enzymes a r e known. S l a c k and Hatch (11) showed t h a t c o r n , s o r -ghum, and sugarcane c o n t a i n e d " m a l i c " enzyme a% l e v e l s much h i g h e r t h a n i t was found i n wheat and s i l v e r b e e t , c o n t r o l p l a n t s which l a c k ^ 3 - c a r b o x y l a t l o n . The r e a c t i o n c a t a l i z e d by " m a l i c " enzyme i s shown be low: COOH C H 2 CH3 + NADP = C=0 +NADPH + CO? I COOH CHOH I COOH The r e a c t i o n i s r e v e r s i b l e . Walker ( 13 ) a rgued t h a t t h i s e n -zyme p r o b a b l y f u n c t i o n s i n m a l i c a c i d breakdown d u r i n g d e a c i d i -f I c a t i o n by Ka lanchoe c r e n a t a , a c r a s s u l a c e a n a c i d p l a n t . I t c o u l d of c o u r s e serve a s i m i l a r f u n c t i o n i n / 3 - c a r b o x y l a t i o n . P - e n o l p y r u v a t e c a r b o x y k i n a s e d e t e c t e d i n c o r n by M a z e l i s and XV Vennes land ( 9 ) c o u l d a l s o s e r v e t h i s f u n c t i o n . RuDP c a r b o x y -l a s e p r e s e n t i n the l e a v e s c o u l d a c c o m p l i s h the r e q u i r e d t r a n s -f e r of c a r b o n i f i t f i x e d C 0 2 d e r i v e d f rom d e c a r b o x y l a t i o n of m a l i c a c i d . T h i s enzyme was d e t e c t e d by S l a c k and Ha tch i n the l e a v e s of t h e s e p l a n t s , but the l e v e l o f a c t i v i t y was found t o be much lower t h a n t h a t p r e s e n t i n the c o n t r o l p l a n t s . More of t h i s enzyme would be r e q u i r e d t o p r o c e s s a l l o f the c a r b o n f i x e d i n p h o t o s y n t h e s i s . I t i s a l s o e v i d e n t t h a t t h i s enzyme and P -e n o l p y r u v a t e c a r b o x y l a s e compete f o r the same s u b s t r a t e , C 0 2 . T h i s c o m p e t i t i o n would i n t e r f e i e w i t h the t r a n s f e r of c a r b o n . C l e a r l y an e x p l a n a t i o n f o r these i n c o n s i s t e n c i e s i s r e q u i r e d . The p o s t u l a t e does however have the advantage t h a t the complex m a t a b o l i c and r e g u l a t o r y f u n c t i o n s of the C a l v i n c y c l e c o u l d be m a i n t a i n e d i n t a c t . Replacement of RuDP c a r b o x y l a s e by a n -o t h e r enzyme would remove a key r e g u l a t o r y enzyme f rom the c y c l e ( 1 ) . I t seemed t o me t h a t the d i f f i c u l t i e s p r e s e n t e d by t h i s p o s t u l a t e c o u l d be overcome by an a p p r o p r i a t e s p a t i a l o r g a n i z a -t i o n of the r e a c t i o n s w i t h i n the l e a f . I was p a r t i c u l a r l y i n -t r i g u e d by the o b s e r v a t i o n t h a t p l a n t s which have the J B - c a r -b o x y l a t l o n pathway have a n unusua l l e a f s t r u c t u r e which appears t o be a s s o c i a t e d w i t h a f u n c t i o n a l s p e c i a l i z a t i o n . Two c h l o r o -p h y l l c o n t a i n i n g t i s s u e s a r e r a d i a l l y a r r a n g e d around the v a s -c u l a r bund les of the l e a f . S u r r o u n d i n g the v a s c u l a r bundle i s a l a y e r of t h i c k w a l l e d c e l l s which c o n t a i n abundant c h l o r o -p l a s t s . T h i s s t r u c t u r e i s termed the bundle o r parenchyma s h e a t h . I t i n t u r n i s sur rounded by p a l i s a d e o r spongy meso-p h y l l c e l l s , which a l s o c o n t a i n c h l o r o p l a s t s . S t u d i e s w i t h the e l e c t r o n m i c r o s c o p e i n d i c a t e t h a t the c h l o r o p l a s t s of the XVi bundle s h e a t h d i f f e r i n u l t r a s t r u c t u r e f rom t h o s e o f the a d -j a c e n t m e s o p h y l l (2). In a much e a r l i e r s tudy Rhoades and C a r v a l h o (10) showed t h a t the m e s o p h y l l and bund le shea th c h l o r o -p l a s t s o f c o r n d i f f e r i n t h e i r f u n c t i o n . Under normal c o n d i -t i o n s s t a r c h accumula ted o n l y i n the c h l o r o p l a s t s o f tfte bund le s h e a t h c e l l s . These workers were a b l e t o show t h a t the s t a r c h s y n t h e s i z e d i n t h e s e c h l o r o p l a s t s was d e r i v e d f rom a p h o t o s y n -t h e t i c p r o d u c t o f the a d j a c e n t m e s o p h y l l . T h u s , a t l e a s t the f i n a l s t e p s i n the r e a c t i o n sequence l i n k i n g p h o t o s y n t h e t i c C0 2 f i x a t i o n t o s t a r c h s y n t h e s i s must be s e p a r a t e d i n t o the bund le s h e a t h c e l l s . T h i s space w i t h i n t h e s e c e l l s might w e l l be i s o l a t e d f rom the e x t e r n a l envi ronment by the t h i c k c e l l w a l l s s u r r o u n d i n g the bundle s h e a t h . The work o f Downton and Tregunna (3) showed t h a t the o c -c u r r e n c e o f c h l o r o p l a s t s w i t h i n the bund le s h e a t h c e l l s , and t h e i r f u n c t i o n a l d i f f e r e n c e f rom m e s o p h y l l c h l o r o p l a s t s i s a c o n s i s t e n t c h a r a c t e r which c o r r e l a t e s e x a c t l y w i t h the p r e s e n c e o f the ^ B - c a r b o x y l a t i o n pathway o f p h o t o s y n t h e s i s . A l l s p e c i e s o f p l a n t s found t o have the ^3 - c a r b o x y l a t i o n pathway a l s o had the c h l o r o p h y l l o u s bundle s h e a t h and s t o r e d s t a r c h i n i t , w h i l e t h o s e which l a c k e d i t a l s o l a c k e d j B - c a r b o x y l a t i o n . Thus i t seems p r o b a b l e t h a t the f u n c t i o n a l d i f f e r e n c e between the two c h l o r o p l a s t t y p e s i s r e l a t e d t o t h i s pathway o f p h o t o s y n t h e s i s . T h i s i m p l i e s a s p a t i a l o r g a n i z a t i o n o f the r e a c t i o n s e q u e n c e . T h i s s p a t i a l o r g a n i z a t i o n might be so a r r a n g e d as t o p reven t l o s s o f COg. and t o p revent c o m p e t i t i o n between the two c a r b o x -y l a t i n g enzymes. x v i i As a work ing h y p o t h e s i s we proposed the f o l l o w i n g : (a) G0 2 f i x a t i o n i s c o n f i n e d t o the m e s o p h y l l c e l l s , and the f i n a l p r o d u c t o f p h o t o s y n t h e s i s by t h e s e c e l l s i s the Ox-a c i d s . (b) The c a r b o x y l t r a n s f e r and the subsequent s t e p s l e a d -i n g t o s t a r c h f o r m a t i o n o c c u r i n the bund le s h e a t h c e l l s , and the c a r b o n u t i l i z e d i n t h e s e r e a c t i o n s i s t r a n s p o r t e d t o t h e s e c e l l s f rom the m e s o p h y l l c e l l s i n the form o f C ^ - a c i d s . Conf inement o f COg f i x a t i o n (^3 -carboxylat ion) t o the meso-p h y l l c e l l s seemed r e a s o n a b l e as t h e s e c e l l s a r e exposed t o the s u b - s t o m a t a l c a v i t i e s and thus a r e i d e a l l y l o c a t e d f o r e f f i -c i e n t gas exchange . The bundle s h e a t h c e l l s a r e c o n t a i n e d w i t h i n t h i c k c e l l w a l l s which c o u l d e f f e c t i v e l y i s o l a t e them from the e x t e r n a l s o u r c e o f CO2. C o n v e r s e l y , COg produced w i t h i n t h e s e c e l l s might be p r e v e n t e d f rom e s c a p i n g by t h e s e t h i c k c e l l w a l l s . F i x a t i o n o f i n t e r n a l l y produced C 0 2 by RuDP c a r b o x y l a s e c o u l d a c c o m p l i s h the c a r b o x y l t r a n s f e r . I f the o t h e r c a r b o x y l a -t i n g enzyme, P - e n o l p y r u v a t e c a r b o x y l a s e , i s c o n f i n e d t o the m e s o p h y l l c e l l s c o m p e t i t i o n between t h e s e two enzymes f o r CO2 would be e l i m i n a t e d . T h i s would enhance the p o t e n t i a l e f f i c i -ency o f the mechanism, and e x p l a i n the f a i l u r e t o d e t e c t f r e e COg as an i n t e r m e d i a t e . T h i s p o s t u l a t e i s complex , and i t ; i s r a d i c a l l y d i f f e r e n t f rom the C a l v i n c y c l e type p h o t o s y n t h e s i s , wh ich o c c u r s w i t h i n the autonomous c o n f i n e s o f a s i n g l e c h l o r o -p l a s t . Some exper iments d e s i g n e d t o t e s t t h i s p o s t u l a t e a r e d e s c r i b e d i n the nex t s e c t i o n o f t h i s t h e s i s . T h i s appears as the t e x t o f a paper p r e s e n t l y i n p r e s s . R e c e n t l y o t h e r x v i l i work which pertains to t h i s topic has been published, and w i l l be discussed i n an epilogue which appears at the end of the t h e s i s . Some information not included i n the text i s presented i n appendices. L i t e r a t u r e C i t e d 1. Bassham, J . A . , and K i r k , M a r t h a . 1968. Dynamic m e t a b o l i c r e g u l a t i o n o f the p h o t o s y n t h e t i c c a r b o n r e d u c t i o n c y c l e . In : S h i b a t a , K . , Takamiya , A . , J a g e n d o r f , A . T . , and F u l l e r , R . C . ( E d s . ) . Comparat ive b i o c h e m i s t r y and b i o p h y s i c s o f p h o t o s y n t h e s i s . U n i v e r s i t y Park P r e s s P e n n s y l v a n i a , p p . 365-378. 2. B i s a l p u t r a , T . , Downton, W . J . S . , and T r e g u n n a , E . B . 1969. The d i s t r i b u t i o n and u l t r a s t r u c t u r e o f c h l o r o p l a s t s i n l e a v e s d i f f e r i n g In p h o t o s y n t h e t i c c a r b o n metab-o l i s m s I. C a n . J . B o t . 47: 15-21. 3. Downton, W . J . S . , and T r e g u n n a , E . B . 1968. Carbon d i o x i d e compensat ion - i t s r e l a t i o n t o p h o t o s y n t h e t i c c a r b o x y -l a t i o n r e a c t i o n s , s y s t e m a t i c s o f t h e Gramineae, and l e a f anatomy. C a n . J . B o t . 46: 207-215. 4. H a t c h , M . D . , and Stumpf , P . K . 196l. F a t metabo l i sm i n h i g h e r p l a n t s . XVI A c e t y l - C o A c a r b o x y l a s e and A c e t y l Co A - m a l o n y l - C o A t r a n s c a r b o x y l a s e f rom wheat germ. J . B i o l . Chem. 236: 2879-2885. 5 . H a t c h , M . D . , and S l a c k , C R . 1966. P h o t o s y n t h e s i s by s u g a r -cane l e a v e s . 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Carbon d i o x i d e f i x a t i o n i n t o o x a l o a c e t a t e i n h i g h e r p l a n t s . P l a n t P h y s i o l . 32: 591 10. Rhoades, M .M. , and C a r v a l h o , A . 1944. The f u n c t i o n and s t r u c t u r e o f the parenchyma shea th p l a s t i d s o f the maize l e a f . B u l l . T o r r e y B o t a n . C l u b 71: 335-346. 11. S l a c k , C R . , and H a t c h , M.D. I967. Comparat ive s t u d i e s on the a c t i v i t y o f c a r b o x y l a s e s and o t h e r enzymes i n r e -l a t i o n t o the new pathway o f p h o t o s y n t h e t i c c a r b o n d i o x i d e f i x a t i o n i n t r o p i c a l g r a s s e s . B iochem. J . 103: 660-665. XX 12. T r e g u n n a , E . B . , K r o t k o v , G . t and N e l s o n , C D . I966. E f f e c t o f oxygen on the r a t e o f p h o t o r e s p i r a t i o n i n de tached t o b a c c o l e a v e s . P h y s i o l . P lan ta rum 19: 723-733. 13. W a l k e r , D . A . i 9 6 0 . P h y s i o l o g i c a l s t u d i e s o f a c i d m e t a b o l i s m . 7 B iochem. J . 74: 213-223. 14 . Wood, H a r l a n d G . , and S t j e r n h o l m , Rune. 1961. T r a n s c a r b o x y -l a s e I I . P u r i f i c a t i o n and p r o p e r t i e s o f m e t h y l m a l o n y l -o x a l o a c e t i c t r a n s c a r b o x y l a s e . P r o c . N . A . S . 47: 289-303. 1 Chapte r I The p h o t o s y n t h e t i c carbon metabo l ism of Zea mays and Gomphrena  g l o b o s a t The l o c a t i o n of the C© 2 f i x a t i o n and the c a r b o x y l t r a n s f e r react ions ." ' " I n t r o d u c t i o n T r o p i c a l g r a s s e s of the p a n i c o i d and c h l o r i d o i d - e r a g r o s -t o i d l i n e s of phy logeny and some d i c o t y l e d o n o u s p l a n t s use a pathway of p h o t o s y n t h e t i c CO2 f i x a t i o n which d i f f e r s , a t l e a s t i n i t s i n i t i a l s t e p s , f rom the C a l v i n c y c l e (7 , 9 , 12, 2 0 , 2 8 ) . 14 In t h e s e p l a n t s most o f the ca rbon from CC>2 i s i n i t i a l l y found i n C ^ - a c i d s ( m a l a t e , a s p a r t a t e , and o x a l o a c e t a t e ) (11, 21 , 2 8 ) . Hatch and S l a c k (11) have proposed a c y c l e l i n k i n g CO2 f i x a t i o n i n t o the C ^ - a c l d s w i th a c a r b o x y l t r a n s f e r r e a c t i o n t o form 3 - P h o s p h o g l y c e r i c a c i d ( 3 - P G A ) . In t h i s pathway, C0 2 i s f i x e d by a ^Decarboxy la t ion of phosphoenol p y r u v i c ( P - e n o l -p y r u v l c ) a c i d t o form o x a l o a c e t i c a c i d . T h i s may be i n t e r c o n -v e r t e d t o o t h e r C ^ - a c i d s by t r a n s a m i n a t i o n o r r e d u c t i o n . One of t h e s e s e r v e s as a c a r b o x y l donor and the remainder o f the c a r b o x y l donor i s p r o b a b l y r e l e a s e d a s p y r u v i c a c i d . T h i s i s c o n v e r t e d t o P - e n o l p y r u v i c a c i d by the enzyme ;:p\yruvate, p h o s -phate d i k i n a s e (14 ) , thus c o m p l e t i n g the c y c l e . Enzymes c a t a l y z -i n g each of the s t e p s of the c y c l e except the c a r b o x y l t r a n s f e r have been d e s c r i b e d (14, 15. 36, 3 9 ) . E x t r a c t s of p l a n t s which 1 T h i s a r t i c l e by J . A . B e r r y , W . J . S . Downton, and E . B . Tregunna was submi t ted t o the Canad ian J o u r n a l of Botany August 21 , 1969. W . J . S . Downton a s s i s t e d i n p r e p a r a t i o n of the r a d i o a u t o g r a p h . E . B . Tregunna s u p e r v i s e d the s t u d y . 2 have the / 3 - c a r b o x y l a t i o n pathway c o n t a i n t h e s e enzymes i n a d e -quate a c t i v i t i e s t o s u s t a i n r a t e s o f p h o t o s y n t h e s i s found i n  v i v o ( 3 6 ) . The c a r b o x y l a c c e p t o r i n the r e a c t i o n above might be r i b u l o s e 1 , 5 - d i p h o s p h a t e (RuDP), the CO^ a c c e p t o r i n p l a n t s which have o n l y the C a l v i n c y c l e ( 1 1 ) . Subsequent metabo l ism o f 3-PGA formed i n t h i s r e a c t i o n appears t o be s i m i l a r t o the normal C a l v i n sequence ( 1 0 ) . One o r more enzymes n e c e s s a r y t o comple te t h e ^ - c a r b o x y l a t i o n c y c l e have n o t been found i n p l a n t s w i t h o n l y the C a l v i n c y c l e ( 1 3 ) . These p l a n t s a l s o c o n t a i n much lower a c t i v i t i e s o f phosphoeno lpyruva te c a r b o x y l a s e (EC 4 .1.1.31), ( 9 , 3 6 ) . P l a n t s which have the J B - c a r b o x y l a t l o n pathway have a c h a r a c -t e r i s t i c arrangement o f the p h o t o s y n t h e t i c a p p a r a t u s . T h i s has been d e s c r i b e d as "Kranz" type l e a f anatomy. I t i s c h a r a c t e r i z e d by a c o n c e n t r i c arrangement o f bund le s h e a t h c e l l s and p a l i s a d e t y p e m e s o p h y l l c e l l s a round the v a s c u l a r bund les ( 3 ) . The c e l l s o f the bund le s h e a t h a r e t i g h t l y packed w i t h c h l o r o p l a s t s . These c h l o r o p l a s t s d i f f e r f rom t h o s e i n the nearby m e s o p h y l l i n t h a t they a r e l a r g e r , s t o r e s t a r c h , and i n some c a s e s l a c k grana ( 2 , 7, 8, 23, 24, 25, 3 3 ) . Both m e s o p h y l l and bund le shea th c h l o r o -p l a s t s o f p l a n t s o f t h i s type p o s s e s s a c h a r a c t e r i s t i c p e r i p h e r a l r e t i c u l u m (24, 34). The bund le shea th t i s s u e , which c o n t a i n s a s i g n i f i c a n t p o r t i o n o f the t o t a l c h l o r o p l a s t s o f t h e l e a f , does n o t a p p e a r t o be as w e l l adapted f o r e f f i c i e n t e x t e r n a l gas exchange as the a d j a c e n t m e s o p h y l l t i s s u e . I t has been sugges ted t h a t the p h o t o s y n t h e t i c sequence may o c c u r i n s p a t i a l l y s e p a r a t e d s i t e s i n l e a v e s o f t h i s type (24). Carbon d i o x i d e f i x a t i o n may o c c u r i n the m e s o p h y l l , w h i l e the f i n a l s t e p l e a d i n g t o s t a r c h f o r m a t i o n seems t o o c c u r i n the 3 bundle shea th c e l l s . I f the s i t e of C0 2 f i x a t i o n i s s p a t i a l l y -s e p a r a t e d f rom the s i t e o f s t a r c h f o r m a t i o n some m e t a b o l i t e must s e r v e t o t r a n s p o r t ca rbon f rom one s i t e to a n o t h e r . T h i s paper d e s c r i b e d , exper iments d e s i g n e d t o t e s t the h y p o t h e s i s t h a t the C ^ - a c i d s s e r v e t h i s f u n c t i o n . The impor tance of the C ^ - a c i d s i n the p h o t o s y n t h e t i c r e a c -14 t i o n sequence was re -examined by use of p u l s e and chase type CO, f e e d i n g s . Rad ioautographs of some of the t i s s u e were p r e p a r e d , 14 and the 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 of C l a b e l l e d compounds was s t u d i e d by nonaqueous c h l o r o p l a s t p r e p a r a t i o n t o de termined the 14 l o c a t i o n of f i x e d C w i t h i n the l e a f . The l o c a t i o n of some p h o t o s y n t h e t i c enzymes was a l s o s t u d i e d a f t e r d i f f e r e n t i a l g r i n d -i n g of the l e a f . Zea mays, a monocoty ledon and Gomphrena g l o b o s a , a d i c o t y l e d o n , were used i n t h e s e s t u d i e s . M a t e r i a l s and Methods P l a n t s . Zea mays L . v a r . p i o n e e r was grown on v e r m i c u l i t e s u p -plemented w i t h n u t r i e n t s o l u t i o n , and Gomphrena g l o b o s a Moq. was grown on s o i l . Growth c o n d i t i o n s were s i x t e e n hour d a y , 22°/i9° ( d a y / n i g h t ) t e m p e r a t u r e , and 22,000 l u x l i g h t i n t e n s i t y p r o v i d e d i n a growth chamber. D e s t a r c h e d l e a v e s of t h r e e - w e e k - o l d c o r n p l a n t s , and the youngest f u l l y expanded l e a v e s of t w o - t o t h r e e -m o n t h - o l d Gomphrena p l a n t s were u s e d . Leaves were d e s t a r c h e d by m a i n t a i n i n g the p l a n t s 36 hours i n d a r k n e s s , f o l l o w e d by 2 h r i l l u m i n a t i o n j u s t p r i o r t o d e t a c h i n g . 14 CO2 f e e d i n g s . Leaves were d e t a c h e d , r e c u t under water and i l -14 l u m i n a t e d f o r a t l e a s t 20 min b e f o r e f e e d i n g . A CO^ f e e d i n g 4 t e c h n i q u e s i m i l a r t o t h a t d e s c r i b e d by K o r t s c h a k et a l . (21) was u s e d . Each l e a f was i n d i v i d u a l l y f e d by i n s e r t i n g i t th rough a s l o t i n the t o p of a g l a s s j a r (200 ml) c o n t a i n i n g 250 j i c u r l e s 14 of C0 2 i n a i r . A f t e r each f e e d i n g the s l o t i n the j a r was 14 12 r e s e a l e d w i t h tape t o p reven t l eakage of COg. The C0 2 chase was p r o v i d e d by w i thdrawing the l e a f f rom the j a r and h o l d i n g i t i n a i r f o r the p r e s c r i b e d t i m e . I l l u m i n a t i o n was m a i n t a i n e d a t about 2 2 , 0 0 0 l u x d u r i n g p r e - i l l u m i n a t i o n , p u l s e and the c h a s e . The temperature range i n t h e s e exper iments was 2 0 - 2 2 ° . Metabo-l i s m was h a l t e d by immersing the l e a f i n l i q u i d n i t r o g e n . The f r o z e n m a t e r i a l was d r i e d o v e r ?2°5 a t - 2 0 ° i n an evacuated d e s i c -c a t o r . A f t e r s e v e r a l days the d e s i c c a t o r was t r a n s f e r r e d t o 4° and s t o r e d u n t i l the d r i e d l e a f m a t e r i a l was r e q u i r e d . R a d l o a u t o g r a p h s . M i c r o r a d i o a u t o g r a p h s were p r e p a r e d f rom t h i n s e c t i o n s of f r e e z e d r i e d l e a v e s embedded d i r e c t l y i n wax. These were mounted on g l a s s s l i d e s and coa ted w i t h I l f o r d K -5 n u c l e a r e m u l s i o n . A f t e r exposure the s l i d e s were deve loped and s t a i n e d w i t h A z u r e B a c c o r d i n g t o J e n s e n ( 1 9 ) . Rad ioautographs of i n -t a c t l e a v e s were p r e p a r e d by p r e s s i n g f r e e z e d r i e d l e a v e s mounted on heavy paper a g a i n s t Kodak No S c r e e n X - r a y F i l m . Nonaqueous F r a c t i o n a t i o n . The g r i n d i n g of f r e e z e d r i e d l e a v e s was done a t 4°. The d r i e d l e a v e s were p l a c e d i n a 250 ml*': Waring b l e n d o r j a r and b l e n d e d f o r 1 minute w i t h 100 m l o f hexane. The c o n t e n t s of the j a r were a l l o w e d t o s e t t l e f o r f i v e m i n u t e s , and the f i n e m a t e r i a l r e m a i n i n g i n s u s p e n s i o n was d e c a n t e d . The sediment was t r a n s f e r r e d t o g e t h e r w i t h a s m a l l amount of hexane t o a p o r c e l a i n m o r t a r , and ground w i th a p e s t l e . F i n e m a t e r i a l r e l e a s e d i n the g r i n d i n g was removed by s u s p e n d i n g i n hexane , 5 a l l o w i n g coarse p a r t i c l e s t o s e t t l e and d e c a n t i n g as i n the p r e v i o u s s t e p . G r i n d i n g was continued u n t i l no r e s i d u e remained. The accumulated suspension of f i n e l y ground m a t e r i a l was pooled and a sample (20% by volume) taken. T h i s i s de s i g n a t e d the "Crude" sample. The remainder of the m a t e r i a l was washed on a 25jti pore s i z e f i l t e r of b o l t i n g s i l k t o remove l a r g e r par-t i c l e s . The f i l t r a t e was concentrated by c e n t r i f u g a t i o n , a t 5000 x g f o r 10 min. The p e l l e t was suspended i n 10-20 ml- of hexane and l a y e r e d on top of a d i s c o n t i n u o u s g r a d i e n t of hexane-carbon t e t r a c h l o r i d e mixtures of i n c r e a s i n g d e n s i t y prepared a c c o r d i n g t o T h e l l a c k e r and Behrens ( 3 7 ) . The g r a d i e n t s were c e n t r i f u g e d a t 5000 x g f o r 30 minutes i n a S o r v a l HB-4 swinging bucket r o t o r . The bottom of the tube was punctured and the f r a c -t i o n s were c o l l e c t e d i n d i v i d u a l l y . The l i g h t e s t c h l o r o p l a s t f r a c t i o n was washed twice with 50/50 v/v, hexane-carbon t e t r a -c h l o r i d e t o remove c h l o r o p l a s t fragments ( 1 6 ) . M i c r o s c o p i c ex-am i n a t i o n of t h i s f r a c t i o n r e v e a l e d no a p p r e c i a b l e contamination of c h l o r o p l a s t or other fragments. Each f r a c t i o n and the crude sample were suspended i n a known volume and subsamples taken 14 f o r p r o t e i n , c h l o r o p h y l l and C d e t e r m i n a t i o n s . The s o l v e n t was removed by c e n t r i f u g a t i o n and the r e s i d u e e x t r a c t e d i n b o i l i n g e t h a n o l : water i n the f o l l o w i n g sequences 80 : 2 0 , 20:80, 0 : 1 0 0 . The accumulated e x t r a c t s f o r each f r a c t i o n were concen-t r a t e d by e v a p o r a t i o n i n an oven a t about 6 0 ° . C a l c u l a t i o n of the p o r t i o n s of l a b e l l e d C^-acids or phosphate e s t e r s l o c a t e d i n the c h l o r o p l a s t s was done on a p r o t e i n b a s i s as proposed by Latzko and Gibbs ( 2 2 ) . The f o l l o w i n g equation was used t o c a l c u l a t e ( y ) , the percent of a l a b e l l e d component 6 l o c a t e d i n the c h l o r o p l a s t s . y = a l b 2 . x a 2 b l Where a-j^  and a 2 a r e the jig p r o t e i n , and and b g the dpmik^C i n a l a b e l l e d component, p r e s e n t i n the crude and the c h l o r o -p l a s t f r a c t i o n s , r e s p e c t i v e l y , f rom a d e n s i t y g r a d i e n t s e p a r a -14 t i o n of C 0 2 f e d l e a f m a t e r i a l . The f a c t o r x i s the mean of s e v e r a l d e t e r m i n a t i o n s of the p e r c e n t of t o t a l l e a f p r o t e i n l o c a t e d i n the c h l o r o p l a s t s of e i t h e r Zea o r Gomphrena. The v a l u e f o r (x)was c a l c u l a t e d from the e q u a t i o n g i v e n i n (22 ) . No c o r r e c t i o n f o r c h l o r o p h y l l l e a c h i n g was a t tempted . T h i s w i l l not a f f e c t the r e l a t i v e d i f f e r e n c e between f e e d i n g t r e a t -ments , but because c h l o r o p h y l l l e a c h i n g may cause (x) t o be too l a r g e , the a b s o l u t e d i s t r i b u t i o n may be s l i g h t l y i n e r r o r . C a r b o n - l 4 was de te rmined i n aqueous s o l u t i o n s o r on c h r o -matography paper w i t h B r a y ' s l i q u i d s c i n t i l l a t i o n s o l u t i o n ( 6 ) . 14 In some c a s e s the r e l a t i v e d i s t r i b u t i o n of c i n compounds of the same sample was de te rmined f rom the r e c o r d of a s t r i p c h a r t chromatogram s c a n n e r . C h l o r o p h y l l was de termined i n 80% ace tone ( 3 8 ) . P r o t e i n was de te rmined a c c o r d i n g t o Lowry ( 2 6 ) . Chromatography The components of each combined e t h a n o l : water e x t r a c t were s e p a r a t e d on 1 § x 24 i n c h Whatman No. 1 paper s t r i p by d e s c e n d i n g chromatography w i th l i q u e f i e d p h e n o l : wa te r : a c e t i c : 1M e t h y l e n e d i a m i n e t e t r a c e t i c a c i d (840: 160: 10: 1) ( 3 0 ) . M a l i c and a s p a r t l c a c i d s chromatographed as two bands of Rp. about 0 .32 and 0 .23 r e s p e c t i v e l y . The monophosphate e s t e r s of s u g a r s and p y r u v i c and g l y c e r i c a c i d s were a t Rp. 0 . 1 . 14 These a r e a s a c c o u n t e d f o r most of the C i n our f e e d i n g s . Some 7 14 l o n g e r term f e e d i n g s showed about of t o t a l C a t R p i 0 . 4 5 , 14 c o r r e s p o n d i n g t o s u c r o s e . In a l l f e e d i n g s i n s o l u b l e C was l e s s t h a n 2% of t o t a l . The sum of m a l i c and a s p a r t i c a c i d s i s r e p r e s e n t e d as the C ^ - a c i d s . O x a l o a c e t i c a c i d was not d e -t e r m i n e d i n t h e s e exper iments because i t i s p r e s e n t i n s m a l l q u a n t i t i e s , and because of the s p e c i a l p r e c a u t i o n s r e q u i r e d t o p r e v e n t i t s d e c o m p o s i t i o n ( 1 2 ) . E x t r a c t i o n of enzymes f rom m e s o p h y l l and bundle sheath t i s s u e . A d i f f e r e n t i a l g r i n d i n g t e c h n i q u e s i m i l a r t o t h a t d e s c r i b e d by Bjbrkman (5) was used t o o b t a i n e x t r a c t s e n r i c h e d i n e i t h e r bundle sheath o r m e s o p h y l l c e l l c o n t e n t s . Leaves ( 1 . 0 g) were cut i n t o 1 cm p i e c e s and ground under l i g h t p r e s s u r e w i t h a p e s t l e i n a mor ta r c o n t a i n i n g 5 ml of g l a s s beads (0.25-0.30mm) and 10 ml of g r i n d i n g medium ( d e s c r i b e d b e l o w ) . G r i n d i n g was c o n t i n u e d u n t i l the l e a f p i e c e s were u n i f o r m l y t r a n s l u c e n t and had l o s t some but not a l l of t h e i r c h l o r o p h y l l . M i c r o s c o p i c i n s p e c t i o n r e v e a l e d t h a t most of the m e s o p h y l l c e l l s c o u l d be r u p t u r e d d u r i n g t h i s t r e a t m e n t , w h i l e most of the bund le sheath c e l l s remained i n t a c t . T h i s e x t r a c t was t h e r e f o r e e n r i c h e d i n the c o n t e n t s of m e s o p h y l l c e l l s . The bundle sheath t i s s u e was washed i n 50 ml, of g r i n d i n g medium; t h e n s u b j e c t e d t o v e r y v i g o r o u s g r i n d i n g w i t h a p e s t l e i n a mor ta r c o n t a i n i n g g l a s s beads ( 0 . 4 5 - 0 . 5 0 mm,) and 10 ml g r i n d i n g medium. S u f f i c i e n t g r i n d i n g p r e s s u r e was used t o break the g l a s s b e a d s , c r e a t i n g a v e r y s h a r p , a n g u l a r g r i n d i n g m a t e r i a l . M o s t , but not a l l , of the bundle shea th c e l l s were r u p t u r e d by t h i s t r e a t m e n t , y i e l d -i n g an e x t r a c t e n r i c h e d i n the c o n t e n t s of bundle shea th c e l l s . A l l s t e p s were c a r r i e d out a t 0 - 4 ° . P l a n t m a t e r i a l was h a r -8 v e s t e d immedia te ly b e f o r e u s e . Enzyme a s s a y s were per formed a t 24-26°C w i t h i n 2 hours o f g r i n d i n g . The g r i n d i n g medium was t h a t used by Bjb'rkman ( 5 ) . I t c o n t a i n e d : 40 mM T r i s - HC1, pH 8 .0 ; 10 mM M g C l 2 ; 5 mM d i t h i o -t h r e i t o l (DTT) ; 5 mM D - a r a b a n o a s c o r b i c a c i d ; 0 .25 mM EDTA. E x t r a c t s were c e n t r i f u g e d f o r 15 min a t 20,000 x g b e f o r e u s i n g them f o r enzyme a s s a y s . T C A - p r e c i p i t a b l e p r o t e i n was de te rmined by the method o f Lowry ( 2 6 ) . Enzyme A s s a y s R i b u l o s e d i p h o s p h a t e c a r b o x y l a s e (EG 4 . 1 . 1 . f ) was a s s a y e d a c c o r d i n g t o Bjorkman (5) i n a t o t a l volume o f 0 .5 ml c o n t a i n i n g : 20 pinoles T r i s - HC1 b u f f e r , pH 8 .0 ; 3 j imoles, M g C l 2 ; 1 . 5 / imoles DTT; 0.1 / imoles, EDTA; 0 . 9 Jimoles o f RuDP; 0.1 ml o f enzyme e x t r a c t ; and 10 pmoles o f NaHCO^ ( lO^dpm/umole ) . Samples o f the a s s a y medium (50 u l l t e r s ) were taken a t 2 minute i n t e r -v a l s and added t o 0 . 5 ml 90$ f o r m i c a c i d i n a s c i n t i l l a t i o n v i a l . These samples were d r i e d under vacuum o v e r NaOH p e l l e t s , and 14 counted as p r e v i o u s l y d e s c r i b e d . Rates o f C f i x a t i o n were l i n e a r f o r a t l e a s t 10 m i n u t e s . " M a l i c " enzyme (EC 1 . 0 . 1 . 4 0 ) was a s s a y e d (35, 40) s p e c t r o -p h o t o m e t r l c a l l y a t 340 nm i n 100 mM T r i s - H C l b u f f e r , pH 8 . 0 . Each 3 .0 ml a s s a y c o n t a i n e d 5 / imole MgCl, , ; 5 / imole DTT; 0 .37 yumole NADP; 0 .05 ml o f e x t r a c t ; and 10 / imole o f L - m a l l c a c i d . NADP - ma la te dehydrogenase , f i r s t d e s c r i b e d by Ha tch and S l a c k (13 ) , was a s s a y e d a t 340 nm i n 100 mM T r i s - HC1 b u f f e r , pH 8 . 0 . Each 3 .0 ml a s s a y c o n t a i n e d 5 / imole o f M g C l 2 ; 5 p n o l e DTT; 0 .35 / imole NADPH; 0 .05 ml enzyme e x t r a c t ; and 3 yumole of o x a l o a c e t i c a c i d . I n i t i a l r a t e s were used i n t h i s 9 a s s a y t o m in imize i n t e r f e r e n c e f rom m a l i c enzyme. Phosphoeno lpyruva te c a r b o x y l a s e (EG 4.1.1.31) was a s s a y e d a t 3^ 0 nm (29t 39) w i t h NAD - malate dehydrogenase which was a l s o p r e s e n t i n the enzyme e x t r a c t s . A s s a y s were per formed i n 100 mM HEPES b u f f e r ( N-2 - H y d r o x y e t h y l p i p e r a z i n e - N 1-2 - e t h a n e s u l f o n i c a c i d ) , pH 7»5» Each 3.0 ml a s s a y c o n t a i n e d : 20 pmole M g C l 2 ; 6 ^umole NaHCOy 0.26 yumole NADH; 0.05 - 0.2 ml o f enzyme e x -t r a c t ; and 0.75 / imole P - e n o l p y r u v i c a c i d . NAD - mala te d e h y -drogenase was a lways i n excess o f the P - e n o l p y r u v a t e c a r b o x y l a s e a c t i v i t y . P r e l i m i n a r y exper iments were per formed t o de te rmine optimum c o n d i t i o n s f o r a s s a y o f P - e n o l p y r u v a t e c a r b o x y l a s e and " m a l i c " enzyme. 14 P r o d u c t i o n o f CCu and CCu d u r i n g l i g h t and dark by l e a v e s o f Zea mays was mon i to red i n n i t r o g e n gas (30 ml /min) which passed t h r o u g h the chamber (45 ml) c o n t a i n i n g the l e a v e s , and then 14 t h r o u g h an i n f r a - r e d C0 2 a n a l y z e r . A t the o u t l e t C0 2 c o u l d be t r a p p e d i n 2 - p h e n y l e t h y l a m i n e . A t 5 min the system was darkened 14 and 50 j i c u r i e s o f C0 2 was i n j e c t e d i n t o the chamber. Plow o f N 2 was i n t e r r u p t e d f o r 1 min t o a l l o w dark f i x a t i o n , and then r e s t a r t e d . F l u s h i n g o f the system was comple te a t 9 - 10 m i n . The COg which appeared a f t e r t h i s t ime was produced by the p l a n t . A t 13 min a sample o f C0 2 was c o l l e c t e d f o r 2 min as a measure 14 of C0 2 p r o d u c t i o n i n dark r e s p i r a t i o n . At 18 min?: a l i g h t was t u r n e d o n , and the r e s u l t i n g b u r s t o f G02 c o l l e c t e d . Gpm. per u n i t a r e a under the c u r v e was taken as a measure o f s p e c i f i c a c t i v i t y . The low gas f l o w r a t e p r o l o n g e d the a p p a r e n t p e r i o d o f G02 r e l e a s e i n the l i g h t , but was n e c e s s a r y t o ge t a c c u r a t e measurements o f C0 2 c o n c e n t r a t i o n . 10 R e s u l t s and D i s c u s s i o n The products formed I n d i v i d u a l detached l e a v e s were exposed a short time to 14 , 12 CO i n the l i g h t (pulse) and then i l l u m i n a t e d i n C0 ? (chase) before k i l l i n g . The l e n g t h of the p u l s e was 2 sec , f o r Zea and , 14 6 s e c : f o r Gomphrena. The d i s t r i b u t i o n of C among the com-pounds separated i s shown i n F i g . 1. At the end of the p u l s e more than 90% of the C present was i n the C^_-acids. T h i s i n d i c a t e s t h a t p - c a r b o x y l a t i o n i s a c t i v e i n both of these p l a n t s . Phosphate e s t e r s are l a b e l l e d 14 a t the end of the p u l s e , but the q u a n t i t y of C present i n these compounds i n c r e a s e s s u b s t a n t i a l l y d u r i n g the chase. 14 Coupled with t h i s i s a decrease i n the c of the C ^ - a c i d p o o l . 14 No CO2 c o u l d be f i x e d d u r i n g the chase; thus compounds which become l a b e l l e d d u r i n g the chase do so a t the expense of com-pounds formed d u r i n g the p u l s e . N e i t h e r Zea nor Gomphrena 14 l o s e s C0 2 i n the l i g h t (7), hence no c should be l o s t from 14 the l e a f d u r i n g the chase. The r a t e of movement of C i n t o the monophosphate e s t e r s seems t o be o n l y s l i g h t l y a f f e c t e d by 14 12 r e p l a c i n g C0 2 with C0 2. T h e r e f o r e , t h i s would i n d i c a t e that the C^-aclds are the p r i n c i p a l source of l a b e l i n c o r p o r a t e d i n t o the monophosphate e s t e r s i n these experiments. These r e s u l t s a r e s i m i l a r t o those p u b l i s h e d f o r sugarcane ( 11) . The r e l a t i v e p o r t i o n Of the C^-acids accounted f o r by a s p a r t a t e or malate d i f f e r e d between the p l a n t s , but not between chase times, i n -d i c a t i n g a very r a p i d e q u i l i b r a t i o n between these two compounds. The chase time r e q u i r e d t o d e p l e t e one h a l f of the C ^ - a c i d p o o l i s 20 sec - f o r Zea and 60 sec f o r Gomphrena. T h i s would i n -d i c a t e 1-3 t u r n o v e r s of t h i s pool per minute. 11 100r 10 2 0 3 0 4 0 C H A S E ( 1 Z C 0 2 ) S E C . • C ^ - A c i d s • P h o s p h a t e e s t e r s C h a p t e r 1. F i g u r e 1. The r e d i s t r i b u t i o n o f C between l a b e l l e d C ^ - a c i d s and phosphate e s t e r s i n l e a v e s d u r i n g p u l s e and chase COg f e e d i n g s . P u l s e t ime f o r Zea mays 2 sec and f o r Gomphrena  g l o b o s a 6 s e c . 12 L o c a t i o n of the p r o d u c t s Leaves of Gomphrena a r e a n a t o m i c a l l y q u i t e s i m i l a r t o Amaranthus e d u l i s , A t r i p l e x l e n t i f o r m i s (24 ) , and A t r i p l e x  r o s e a ( 8 ) . The v a s c u l a r bund les a r e sur rounded by two c o n c e n -t r i c l a y e r s of t i s s u e : a bundle s h e a t h , and a l a y e r of meso-p h y l l r e f e r r e d t o by L a e t s c h (24) as a c o n c e n t r i c a l l y a r r a n g e d p a l i s a d e t i s s u e . The r e m a i n i n g i n t e r v a s c u l a r t i s s u e i s com-posed of c e l l s which do not c o n t a i n much c h l o r o p h y l l . The v i s u a l c o n t r a s t between t h e s e c e l l s and the t i s s u e s s u r r o u n d i n g the v a s c u l a r b u n d l e s appears as a g r e e n r e t i c u l u m which i s c h a r -a c t e r i s t i c of t h i s type of l e a f . C h l o r o p h y l l o u s t i s s u e l a y e r s s i m i l a r t o those d e s c r i b e d above f o r Gomphrena sur round the p a r a l l e l v a s c u l a r bund les of the l e a v e s of Zea mays. A r a d i o a u t o g r a p h of a f r e e z e d r i e d Gomphrena l e a f i s p r e s -14 ented i n F i g u r e 3. T h i s l e a f was f e d CO^ f o r 6 s e c , and f r o z e n (no c h a s e ) . More t h a n 90% of the r a d i o a c t i v i t y was i n Cjj , -acids i n t h i s l e a f . The darkened a r e a s c o r r e s p o n d t o the d i s t r i b u t i o n of c h l o r o p h y l l , and t o the l o c a t i o n of the bundle shea th and the c o n c e n t r i c a l l y a r r a n g e d m e s o p h y l l t i s s u e ' . I t i s c l e a r t h a t the remainder of the t i s s u e i s not l a b e l l e d . C a r -bon d i o x i d e f i x a t i o n must be c o n f i n e d t o the t i s s u e s ( p a l i s a d e m e s o p h y l l and bund le sheath) which sur round the v a s c u l a r b u n d l e s . 14 A m i c r o - r a d i o a u t o g r a p h of a s e c t i o n e d Gomphrena l e a f f e d cOg f o r 6 sec f o l l o w e d by a 30 sec chase b e f o r e f r e e z i n g i s shown i n F i g u r e 2 . The d i s t r i b u t i o n of l a b e l among the compounds a t t h i s t ime i s shown i n F i g u r e 1. Most of the g r a i n s c o r r e s p o n d -i n g t o the l o c a t i o n of r a d i o a c t i v i t y o v e r l i e the bundle sheath c e l l s . The C ^ - a c i d s must be i n the bundle s h e a t h . O t h e r s i m i -C h a p t e r I F i g u r e 2 . M l c r o r a d l o a u d i o a u t o g r a p h o f a t h i n s e c t i o n o f a Gomphrena g l o b o s a l e a f (x500) f e d i^CCu f o r 6 sec f o l l o w e d by 30 sec i n -"-^CO?. F i g u r e 2a shows the d i s t r i b u t i o n o f g r a i n s . F i g u r e 2 D i s a m i c r o g r a p h f o c u s e d on the t i s s u e . V a s c u l a r bund le (v) c e l l s o f the bund le s h e a t h (B) and the r a d i a l l y a r r a n g e d m e s o p h y l l a r e shown. C h a p t e r I F i g u r e 3 . A whole l e a f r a d i o a u t o g r a p h o f a l e a f o f Gomphrena  g l o b o s a x 5 , f e d ^ C O p f o r 6 s e c . 1% 15 14 l a r s t u d i e s of the l o c a t i o n of l a b e l i n c o r n l e a v e s f e d CO 2 f o r l o n g e r t imes (2 - 10 m i n , ) (27, 3 2 ) , when the l a b e l would be i n c a r b o h y d r a t e , a l s o show most of the l a b e l t o be i n the bund le shea th c e l l s . T h i s i n d i c a t e s t h a t these c e l l s a r e p r o b a b l y the s i t e of the c a r b o x y l t r a n s f e r r e a c t i o n and the C a l v i n c y c l e . The s i t e of f i x a t i o n The above s t u d i e s i n d i c a t e t h a t COg may be f i x e d i n the bundle s h e a t h , or a l t e r n a t i v e l y , the p r o d u c t s of f i x a t i o n may be t r a n s l o c a t e d f rom the s u r r o u n d i n g m e s o p h y l l t i s s u e t o the bundle shea th v e r y r a p i d l y a f t e r f i x a t i o n . The s i t e of f i x a -t i o n s h o u l d c o r r e s p o n d t o the l o c a t i o n of the enzyme r e s p o n -s i b l e f o r the i n i t i a l C 0 2 f i x a t i o n r e a c t i o n , P - e n o l p y r u v a t e c a r b o x y l a s e . Bjorkman (5) has r e p o r t e d t h a t most of the P - e n o l p y r u v a t e c a r b o x y l a s e a c t i v i t y of A t r i p l e x r o s e a l e a v e s i s l o c a t e d i n the m e s o p h y l l t i s s u e . The s p e c i f i c a c t i v i t i e s of P - e n o l p y r u v a t e c a r b o x y l a s e and s e v e r a l o t h e r enzymes f rom the t i s s u e s of Zea and Gomphrena a r e p r e s e n t e d i n T a b l e I. E x t r a c t s e n r i c h e d i n the c o n t e n t s of e i t h e r m e s o p h y l l o r bundle sheath t i s s u e were p r e p a r e d by d i f f e r e n t i a l g r i n d i n g . The t o t a l p r o t e i n e x t r a c t e d f rom the l e a f was a p p r o x i m a t e l y e q u a l l y d i v i d e d between the two e x t r a c t s . Most of the P - e n o l p y r u v a t e c a r b o x y l a s e a c t i v i t y i s l o c a t e d i n the e x t r a c t of the m e s o p h y l l c e l l s . The impor tance of t h i s enzyme i n the i n i t i a l f i x a t i o n of C 0 2 , ( P i g . 1 ) , the h i g h P - e n o l p y r u v a t e c a r b o x y l a s e a c t i v i t y i n the e x t r a c t of the l e a f m e s o p h y l l (Tab le I) and the i n i t i a l l o -c a t i o n of x ^ c f i x e d , ( F i g . 3) i n d i c a t e t h a t the r a d i a l l y a r r a n g e d 16 m e s o p h y l l c e l l s a r e the s i t e o f f i x a t i o n o f C 0 2 l e a v e s o f Zea and Gomphrena. The c e l l s o f the bund le shea th c o u l d no t f i x v e r y much C 0 2 i n t o C ^ - a c i d s . Rap id t r a n s p o r t o f the p r o d u c t s o f f i x a t i o n , n o t CC»2 f i x a t i o n i n the bundle s h e a t h , p r o b a b l y 14 a c c o u n t s f o r the a c c u m u l a t i o n of C i l l u s t r a t e d i n F i g u r e 2 . The s i t e o f c a r b o x y l t r a n s f e r P l a n t s w i t h the ^ 3 - c a r b o x y l a t i o n pathway c o n t a i n normal RuDP c a r b o x y l a s e a c t i v i t i e s (5)» D u r i n g s h o r t term f e e d i n g s , 14 however, v e r y l i t t l e C 0 2 i s f i x e d i n the r e a c t i o n c a t a l y z e d by t h i s enzyme. T h i s may be due tot*the f a c t t h a t much o f t h i s enzyme i s l o c a t e d i n the c e l l s o f the bund le s h e a t h . T h i s was f i r s t r e p o r t e d by Bjorkman f o r A t r l p l e x r o s e a (5). The d a t a p r e s e n t e d i n T a b l e I i n d i c a t e t h a t most o f the RuDP c a r b o x y l a s e o f Zea and Gomphrena i s l o c a t e d i n the bund le s h e a t h . " M a l i c " enzyme, which c a t a l y z e s the o x i d a t i v e d e c a r b o x y l a -t i o n ' o f m a l i c a c i d , i s a l s o p r e s e n t i n the bund le s h e a t h ( T a b l e I ) , and c o u l d f u n c t i o n t o g e n e r a t e G 0 2 f rom m a l i c a c i d . I f the r e a c t i o n s i n v o l v i n g " m a l i c " enzyme*and RuDP c a r b o x y l a s e a r e c o u p l e d , C - ^ - l a b e l l e d 3-PGA would be formed f rom C ^ - l a b e l l e d m a l i c a c i d . T h i s i s c o n s i s t e n t w i t h the d a t a o f Ha tch and S l a c k ( 1 1 ) . T a b l e II p r e s e n t s a compar ison o f the i n v i v o p h o t o s y n t h e t i c r a t e s t o the a c t i v i t i e s o f RuDP c a r b o x y l a s e , " m a l i c " enzyme, and P - e n o l p y r u v a t e c a r b o x y l a s e e x t r a c t e d f rom l e a v e s o f Z e a . Gomphrena and wheat . The e x t r a c t s o f Zea and Gomphrena c o n t a i n e d " m a l i c " enzyme a c t i v i t y g r e a t e r than the i n v i v o r a t e o f p h o t o -s y n t h e s i s . The a c t i v i t y o f RuDP c a r b o x y l a s e approach the p h o t o -s y n t h e t i c r a t e . The a c t i v i t i e s , o f t h e s e enzymes a r e , t h e r e f o r e , C h a p t e r I TABLE I. A c t i v i t i e s of enzymes i n e x t r a c t s of m e s o p h y l l and bund le sheath t i s s u e s of Zea mays and Gomphrena g l o b o s a . Enzyme Zea mays mesophy l l bundle sheath Gomphrena g l o b o s a mesophy l l bundle sheath a c t i v i t i e s expressed i n ,umoles/mg p r o t e i n x m in . PEP c a r b o x y l a s e 0 . ? 1 * , 0 . 8 3 * 0 .04 , 0.10 1 .25, 1.51 0 .04 , 0.12 RuDP c a r b o x y l a s e 0 . 1 3 , 0 .08 0 . 2 2 , 0.29 0 . 0 6 , 0.11 0 . 3 4 , 0.26 " M a l i c " enzyme 0 . 1 3 , 1.01 2 . 6 , 7.7 0 . 3 8 , 0.74 2 . 08 2 . 4 4 NADP-malate dehydrogenase 0.96 0.10 U.20- 0.07 R e p l i c a t e s 18 C h a p t e r I TABLE I I . Compar ison of the i n v i v o p h o t o s y n t h e t i c r a t e t o the e x t r a c t a b l e a c t i v i t i e s of some p h o t o s y n t h e t i c enzymes. Wheat Zea Gomphrena P r o c e s s ^ m o l e s / m i n . gram f r e s h wt. In v i v o r a t e of of p h o t o s y n t h e s i s 3 . 2 * 3 . 5 1.4 RuDP C a r b o x y l a s e 4 .6 2 .2 1.0 P - e n o l p y r u v a t e C a r b o x y l a s e 0.12 10 .3 7.2 " M a l i c " enzyme n i l 24.1 9.2 Measured i n 2% 0 c o m p a t i b l e w i t h t h e i r p roposed f u n c t i o n . " M a l i c " enzyme a c t i v i t y i s i n excess of the RuDP c a r b o x y l a s e a c t i v i t y . The r a t e of t h i s r e a c t i o n c o u l d be c o n t r o l l e d by the a v a i l a b i l i t y of NADP. NADPH g e n e r a t e d i n t h i s r e a c t i o n might p a r t i c i p a t e i n the subsequent r e d u c t i o n of 3 -PGA. RuDP c a r b o x y l a s e and " m a l i c " enzyme a l s o o c c u r i n the e x -t r a c t s of the m e s o p h y l l c e l l s . T h i s may be due t o breakage of some bundle shea th c e l l s d u r i n g the f i r s t g r i n d i n g , o r the enzyme may a l s o be p r e s e n t i n m e s o p h y l l c e l l s . S t a r c h i s formed i n the m e s o p h y l l of t h e s e p l a n t s under some c o n d i t i o n s . Carboxy l t r a n s -f e r , o r normal C a l v i n c y c l e f i x a t i o n , might a l s o o c c u r t o some ex ten t i n t h i s t i s s u e . I t would thus seem t h a t C 0 2 f i x a t i o n and the c a r b o x y l t r a n s f e r r e a c t i o n s a r e a t l e a s t p a r t i a l l y s e p -a r a t e d . J B - c a r b o x y l a t i o n o c c u r s i n the r a d i a l l y a r r a n g e d meso-19 p h y l l c e l l s while the c a r b o x y l t r a n s f e r i s predominantly i n the c e l l s of the bundle sheath. The movement of C^-acids t o the bundle sheath a l s o i n d i c a t e s the sheath i s the major s i t e f o r the c a r b o x y l t r a n s f e r . S l a c k and Hatch (15, 36) r e p o r t t h a t P-enolpyruvate c a r b o x y l a s e , RuDP ca r b o x y l a s e , " m a l i c " en-zyme and NADP-specific malate dehydrogenase are l o c a t e d i n the c h l o r o p l a s t s of Zea mays. Evidence presented e a r l i e r i n t h i s paper i n d i c a t e s t h a t the enzymes a r e predominantly i n one c e l l type. C h l o r o p l a s t s of the bundle sheath t h e r e f o r e c o n t a i n RuDP carboxylase and " m a l i c " enzyme, w h i l e c h l o r o p l a s t s of the mesophyll c o n t a i n P-enolpyruvate c a r b o x y l a s e , and are the s i t e of ^ - c a r b o x y l a t i o n . The product of ^3-carboxylation, oxalo-a c e t i c a c i d may be reduced t o m a l i c a c i d by NADPH produced i n the mesophyll c h l o r o p l a s t or transaminated t o produce a s p a r t i c a c i d . These form the C ^ - a c i d p o o l . The p o s t u l a t e d " m a l i c " enzyme - RuDP car b o x y l a s e mediated s y n t h e s i s of 3-PGA withdraws m a l i c a c i d from t h i s p o o l . T r a n s p o r t of m e t a b o l i t e s between s i t e s The s i t e s of s y n t h e s i s and u t i l i z a t i o n of the C^-acids ap-pear t o be s p a t i a l l y separate. Net movement of C ^ - a c i d must occur from the mesophyll t o the bundle sheath i n order t o sup-po r t the s y n t h e s i s of 3-PGA. Plasmodesmata which l i n k the c y t o -plasm of the two c e l l l a y e r s (25) c o u l d supply the path f o r t h i s t r a n s p o r t p r o v i d e d t h a t the C ^ - a c i d s , i n i t i a l l y s y n t h e s i z e d i n the c h l o r o p l a s t , a r e r e l e a s e d i n t o the cytoplasm. The nonaqueous c h l o r o p l a s t i s o l a t i o n technique has been used by o t h e r i n v e s t i g a t o r s t o t r a c e the d i s t r i b u t i o n and move-ment of p h o t o s y n t h e t i c m e t a b o l i t e s between the cytoplasm and the 20 c h l o r o p l a s t s (16, 17 , 1 8 ) . We a p p l i e d t h i s t e c h n i q u e t o Gom-phrena and Zea i n o r d e r t o de te rmine the d i s t r i b u t i o n and move-ment o f C ^ - a c i d s and p h o s p h o r y l a t e d compounds i n t h e s e p l a n t s . The r e s u l t s a r e p r e s e n t e d i n T a b l e I I I f o r s e v e r a l p u l s e - c h a s e type f e e d i n g s . The v a l u e s (y) r e p r e s e n t the p e r c e n t a g e s o f the t o t a l l e a f complement o f e i t h e r C ^ - a c i d s o r phosphate e s t e r s t h a t a r e i s o l a t e d w i t h the c h l o r o p l a s t s . Thus (100-y) i s the p e r c e n t l o c a t e d i n the remainder o f the l e a f ( c y t o p l a s m , and o t h e r o r g a n e l l e s ) . The p e r c e n t o f l e a f p r o t e i n a s s o c i a t e d w i t h the c h l o r o p l a s t s (x) i s a l s o g i v e n t o g e t h e r w i t h the s t a n d a r d e r r o r o f the mean. In our hands r e p r o d u c i b i l i t y was poor compared t o o t h e r p u b l i s h e d r e s u l t s (a t l e a s t + 20%). We have some r e s e r v a t i o n s c o n c e r n i n g t h e a p p l i c a t i o n o f t h i s t e c h n i q u e t o p l a n t s o f t h i s t y p e . The i s o l a t e d c h l o r o p l a s t f r a c t i o n s c o n t a i n e d bo th meso-p h y l l and bund le shea th c h l o r o p l a s t s . The bund le shea th c h l o r o -p l a s t s a r e more d i f f i c u l t t o r e l e a s e by g r i n d i n g i n nonaqueous medium. A l s o the bund le shea th c h l o r o p l a s t s o f Zea c o n t a i n few o v e r l a p p i n g i n t e r n a l membranes and may f ragment more e a s i l y i n nonaqueous s o l v e n t s . Both o f t h e s e f a c t o r s would reduce the y i e l d o f bund le s h e a t h c h l o r o p l a s t s r e l a t i v e t o m e s o p h y l l c h l o r o p l a s t s . In a d d i t i o n the p e r i p h e r a l r e t i c u l u m , which i s a membrane e l a b o r a -t i o n p e c u l i a r t o c h l o r o p l a s t s o f p l a n t s w i t h ^ B - c a r b o x y l a t i o n , may-be s t r i p p e d away f rom the c h l o r o p l a s t body i n p r e p a r a t i o n . In s p i t e o f the poor r e p r o d u c i b i l i t y two t r e n d s a r e e v i d e n t i n the r e s u l t s , T a b l e I I I . The C ^ - a c l d s o f Gomphrena and Zea a r e not c o n f i n e d t o the c h l o r o p l a s t s . A l a r g e percen tage (50 -75%) o f t h e s e compounds o c c u r i n the c y t o p l a s m . Thus i t appears 21 C h a p t e r I TABLE I I I . F r a c t i o n (y) o f l a b e l l e d m a t e r i a l c o n t a i n e d i n the c h l o r o p l a s t s . F e e d i n g _ _ Phosphate C0 2 sec - C0 2 s e c C ^ - a c l d s e s t e r s Gomphrena g l o b o s a 6 - 0 30% 92% 6 - 15 27 93 6 - 30 37 95 P r o t e i n i n c h l o r o p l a s t s = 57+ 11$ Zea mays 2 - 0 3k% 29% 2 - 5 34 44 2 - 20 51 25 P r o t e i n i n c h l o r o p l a s t s = 51+_ 9% t h a t t r a n s p o r t v i a the c y t o p l a s m i s p o s s i b l e . Even a f t e r v e r y s h o r t f e e d i n g s ( e g . Zea f e d ^ C O g f o r 2 s e c ) , most o f the l a b e l l e d C ^ - a c i d s were i n the c y t o p l a s m . I f the s y n t h e s i s o f the C ^ - a c i d s o c c u r s w i t h i n the c h l o r o p l a s t s then they s h o u l d appear i n t h e c h l o r o p l a s t s b e f o r e t h e c y t o p l a s m . Our r e s u l t s i n d i c a t e t h a t e i t h e r movement out o f t h e c h l o r o p l a s t o c c u r s v e r y r a p i d l y , o r t h a t t h e s i t e o f s y n t h e s i s i s no t i n the c h l o r o -p l a s t . T h i s w i l l r e q u i r e f u r t h e r s t u d y . The phosphate e s t e r s o f Gomphrena a r e l o c a t e d i n the c h l o r o -p l a s t . F o r chase t ime up t o 30 seconds f o l l o w i n g the "^COg p u l s e a l l o f t h e l a b e l l e d phosphates o f the l e a f were i n t h e c h l o r o -p l a s t s . Wi th l o n g e r chase t imes (not r e p o r t e d he re ) the p r o p o r -22 t l o n of l a b e l l e d phosphates i n the c h l o r o p l a s t s tended t o d e -c l i n e . The f a c t t h a t the phosphate e s t e r s show a d i f f e r e n t d i s t r i b u t i o n t h a n do the C ^ - a c i d s i n t h e s e exper iments l e n d s suppor t t o the v a l i d i t y of our t e c h n i q u e . The d i s t r i b u t i o n of phosphate e s t e r s i n Zea i s v e r y d i f -f e r e n t f rom t h a t of Gomphrena, and of o t h e r p l a n t s (16) . P h o s -phate e s t e r s do not appear t o be c o n f i n e d t o the c h l o r o p l a s t s . F u r t h e r i n v e s t i g a t i o n s a r e n e c e s s a r y t o determine the s i g n i f -i c a n c e of t h i s o b s e r v a t i o n . These s t u d i e s show t h a t C ^ - a c i d s a r e a v a i l a b l e i n the c y t o p l a s m f o r t r a n s p o r t between c h l o r o p l a s t s . The d i s t r i b u t i o n of enzymes show t h a t the C ^ - a c i d s a r e s y n t h e s i z e d i n the meso-p h y l l c e l l s . The a u t o r a d i o g r a p h s i n d i c a t e t h a t these compounds a r e t r a n s p o r t e d f rom the mesophy l l t o the bundle sheath c e l l s . The e x i s t e n c e of abundant plasmodesmata (2 , 24 , 2$) and the c y t o p l a s m i c l o c a t i o n of C ^ - a c i d s p r o v i d e a mechanism t o e x p l a i n t h i s t r a n s p o r t . The d i s t a n c e s e p a r a t i n g the s i t e s i s s m a l l . T r a n s p o r t would be f rom one c e l l of the c o n c e n t r i c a l l y a r r a n g e d p a l i s a d e t i s s u e t o a bundle sheath c e l l l o c a t e d c e n t r i p e t a l t o i t . The p a l i s a d e c e l l s c o n t a i n a l a r g e c e n t r a l v a c u o l e , and a t h i n p e r i p h e r a l l a y e r of p r o t o p l a s m . C y c l o s i s c o u l d r a p i d l y pass a l l of t h i s p r o t o p l a s m pas t the w a l l s e p a r a t i n g the p a l i -sade c e l l f rom the bundle sheath c e l l . Very r a p i d communica-t i o n s between t h e s e c e l l s might t h e r e f o r e be e x p e c t e d . Carbon d i o x i d e exchange I f COg i s an i n t e r m e d i a t e i n the c a r b o x y l t r a n s f e r s t e p 14 some CO,, might be expected t o escape f rom the l e a f a f t e r the 14 l e a f i s removed f rom a CO env i ronment . T h i s a p p a r e n t l y does 23 not happen (11, 41). The r e a c t i o n s may be v e r y t i g h t l y c o u p l e d , o r c o n t a i n e d . A b u r s t of CO^ from Z e a , Sorghum and Gomphrena l e a v e s does o c c u r d u r i n g the onset of p h o t o s y n t h e s i s f o l l o w i n g a s h o r t d a r k p e r i o d . Bjorkman ( r e f . 4 P i g ; . 47) a l s o r e p o r t s such a b u r s t f o r Z e a . The source of t h i s C 0 2 was i n v e s t i g a t e d . 14 Data f o r C 0 2 p r o d u c t i o n , and C 0 2 p r o d u c t i o n by Zea l e a v e s a r e p r e s e n t e d i n F i g u r e 4. Carbon-l4 which was f i x e d by the l e a v e s d u r i n g the b e g i n n i n g of the dark p e r i o d appeared i n l a r g e q u a n -14 t i t y d u r i n g the b u r s t . The s p e c i f i c a c t i v i t y of CO which 2 appeared d u r i n g the b u r s t was 17 t imes t h a t produced i n the p r e -c e d i n g dark p e r i o d . T h i s b u r s t t h e r e f o r e r e p r e s e n t s d e c a r b o x y l a t i o n of a p roduc t of the p r e v i o u s f i x a t i o n . More t h a n 95$ of 14 the c f i x e d i n a s i m i l a r f e e d i n g was found i n the C ^ - a c i d s . A c t i v i t y of RuDP c a r b o x y l a s e i s l i g h t - s t i m u l a t e d and d e -c r e a s e s r a p i d l y i n da rkness (1). The ^ 3 - c a r b o x y l a t i o n pathway i s a p p a r e n t l y a c t i v e f o r a l o n g e r t ime a f t e r darkness ( 3 1 ) . but a l s o becomes i n a c t i v e . I t appears t h a t the d e c a r b o x y l a t i o n of m a l i c a c i d i s l i g h t - s t i m u l a t e d , and some of the C 0 2 produced escapes f rom the l e a f d u r i n g the t ime t h a t the C 0 2 - f i x i n g e n -zymes a r e r e c o v e r i n g i n a c t i v i t y . The b u r s t has not been o b -s e r v e d w i t h p l a n t s which l a c k the ^ - c a r b o x y l a t i o n pathway. The o c c u r r e n c e of t h i s C 0 2 l e a k i n the l i g h t i s c o m p a t i b l e w i t h our p o s t u l a t e t h a t C 0 2 i s a n i n t e r m e d i a t e i n the s y n t h e s i s of phosphates f rom C ^ - a c i d s , but i t has not been de te rmined t h a t the b u r s t shown i n F i g u r e 4 r e p r e s e n t s the c a r b o x y l t r a n s f e r s t e p . The a b i l i t y of t h e s e p l a n t s t o c a r r y out net p h o t o s y n t h e s i s a t v e r y low C 0 ? c o n c e n t r a t i o n s i s w e l l known. The l o c a t i o n of 24 P H O T O -SY N. F E E D I N G D A R K RESPI RATI 0 N BURST U C O T R A P P E D - C p m 2 1 5 6 2 2 6 6 5 0 t o CJ ZD O o CL O CJ 5 0 /J c \ U C 0 1 2 1 L I G H T LI G HT M i n . 5 10 15 20 C h a p t e r I , F i g u r e 4. P r o d u c t i o n o f CO? and C 0 2 by c o r n l e a v e s i n l i g h t and d a r k . See Methods f o r d e t a i l s . 2 5 ^ 3 - c a r b o x y l a t i o n i n the p a l i s a d e m e s o p h y l l i s w e l l s u i t e d f o r gas exchange w i t h t h e s t o m a t a l c a v i t y . T h i s l a y e r of c e l l s may a l s o s e r v e as an e f f i c i e n t t r a p which would p r e v e n t t h e l o s s of C 0 2 from t h e l e a f . Any C 0 2 w h i c h might escape from t h e b undle s h e a t h d u r i n g a c t i v e p h o t o s y n t h e s i s would v e r y l i k e l y be r e - f i x e d b e f o r e i t c o u l d escape from t h e p l a n t . These f a c t o r s may c o n t r i b u t e t o t h e l o w e r C 0 2 compensation p o i n t (7). The m e t a b o l i c a c t i v i t i e s of t h e m e s o p h y l l might a l s o i n c r e a s e t h e c o n c e n t r a t i o n of s u b s t r a t e s a v a i l a b l e t o t h e subsequent r e a c -t i o n s , of t h e b u n d l e s h e a t h . The mechanism p o s t u l a t e d t o e x p l a i n t h e c a r b o x y l t r a n s f e r i n t h e b u n d l e s h e a t h f o r Zea and Gomphrena i s s i m i l a r t o t h a t p o s t u l a t e d f o r t h e d e - a c i d i f i c a t i o n p r o c e s s which o c c u r s i n p l a n t s ' w i t h c r a s s u l a c e a n a c i d m e t a b o l i s m (39. 4 0 ) . I n p l a n t s w i t h t h e B - c a r b o x y l a t i o n pathway, C 0 2 f i x a t i o n ( a c i d i f i c a t i o n ) and t h e c a r b o x y l t r a n s f e r ( d e a c i d i f i c a t i o n ) o c c u r a t t h e same t i m e , w h i l e t h e s e o c c u r a t d i f f e r e n t t i m e s of t h e day i n C r a s -s u l a c e a n p l a n t s . The o c c u r r e n c e of two C 0 2 - f i x i n g r e a c t i o n s s i m u l t a n e o u s l y c o u l d r e s u l t i n c o m p e t i t i o n f o r s u b s t r a t e . Re-f i x a t i o n of G0 2 by P - e n o l p y r u v a t e c a r b o x y l a s e i n t h e bundle s h e a t h would i n t e r f e r e w i t h t h e c a r b o x y l t r a n s f e r r e a c t i o n . T h i s w a s t e f u l c o m p e t i t i o n i s a p p a r e n t l y e l i m i n a t e d by s e p a r a t e c o m p a r t m e n t a t i o n of t h e enzymes. Summary These e x p e r i m e n t s s u p p o r t t h e c o n c l u s i o n t h a t ^ - c a r b o x y l a -t i o n i s t h e i m p o r t a n t C 0 2 f i x a t i o n r e a c t i o n d u r i n g p h o t o s y n t h e s i s by Zea mays and Gomphrena g l o b o s a . S t u d i e s of t h e l o c a t i o n of 26 the c a r b o x y l a t i o n enzyme and of l a b e l l e d products i n d i c a t e t h a t t h i s r e a c t i o n probably i s c o n f i n e d t o the r a d i a l l y arranged p a l i -sade mesophyll c e l l s which surround the bundle sheath c e l l s . 14 Pulse and chase type CO,, fe e d i n g s i n d i c a t e t h a t the C ^ - a c i d products of ^ - c a r b o x y l a t i o n a re o b l i g a t o r y i n t e r m e d i a t e s i n the normal f o r m a t i o n of l a b e l l e d phosphorylated compounds which are a l s o formed d u r i n g p h o t o s y n t h e s i s . These compounds become l a b e l l e d v i a a c a r b o x y l t r a n s f e r r e a c t i o n which appears t o oc-cur i n the bundle sheath c e l l s . Enzymes, " m a l i c " enzyme and RuDP carboxylase which may be i n v o l v e d i n the c a r b o x y l t r a n s f e r r e a c t i o n ( s ) a re l o c a t e d i n the c e l l s of the bundle sheath. L a b e l l e d photosynthate, and u l t i m a t e l y s t a r c h accumulate i n these c e l l s . T r a n s p o r t of carbon from the s i t e of CO,, f i x a t i o n i n the p a l i s a d e mesophyll t i s s u e t o the s i t e of the c a r b o x y l - t r a n s f e r i n the c e l l s of the bundle sheath occurs between adjacent c e l l s c o n c e n t r i c a l l y arranged around the v a s c u l a r bundle. These c e l l s a r e l i n k e d by abundant plasmodesmata. The C.^ a c i d s , which may be the t r a n s p o r t m e t a b o l i t e s , a re not r e t a i n e d i n the c h l o r o -p l a s t s . The model presented here i s th a t the C^-acids pass v i a the c y t o p l a s m i c continuum from the p a l i s a d e c e l l t o the c e l l of the bundle sheath ad j a c e n t t o i t . W i t h i n the c h l o r o p l a s t s of the bundle sheath c e l l s , COg p a r t i c i p a t e s i n the c a r b o x y l t r a n s f e r . T h i s CO^ i s generated from m a l i c a c i d by "m a l i c " en-zyme, and r e f i x e d t o 3-PGA by RuDP car b o x y l a s e . NADPH a l s o generated i n the d e c a r b o x y l a t i o n of m a l i c a c i d could c o n t r i b u t e energy t o the r e a c t i o n s o c c u r r i n g i n the bundle sheath c h l o r o -p l a s t s . The l i g h t r e a c t i o n s of the mesophyll c h l o r o p l a s t s may 27 have i n i t i a l l y c o n t r i b u t e d the energy r e p r e s e n t e d by t h i s i n -termediate through NADP-malate dehydrogenase. The p a t t e r n of l a b e l l i n g of p h o t o s y n t h e t i c products, the d i s t r i b u t i o n of en-zymes w i t h i n the l e a f , and the gas exchange p r o p e r t i e s of p l a n t s with t h i s pathway appear t o be c o n s i s t e n t with t h i s pro-posed sequence of r e a c t i o n s . 28 References 1 . Bassham, J.A., and K i r k , Martha. I 9 6 8 . Dynamic me t a b o l i c r e g u l a t i o n of the p h o t o s y n t h e t i c carbon r e d u c t i o n c y c l e . I n : S h i b a t a , K., Takamiya, A., Jagendorf, A.T., and P u l l e r , R.C. ( E d s . ) . Comparative b i o c h e m i s t r y and b i o p h y s i c s of p h o t o s y n t h e s i s . U n i v e r s i t y Park Press P e n n s y l v a n i a , pp. 3 6 5 - 3 7 8 . 2 . 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Unter-suchungen zur i n t r a z e l l u r M r e n V e r t e i l u n g von Enzymen und S u b s t r a t e n i n der B l a t t z e l l e . I I . Z. Na t u r f o r s c h g . 22b: 1200-1215. 19. Jensen, W.A. I962. B o t a n i c a l H i s t o c h e m i s t r y . W.H. Freeman and Co. San F r a n c i s c o . 20. Johnson, H i l a r y S., and Hatch, M.D. 1968. D i s t r i b u t i o n of the C ^ - d i c a r b o x y l i c a c i d pathway of ph o t o s y n t h e s i s and i t s occurrence i n d i c o t y l e d o n o u s p l a n t s . Phytochem ?: 375-380. 21. Kortschak, Hugo P., H a r t t , Constance E., Burr, George 0. 1965. Carbon d i o x i d e f i x a t i o n i n sugar cane l e a v e s . P l a n t P h y s i o l . 40: 209-213. 22. Latzko, E., and Gibbs, M a r t i n . 1968. D i s t r i b u t i o n and ac-t i v i t y of enzymes of the r e d u c t i v e pentose phosphate c y c l e i n spinach l e a v e s and i n c h l o r o p l a s t s i s o l a t e d by d i f f e r e n t methods. Z. P f l a n z e n p h y s i o l . 59: 184-194. 23. L a e t s c h , W.M., S t e t l e r , D.A., and V l i t o s , A . J . 1966. The u l t r a s t r u c t u r e of sugar cane c h l o r o p l a s t s . Z. P f l a n z e n -p h y s i o l . 54: 472-474. 30 24. L a e t s c h , W.M. 1968. C h l o r o p l a s t s p e c i a l i z a t i o n i n d i c o t y l e -dons p o s s e s s i n g the C ^ - d l c a r b o x y l i c a c i d pathway of p h o t o s y n t h e t i c COp f i x a t i o n . Amer. J . Botany 55• 875-883. 25. L a e t s c h , W.M., and P r i c e , Ian. 1969. Development of the dimorphic c h l o r o p l a s t s of sugar cane. Amer. J . Bot. 56: 77-87. 26. Lowry, O.H., Rosenbrough, N.J., F a r r , A.L., and R a n d a l l , R.J. 1951. P r o t e i n measurement with the F o l i n phenol reagent. J . B i o l . Chem. 193s 265-275-27. Moss, Dale N. and Rasmussen, H. P a u l . 1969. T r a n s l o c a t i o n and c e l l u l a r l o c a l i z a t i o n of C0 2 f i x a t i o n . P l a n t P h y s i o l . 4 4 : 1063-1068. 28. Osmond, B. 1967. ^ - c a r b o x y l a t i o n d u r i n g p h o t o s y n t h e s i s i n A t r l p l e x . Biochim. Biophys. A c t a 141: 197-199. 2 9 . Osmond, C.B. 1969. ^ - c a r b o x y l a t i o n 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 o n i n h i g h e r p l a n t s . Biochim. Biophys. A c t a 172: 144-149. 30. Pedersen, P.A., K i r k , Martha, and Bassham, J.A. 1966. I n h i b i t i o n of p h o t o p h o s p h o r y l a t i o n and p h o t o s y n t h e t i c carbon c y c l e r e a c t i o n s by f a t t y a c i d s and e s t e r s . Biochim. Biophys. A c t a 112: 189-203. 31. Poskuta, J . 1969. P h o t o s y n t h e s i s , r e s p i r a t i o n and p o s t -i l l u m i n a t i o n f i x a t i o n of C0 2 by Corn l e a v e s as i n f l u -enced by l i g h t and oxygen c o n c e n t r a t i o n . P h y s i o l o g i a Plantarum 2 2 : 7 6 - 8 5 . 32. P r l s t u p a , N.A. 1964. R e d i s t r i b u t i o n of r a d i o a c t i v e a s s i m i - r l a t e s i n the l e a f of c e r e a l s . S o v i e t P l a n t Physio. 11: 31-36. 33. Rhoades, M.M., and Carvalho, A. 19^4. The f u n c t i o n and s t r u c t u r e of the parenchyma sheath p l a s t i d s of the maize l e a f . B u l l . T o r r e y Botan. Club 71s 335-3^6. 34. Rosado-Alberio, Jaime, Weier, T.E., and S t o c k i n g , C R . 1968. C o n t i n u i t y of the c h l o r o p l a s t membrane systems i n Zea  mays L. P l a n t P h y s i o l . 4 3 : 1325-1331. 35. R u t t e r , W.J., and Lardy, H.A. 1958. P u r i f i c a t i o n and prop-e r t i e s of pigeon l i v e r m a l i c enzyme. J . B i o l . Chem. 213: 374-382. 3 6 . S l a c k , C.R., and Hatch, M.D. I 9 6 7 . Comparative s t u d i e s on the a c t i v i t y of c a r b o x y l a s e s and other enzymes i n r e -l a t i o n t o the new pathway of p h o t o s y n t h e t i c carbon d i o x i d e f i x a t i o n i n t r o p i c a l g r a s s e s . Biochem. J . 103: 660-665. 31 3 7 . T h a l a c k e r , R . , u n d B e h r e n s , M. 1959 . Uber den R e i n h e l t s g r a d d e r i n einem n i c h t w a s s r i g e n s p e z i f i s c h e n G e w i c h t s g r a d i e n -t e n gewonnenen C h l o r o p l a s t e n . Z . N u t r f o r s c h . 146: 4 4 3 . 3 8 . S t r a i n , H a r o l d H . , and S v e c , W a l t e r A . i 9 6 0 . E x t r a c t i o n , s e p a r a t i o n , and i s o l a t i o n of the c h l o r o p h y l l s . I n : V e r n o n , L . P . , and S e e l y , G .R . ( E d s . ) . The C h l o r o p h y l l s . Academic P r e s s . New Y o r k , London , p p . 2 1 - 6 6 . 3 9 . Wa lker , D . A . , and Brown, J . M . A . 1 9 5 7 . P h y s i o l o g i c a l s t u d i e s on a c i d m e t a b o l i s m . 5 B iochem. J . 6 7 : 7 9 - 8 3 . 40. Wa lker , D . A . i 9 6 0 . P h y s i o l o g i c a l s t u d i e s of a c i d m e t a b o l i s m . 7 B iochem. J . ?ki 2 1 3 - 2 2 3 . 41 . Z e l i t c h , I s r a e l . 1 9 6 8 , I n v e s t i g a t i o n s on p h o t o r e s p i r a t i o n w i t h a s e n s i t i v e J - ^ c - a s s a y . P lan t . P h y s i o l . 4 3 : 1 8 2 9 - 1 8 3 7 . 32 C h a p t e r I I Assay p r o c e d u r e s , K i n e t i c p r o p e r t i e s , and the i s o l a t i o n o f • •mal ic" enzyme f rom Gomphrena g l o b o s a and Zea mays. In the p r e v i o u s c h a p t e r we sugges ted t h a t two c o n s e c u t i v e c a r b o x y l a t i o n s may be i n v o l v e d i n the J3-carboxylation pathway o f p h o t o s y n t h e s i s which o c c u r s i n Zea mays and Gomphrena g l o b o s a . C o n s i s t e n t w i t h t h i s h y p o t h e s i s we found a " b u r s t " o f C0 2 f rom l e a v e s o f Zea and Gomphrena which was l i g h t - s t i m u l a t e d . I t was not O g - s e n s i t i v e , hence i t i s u n l i k e l y t h a t i t was d e r i v e d from p h o t o r e s p i r a t i o n . We showed t h a t COg e v o l v e d d u r i n g t h i s b u r s t i s p r o b a b l y d e r i v e d f rom a p roduc t o f ^5 -carboxy la t lon . A p o s -s i b l e s o u r c e o f t h i s C0 2 i s the o x i d a t i v e d e c a r b o x y l a t i o n o f m a l i c a c i d brought about by " m a l i c " enzyme. There i s however l i t t l e r e a s o n t o expect t h a t t h i s enzyme i s l i g h t - a c t i v a t e d . The o x i d i z e d c o f a c t o r NADP i s r e q u i r e d . T h i s s h o u l d be more a v a i l a b l e i n t h e dark t h a n i n the l i g h t . The s t u d i e s o f Ha tch and S l a c k (3), however i n d i c a t e t h a t m a l i c and a s p a r t i c a c i d s remain i n the l e a v e s o f sugarcane i n the dark f o r s e v e r a l m i n -u t e s a f t e r t h e i r f o r m a t i o n . E i t h e r m a l i c a c i d i s n o t a v a i l a b l e t o the enzyme i n the d a r k , o r the enzyme i s i n a c t i v e . The s t u d i e s o f S l a c k and Hatch (7) i n d i c a t e d t h a t " m a l i c " enzyme o f Zea i s l o c a t e d In the c h l o r o p l a s t s . Recent s t u d i e s have I n d i c a t e d t h a t some o t h e r c h l o r o p l a s t enzymes a r e l i g h t a c t i v a t e d . These a r e f r u c t o s e d i p h o s p h a t e phosphatase (5) and r i b u l o s e d i p h o s p h a t e c a r b o x y l a s e (2). The mechanism o f a c t i v a t i o n o f t h e s e enzymes may be s i m i l a r . Both enzymes show a s h i f t i n t h e i r pH optimum t o lower pH v a l u e s w i t h i n c r e a s i n g 33 +2 Mg c o n c e n t r a t i o n . I t has been argued t h a t c o n t r o l o f t h e s e enzymes i n v i v o i s e f f e c t e d by l i g h t - s t i m u l a t e d changes i n the c o n c e n t r a t i o n s o f M g + 2 and H + i n the c h l o r o p l a s t s t r o m a , which i s presumed t o be the l o c a t i o n o f t h e s e enzymes. Magnesium o r manganese i o n s a r e r e q u i r e d f o r " m a l i c " e n -zyme a c t i v i t y (6) , but t h e r e i s no e v i d e n c e o f a r e g u l a t o r y + f u n c t i o n o f e i t h e r H o r d i v a l e n t c a t i o n s . The k i n e t i c p r o p e r -t i e s o f t h i s enzyme were I n v e s t i g a t e d i n - o r d e r t o t e s t the p o s -s i b i l i t y t h a t a c t i v a t i o n o f t h i s c h l o r o p l a s t enzyme by l i g h t f o l l o w s a s i m i l a r mechanism. I n v e s t i g a t i o n s were a l s o per formed t o de te rmine the o p -timum c o n d i t i o n s f o r s p e c t r o p h o t o m e t r i c a s s a y o f t h i s enzyme i n l e a f e x t r a c t s , and t o v e r i f y t h a t t h e r e a c t i o n a s s a y e d r e -s u l t s i n the p r o d u c t i o n o f COg. P l a n t s o f Gomphrena g l o b o s a and Zea mays were grown as d e s c r i b e d p r e v i o u s l y on page 3» Assay o f " m a l i c " enzyme was as d e s c r i b e d on page 8. Each a s s a y c u v e t t e c o n t a i n e d the f o l l o w i n g : 2 .88 ml o f b u f f e r p l u s an a p p r o p r i a t e q u a n t i t y o f enzyme e x t r a c t ; 50 / i l NADP 3 mg/0.5 ml H 2 0 ; 50 p.1 MgClg . When the O D ^ 0 had become s t a b l e the r e a c t i o n was s t a r t e d by a d d i t i o n o f 10 ^ i l o f IM L - m a l i c a c i d (sodium s a l t pH 7 .0) T r i s - H C l , 0.1 M was used t o b u f f e r a t pH 7 . 7 , 8 . 0 , and 9 . 0 HEPES, 0.1 M was used t o b u f f e r a t pH 7 . 0 , 7 . 3 . and 7 . 5 . In the i s o l a t i o n p rocedure a c t i v i t y o f " m a l i c " enzyme was measured a t pH 9 . 0 . In p l a n t e x t r a c t s a s s a y s were r o u t i n e l y per formed a t pH 8 . 0 . D e c a r b o x y l a t i o n o f m a l i c a c i d was a s s a y e d r a d i o c h e m i c a l l y i n 0 . 5 ml t o t a l volume c o n t a i n i n g 50 p. moles T r i s - H C l pH 8 . 0 ; 1.5 p- moles L - m a l i c a c i d -k-^G; 1 ja mole DTT; 2 u moles NADP; 30 1 j i mole M g C l 2 ; and 0 .25 ml o f enzyme. A l l q u o t s o f 50 pi each were taken a t one minute i n t e r v a l s a f t e r a d d i t i o n o f the m a l i c a c i d . The a c i d s t a b l e C was de te rmined as i n the RuDP c a r -b o x y l a s e a s s a y d e s c r i b e d on page 8. I s o l a t i o n o f " m a l i c " enzyme s t a r t e d f rom an e x t r a c t o f l e a v e s o f Gomphrena g l o b o s a . T h i r t y t h r e e grams o f l e a v e s were ground i n a Waring b l e n d o r w i t h 300 ml o f 0 . 0 5 M T r i s - H C l pH8.3; c o n t a i n i n g 8 g p o l y v i n y l p y r o l l i d o n e : and 0.16 g DTT. The r e s i -due was removed by f i l t r a t i o n w i t h cheese c l o t h and ground w i t h sand and g r i n d i n g medium in^mortar- and p e s t l e . The com-b i n e d e x t r a c t s were c e n t r i f u g e d 10 min a t 20.000 x g . F i n a l volume was 375 m l ; 78 g o f s o l i d (NH^gSO^ was added t o b r i n g the s u s p e n s i o n t o 0 .35 s a t u r a t i o n . The p r e c i p i t a t e was s e p a -r a t e d by c e n t r i f u g a t i o n and d i s c a r d e d . The s u p e r n a t a n t was brought t o 0.46 s a t u r a t i o n by a d d i t i o n o f 25 g o f s o l i d (NH^JgSO^. The p r e c i p i t a t e c o n t a i n e d most o f the " m a l i c " enzyme a c t i v i t y and was r e t a i n e d . T h i s was d i s s o l v e d i n 20 ml o f 20 mM Na^PgOr, b u f f e r pH 6 . 2 , and d i a l i z e d f o r 3 h r . C a l c i u m phosphate g e l (25 mg/ml) was added i n s m a l l ba tches t o the d i a l i z e d p r e p a r a t i o n and removed by c e n t r i f u g a t i o n . T o t a l a c t i v i t y o f " m a l i c " enzyme began t o d e c l i n e a f t e r 30 ml o f g e l had been a d d e d . V i r -t u a l l y no a c t i v i t y remained i n s o l u t i o n a f t e r the next 8 ml b a t c h o f g e l was removed. The ba tches o f g e l c o n t a i n i n g the a b s o r b e d enzyme were washed w i t h 5 ml HgO; 5 ml o f 50 mM T r i s -H C l b u f f e r pH 8.3; and t w i c e w i t h 5 ml T r i s - H C l pH 8.3 c o n t a i n -i n g 10% (NHj^^SO^. The l a s t two washes c o n t a i n e d the enzyme. T h i s was d i a l i z e d o v e r n i g h t a g a i n s t two changes o f b u f f e r : 0 . 0 5 M T r i s - H C l pH8.3; 1 mM EDTA; and 1 mM DTT. The i s o l a t i o n p r o c e -35 Chapter II Table I Summary of the isolation of "malic" enzyme. Step Total activity umole min - 1 Specific Activity /imole min mg protein Yield % I Crude extract 6 3 0 . 3 0 100 II Ammonium sulfate precipitation 37 59 III Dialysis 16 hours 33 52 IV Calcium phosphate gel eluate 18.5 19.7 29 V Dialysis 16 hours 14.7 23 dure i s summarized in Table I. "Malic" enzyme was also pre-pared from leaves of Zea mays through step III by the procedure outlined above. The f i n a l enzyme preparation was unstable and lost about 50$ of i t s activity upon storage 24 hr at 4°. It was essen-t i a l l y free of MADP-malate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase. No "malic" enzyme activity was detected in the presence of NAD in place of NADP. Figure 1 shows the substantial effect of pH upon the af-f i n i t y of the isolated "malic" enzyme for malic acid. The -4 K g (malate) i s 6.4 x 10 -'M at pH 7 . 5 ; 1.7 x 10 M at pH 7 .7; -4 - 3 5 x 10 M at pH 8 . 0 ; and 5 x 10 H at pH 9 . 0 . Rutter and -4 Lardy (6) report K„ (malate) of 3 . 9 x 10 M at pH 7 . 5 . and 36 3.3 x 10~^M a t pH 8.5 f o r " m a l i c " enzyme p r e p a r e d f rom p i g e o n l i v e r . The " m a l i c " enzyme o f Ka lanchoe c r e n a t a . a c c o r d i n g t o Walker (8) has a K (malate) o f 5.5 x 10"%! a t pH 7 .0. The s s u b s t a n t i a l e f f e c t o f pH upon the a f f i n i t y o f the enzyme f o r m a l i c a c i d was r e p o r t e d by R u t t e r and L a r d y . They a t t r i b u t e i t t o the p r e s e n c e o f an i o n i z a b l e group i n v o l v e d i n b i n d i n g t h e s u b s t r a t e . C l e a r l y a s i m i l a r group i s i n v o l v e d i n the c a t a l y t i c a c t i v i t i e s o f " m a l i c " enzyme f rom Gomphrena and p i g -eon l i v e r . However the enzyme f rom Gomphrena has a s l i g h t l y g r e a t e r a f f i n i t y f o r m a l a t e . The f i n a l d i a l i z e d p r e p a r a t i o n had a n a b s o l u t e requ i rement f o r added d i v a l e n t c a t i o n s . T h i s c o u l d be s a t i s f i e d by the a d d i t i o n o f Mg o r Mn . The requ i rement f o r Mn was lower +2 t h a n t h a t f o r Mg . There was v e r y l i t t l e e f f e c t o f pH upon +2 +2 t h e requ i rement f o r Mg . The K e ( M g ) was de te rmined t o be s - i i . 3-5 x 10 M o v e r the range f rom pH 7.3 t o 8 .5 . There was no +2 e v i d e n c e t h a t Mg was i n t e r a c t i n g i n any way w i t h the a f f i -n i t y o f t h e enzyme f o r m a l a t e . Changes i n the pH c o u l d e f f e c t some c o n t r o l o v e r the enzyme a c t i v i t y a t v e r y low mala te c o n -c e n t r a t i o n s . The p o s t u l a t e d pH s h i f t o f the c h l o r o p l a s t s t roma i s however toward more a l k a l i n e v a l u e s upon i l l u m i n a t i o n . T h i s would reduce enzyme a c t i v i t y . There was a g e n e r a l d e p r e s s i o n o f c a t a l y t i c a c t i v i t y w i t h the a d d i t i o n o f 10 j imole of AMP, ADP o r ATP t o the a s s a y m i x -t u r e . T h i s n o n s p e c i f i c e f f e c t was p r o b a b l y due t o b i n d i n g o f +2 Mg r a t h e r t h a n a s p e c i f i c e f f e c t o f t h e s e compounds on the enzyme. M a l o n i c a c i d , which i s r e p o r t e d by K o r t s c h a k (0) t o i n h i b i t p h o t o s y n t h e s i s by s u g a r c a n e , appears t o c o m p e t i t i v e l y i n h i b i t " m a l i c " enzyme. Ma lon ic a c i d i s a l s o r e p o r t e d t o 37 C h a p t e r II F i g u r e 1. The e f f e c t o f pH and mala te c o n c e n t r a t i o n on the r a t e o f ' ' m a l i c " enzyme c a t a l y z e d r e d u c t i o n o f NADP. The pH i s i n d i c a t e d i n ( ) . 70 L 1 2 3 ^ 5 6 7^ 8 9 1 0 * * 8 0 8 4 M x 10 mala te 38 Chapter II ^ Figure 2. Malate-4- C remaining a f t e r incubation with "malic" enzyme. 3 9 14 i n h i b i t i n c o r p o r a t i o n o f G i n t o m a l i c a c i d d u r i n g p h o t o s y n -t h e s i s by Scenedesmus ( 1 ) . These p r e l i m i n a r y exper iments i n d i c a t e t h a t r e g u l a t i o n o f " m a l i c " enzyme a c t i v i t y i f i t does o c c u r must be by a mechanism o t h e r than t h o s e t e s t e d . One p o s s i b i l i t y i s c o n t r o l th rough t h e a v a i l a b i l i t y o f s u b s t r a t e . M a l i c a c i d i s p r o b a b l y s y n t h e -s i z e d i n a d i f f e r e n t s i t e w i t h i n the l e a f f rom the s i t e o f d e -c a r b o x y l a t i o n . I f d e c a r b o x y l a t i o n i s t o be s u s t a i n e d t r a n s p o r t o f m a l i c a c i d must o c c u r between s i t e s . I f the t r a n s p o r t mecha-n i s m i s l i g h t - a c t i v a t e d t h i s c o u l d s e r v e t o c o n t r o l the s u p p l y o f s u b s t r a t e t o the enzyme. O x i d a t i o n d e c a r b o x y l a t i o n : In o r d e r f o r " m a l i c " enzyme t o f u n c t i o n as proposed i n t h e c a r b o x y l t r a n s f e r mechanism i t must make COg a v a i l a b l e t o RuDP c a r b o x y l a s e f o r f i x a t i o n . F o r t h i s s tudy " m a l i c " enzyme was p r e p a r e d th rough s t e p I I I (ammonium s u l f a t e f r a c t i o n a t i o n f o l l o w e d by d i a l y s i s ) f rom l e a v e s o f Zea mays. D e c a r b o x y l a t i o n 14 was a s s a y e d by the d e c l i n e i n a c i d s t a b l e C as d e s c r i b e d a b o v e . The c o u r s e o f d e c a r b o x y l a t i o n i s p l o t t e d i n F i g u r e 2; ? A p p r o x i m a t e l y 1 umole o f m a l i c a c i d was d e c a r b o x y l a t e d i n the f i r s t m i n u t e . The d e c a r b o x y l a t i o n d i d no t go t o c o m p l e t i o n but r e a c h e d e q u i l i b r i u m . The r a t e and t ime c o u r s e o f the r e -a c t i o n were s i m i l a r when a s s a y e d s p e c t r o p h o t o m e t r i c a l l y . T h i s v e r i f i e s t h a t C 0 2 i s produced by the enzyme a s s a y e d i n t h e s e s t u d i e s . The r o u t i n e a s s a y o f t h i s enzyme i n p l a n t e x t r a c t s was per formed as i n d i c a t e d i n the methods s e c t i o n . The enzyme was s t a b l e f o r 24 hours a t 0 ° i n c rude e x t r a c t s . The s p e c i f i c a c -t i v i t y o f some e x t r a c t s i n c r e a s e d upon t rea tment w i t h G-25 40 Sephadex. This may indicate the presence of a low molecular weight i n h i b i t o r . The i n i t i a l rate of reaction was used, as the rate declined upon approach to equilibrium. Conditions within the plant may be much clo s e r to equilibrium than those used i n the assay thus the net rate of reaction i n vivo may be lower than that indicated by these assays. 01 L i t e r a t u r e C i t e d 1. Bassham, J . A . , B e n s e n , A . A . , and C a l v i n , M. 1 9 5 0 . Pa th of ca rbon i n p h o t o s y n t h e s i s V I I I . The r o l e of m a l i c a c i d . J . B i o l . Chem. 1 8 5 : 7 8 1 - 7 8 7 . 2 . Bassham, J . A . , S h a r p , Pamela , and M o r r i s , I an . I 9 6 8 . The e f f e c t of Mg2+ c o n c e n t r a t i o n on the pH optimum and M i c h a e l i s c o n s t a n t s of the s p i n a c h c h l o r o p l a s t r i b u l o s e d i p h o s p h a t e c a r b o x y l a s e ( c a r b o x y d i s m u t a s e ) . B i o c h i m . B i o p h y s . A c t a 1 5 3 : 8 9 8 - 9 O O . 3. H a t c h , M . D . , and S l a c k , C R . 1 9 6 6 . P h o t o s y n t h e s i s by s u g a r -cane l e a v e s . A new c a r b o x y l a t i o n r e a c t i o n and the p a t h -way of sugar f o r m a t i o n . B iochem. J . 101s 1 0 3 - 1 1 1 . 4. K o r t s c h a k , Hugo P . , H a r t t , Constance E . , B u r r , George 0. 1 9 6 5 . Carbon d i o x i d e f i x a t i o n i n sugar cane l e a v e s . P l a n t P h y s i o l . 40: 2 0 9 - 2 1 3 . 5 . P r e i s s , J . , B i g g s , M . L . , and G r e e n b e r g , E . I 9 6 7 . The e f f e c t of magnesium i o n c o n c e n t r a t i o n on the pH optimum of the s p i n a c h l e a f a l k a l i n e f r u c t o s e d i p h o s p h a t a s e . J . B i o l . Chem. 242: 2 2 9 2 . 6 . R u t t e r , W . J . , and L a r d y , H . A . 1 9 5 8 . P u r i f i c a t i o n and p r o p -e r t i e s of p i g e o n l i v e r m a l i c enzyme. J . B i o l . Chem. 2 1 3 : 370 - 3 8 2 . 7. S l a c k , C R . , and H a t c h , M.D. 1 9 6 7 . Comparat ive s t u d i e s on the a c t i v i t i e s of c a r b o x y l a s e s and o t h e r enzymes i n r e -l a t i o n t o the new pathway of p h o t o s y n t h e t i c ca rbon d i o x i d e f i x a t i o n i n t r o p i c a l g r a s s e s . Biochem. J . 1 0 3 : 6 6 0 - 6 6 5 . 8 . W a l k e r , D .A . i 9 6 0 . P h y s i o l o g i c a l s t u d i e s of a c i d m e t a b o l i s m . 7 B iochem. J . 70: 2 1 3 - 2 2 3 . 42 Chapte r I I I L o c a t i o n of P - e n o l p y r u v a t e c a r b o x y l a s e . In v iew of the f i n d i n g r e p o r t e d (p . 21 ) t h a t much of 14 the c found i n C ^ - a c i d s i s l o c a t e d o u t s i d e of the c h l o r o -p l a s t s even a f t e r v e r y s h o r t - t e r m f e e d i n g s i t seemed a p p r o p r i -a t e t o re -examine the r e p o r t of S l a c k and Hatch ( 2 ) t h a t P - e n o l p y r u v a t e c a r b o x y l a s e was l o c a t e d i n the c h l o r o p l a s t s . F o r t h i s s tudy l e a v e s were d e s t a r c h e d , f r e e z e d r i e d , ground and f r a c t i o n a t e d as d e s c r i b e d on page 4 . T h e g i s o l a t e d f r a c -t i o n s were d r i e d under vacuum and e x t r a c t e d w i t h medium c o n -t a i n i n g : 50 mM T r i s - H C l pH 8 . 3 ; 10 mM, M g C l 2 ; 5 mM, d l t h i o t h r e -i t o l . T h i s e x t r a c t was subsampled f o r c h l o r o p h y l l d e t e r m i n a -t i o n , and t h e n a s s a y e d f o r P - e n o l p y r u v a t e c a r b o x y l a s e a c t i v i t y by the r a d i o c h e m i c a l t e c h n i q u e d e s c r i b e d on page 46 . Repreg s e n t a t i v e r e s u l t s of two s e p a r a t i o n s a r e shown i n T a b l e 'I. These i n d i c a t e t h a t P - e n o l p y r u v a t e c a r b o x y l a s e i s found i n both the c y t o p l a s m and the c h l o r o p l a s t s . The r e l a t i v e d i v i s i o n of the a c t i v i t y between the c h l o r o p l a s t s and the c y t o p l a s m d i f -f e r e d f rom p r e p a r a t i o n t o p r e p a r a t i o n , even w i t h the same l e a f m a t e r i a l . I t was c o n c l u d e d t h a t P - e n o l p y r u v a t e c a r b o x y l a s e was l o c a t e d i n some s t r u c t u r e which was not c o n s i s t e n t l y i s o l a t e d w i t h the c h l o r o p l a s t s . T h i s seems t o r u l e out the c h l o r o p l a s t s t roma . B a l d r y et a l . ( 1 ) r e p o r t t h a t a h i g h s p e c i f i c a c -t i v i t y f r a c t i o n of P - e n o l p y r u v a t e c a r b o x y l a s e was sedimented a t 140,000 x g . T h i s may i n d i c a t e t h a t the enzyme i s bound t o membranes. R e c e n t l y S l a c k et a l . ( 3 ) r e p o r t a r e - e x a m i n a t i o n of t h e i r p r e v i o u s s tudy of the l o c a t i o n of P - e n o l p y r u v a t e c a r -03 boxylase. T h e i r r e s u l t s a r e v a r i a b l e l i k e these r e p o r t e d here. They conclude t h a t the enzyme i s probably a s s o c i a t e d with the c h l o r o p l a s t p e r i p h e r a l r e t i c u l u m . P a r t i a l or com-p l e t e removal of t h i s s t r u c t u r e from the c h l o r o p l a s t body dur-i n g i s o l a t i o n c o u l d e x p l a i n the v a r i a b l e r e s u l t s of nonaqueous f r a c t i o n a t i o n . Fragments of t h i s membrane system might a l s o be recovered by u l t r a c e n t r i f u g a t i o n of aqueous e x t r a c t s of l e a v e s as r e p o r t e d by B a l d r y et a l . ( 1 ). While t h i s pos-t u l a t e e x p l a i n s some ob s e r v a t i o n s i t i s s t i l l not supported by adequate experimental evidence. More work w i l l be r e q u i r e d to s p e c i f y the l o c a t i o n of t h i s enzyme. C h a p t e r I I I TABLE I. P - e n o l p y r u v a t e c a r b o x y l a s e a c t i v i t y i n f r a c t i o n s i s o l a t e d by nonaqueous 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 . Gomphrena g l o b o s a d e n s i t y range C h l o r o p h y l l (jig) IK ENZYME ACTIVITY f i x e d S p e c i f i c a c t i v i t y dpm/mln dpm/ug C h i min l e s s t h a n 1 .32 1.32 - 1.37 1.37 - 1.45 g r e a t e r t h a n 1.45 Crude sample 2.59 6.22 4.16 2.15 6.68 734 2084 2073 638 2729 284 335 498 297 410 Zea mays d e n s i t y range C h l o r o p h y l l (ug) ENZYME ACTIVITY 14c f i x e d S p e c i f i c a c t i v i t y dpm/mln dpm/ jag C h i min l e s s than 1 .34 1.34 - 1.45 g r e a t e r t h a n 1.45 10.29 8.72 6.40 667 2500 2116 65 286 303 45 L i t e r a t u r e C i t e d 1 . B a l d r y , C W . , B u r k e , C , and Coombs, J . I 9 6 9 . L i g h t / p h o s p h o -e n o l p y r u v a t e dependent ca rbon d i o x i d e f i x a t i o n by i s o -l a t e d sugar cane c h l o r o p l a s t s . B iochem. B i o p h y s . R e s . Comm. 3?: 8 2 8 - 8 3 2 . 2 . S l a c k , C R . , and H a t c h , M.D. 1 9 6 7 . Comparat ive s t u d i e s on the a c t i v i t y of c a r b o x y l a s e s and o t h e r enzymes i n r e -l a t i o n t o the new pathway of p h o t o s y n t h e t i c carbon d i o x i d e f i x a t i o n i n t r o p i c a l g r a s s e s . B iochem. J . 1 0 3 : 6 6 O - 6 6 5 . 3 . S l a c k , C R . , H a t c h , M . D . , and G o o d c h i l d , D . J . I 9 6 9 . D i s t r i -b u t i o n of enzymes i n m e s o p h y l l and parenchyma-sheath c h l o r o p l a s t s of maize l e a v e s i n r e l a t i o n t o the C ^ -d i c a r b o x y l i c a c i d pathway of p h o t o s y n t h e s i s . B iochem. J . 114: 4 8 9 - 4 9 8 . 06 Appendix I E x t r a c t i o n and Assay o f P - e n o l p y r u v a t e c a r b o x y l a s e . Two a s s a y p rocedures were used i n t h e s e s t u d i e s . One was a s p e c t r o p h o t o m e t r i c a s s a y s i m i l a r t o t h o s e o f Walker ( 9 ) . Muker j i st al* (5) and Osmond ( 6 ) . The p r o d u c t o f c a r b o x y l a -t l o n o f P - e n o l p y r u v a t e , o x a l o a c e t a t e , i s reduced i n the p resence o f malate dehydrogenase and NADH t o m a l i c a c i d . The consumpt ion of NADH was f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y a t 300 nm. A l t e r n a -t i v e l y o x a l o a c e t a t e may be c o n v e r t e d t o a s p a r t a t e i n the p r e s e n c e o f a s p a r t a t e - g l u t a m a t e amino t r a n s f e r a s e i n the p r e s e n c e o f e x -c e s s g l u t a m a t e . T h i s may be f o l l o w e d by a s s a y i n g the f o r m a t i o n o f " ^ C - a s p a r t a t e f rom H^COcj. T h i s p rocedure i s used i n the a s s a y o f S l a c k and Hatch (7) and Bjorkman ( 1 ) . Both the s p e c t r o -p h o t o m e t r i c and the r a d i o c h e m i c a l t e c h n i q u e s were used i n p r e l i m i ' n a r y exper iments f o r t h i s s tudy and t o examine d i f f e r e n c e s b e t -ween subgenera o f Panlcum( 3 T h e p r o c e d u r e s used a r e d e s c r i b e d be low. R a d i o c h e m i c a l a s s a y ; A s t o c k s o l u t i o n was p r e p a r e d by a d d i n g i n the f o l l o w i n g sequence , 10 ml H 2 0 ; 2 . 0 ml 1 M, T r i s - H C l (pH= 0 . 0 ) ; 290 mg o f g l u t a m i c a c i d ; s a t u r a t e d KOH s o l u t i o n t o a d j u s t i it the pH t o 8 . 3 ; 305 mg M g C l 2 ; 8 . 0 mg NaHC03; 20 / ac , N a 2 G 0 3 * and HgO t o 20 ml f i n a l vo lume. The a s s a y was per formed i n a f i n a l volume o f 0 . 5 ml c o n t a i n i n g 0 . 1 5 ml o f the s t o c k s o l u t i o n , 0 . 3 ml o f 100 mM T r i s - H C l b u f f e r pH 8 . 0 and enzyme e x t r a c t . The r e a c t i o n was s t a r t e d by a d d i n g 0 . 1 ml o f 50 mM P - e n o l p y r u v a t e and s t o p p e d by a d d i n g 0 . 5 ml 10$ T r i c h l o r o a c e t i c a c i d . A 0 . 2 5 ml a l l q u o t e o f the s topped r e a c t i o n m i x t u r e was d r i e d and counted w i t h B r a y ' s l i q u i d s c i n t i l l a t o r ( 2 ) . The r e a c t i o n r a t e was %7 c o n s t a n t f o r a t l e a s t 5 m i n u t e s . I t i s assumed t h a t P - e n o l p y r u v a t e c a r b o x y l a s e l i m i t s the 14 r a t e o f G - a s p a r t a t e s y n t h e s i s i n the above r e a c t i o n m i x t u r e . A s p a r t a t e - g l u t a m a t e amino t r a n s f e r a s e was p r e p a r e d f rom p i g h e a r t by the method o f J e n k i n s et a l . ( 4 ) . In no c a s e d i d the 14 a d d i t i o n o f t h i s enzyme a l t e r the r a t e o f C - a s p a r t a t e s y n t h e s i s w i t h enzymes e x t r a c t e d f rom e i t h e r Zea o r Gomphrena l e a v e s . 14 S p e c i f i c a c t i v i t y o f the H CO^ p r e s e n t e d i n t h i s a s s a y was found t o v a r y due t o a b s o r p t i o n o f C 0 2 by the t r i s b u f f e r u s e d . S p e c i f i c a c t i v i t y was de te rmined by c o u n t i n g an a l i q u o t o f the a s s a y and the t o t a l HCO^ was de termined on a n o t h e r a l i -quot 1 by the a c i d r e l e a s e method o f Tregunna and Thomas (8). The s p e c t r o p h o t o m e t r y a s s a y was per formed In a c u v e t t e c o n t a i n i n g the f o l l o w i n g : 2 . 6 0 ml 0.1 M HEPES b u f f e r pH 7 . 5 ; 0.2 ml NADH l m g / m l ; 20u l M*MgCl2; 6pl M NaHCOy l O O u l enzyme e x t r a c t ; 20ji l 0.2M P - e n o l p y r u v a t e . The r e a c t i o n was s t a r t e d by a d d i t i o n o f P - e n o l p y r u v a t e , and the O ° ^ 0 m o n i t o r e d . The o p t i c a l " ; d e n s i t y was e i t h e r r e c o r d e d c o n t i n u o u s l y , o r measured a t h a l f minute i n t e r v a l s . The endogenous l e v e l o f m a l i c dehydrogenase was a s s a y e d by a d d i n g 5pl o f M o x a l o a c e t i c a c i d i n p l a c e o f the P - e n o l p y r u v a t e . The s p e c t r o p h o t o m e t r i c a s s a y was l e s s s e n s i t i v e than the r a d i o c h e m i c a l a s s a y but was used i n p r e f e r e n c e t o i t i n the l a t e r p a r t o f t h i s s tudy as o t h e r s p e c t r o p h o t o m e t r i c a s s a y s were a l s o b e i n g u s e d . P - e n o l p y r u v a t e c a r b o x y l a s e was a lways u n s t a b l e when e x -48 t r a c t e d f rom the p l a n t . S e v e r a l g r i n d i n g media and methods of e x t r a c t i o n were t e s t e d . G r i n d i n g i n a m o r t a r . and p e s t l e was s u p e r i o r t o b l e n d i n g . P o l y v i n y l p y r o l l i d o n e (PVP) (20$ o f f r e s h t i s s u e weight ) was used i n some p r e p a r a t i o n s t o b i n d p h e n o l i c compounds r e l e a s e d i n g r i n d i n g . An i n h i b i t o r y substance a c -cumulated i n the e x t r a c t i n g medium w i t h s t o r a g e , hence o n l y f r e s h l y p r e p a r e d g r i n d i n g medium was u s e d . Treatment of an a c -t i v e p r e p a r a t i o n w i t h Sephadex G-25 removed much of the P - e n o l -p y r u v a t e c a r b o x y l a s e a c t i v i t y . No e x t r a c t i o n medium was found which c o m p l e t e l y s t a b i l i z e d P - e n o l p y r u v a t e c a r b o x y l a s e . T h i s i n s t a b i l i t y of the e x t r a c t e d enzyme i s a s i g n i f i c a n t s o u r c e of e r r o r i n s t u d i e s of t h i s enzyme. In one exper iment an e x t r a c t was p r e p a r e d f rom 2 g of c o r n l e a v e s by g r i n d i n g w i t h : 10 ml. 50 mM T r i s H C l pH 8.4, 10 mM M g C l 2 , 5 mM . 2 - m e r c a ^ t o ^ t h a n o l ; and 0.4 g PVP. I t was assayed by the s p e c t r o p h o t o m e t r i c method immedia te ly a f t e r p r e p a r a t i o n , and s t o r e d a t 0 ° . A f t e r one hour i t had l o s t 38$, and a f t e r 24 hours 62$ of i t s o r i g i n a l a c t i v i t y . The e x t r a c t was most a c t i v e when a s s a y e d a t pH 7.3 to 7.5. A t pH 7 .0 the r a t e was 38$ of maximum, and a t pH 7.7 the r a t e was 45$ of maximum. HEPES b u f f e r s i n t h i s pH r e g i o n and was s e l e c t e d t o b u f f e r the a s s a y s t o pH 7.5* There was no e f f e c t of i n c l u d i n g a s u l f h y d r y l reagent i n the a s s a y m i x t u r e . P r e p a r a t i o n s were a lways a s s a y e d as q u i c k l y as p o s s i b l e a f t e r p r e p a r a t i o n , but due t o the i n -s t a b i l i t y of the enzyme i t s _in v i v o a c t i v i t y i s p r o b a b l y u n d e r -e s t i m a t e d . 49 L i t e r a t u r e C i t e d 1. Bjorkman, O l l e , and Eckard Gauhl. I969. Carboxydismutase a c t i v i t y i n p l a n t s w i t h and without jB-carboxylation photosynthesis. P l a n t a 88: 197-203. 2. Bray, G.A. i960. A simple e f f i c i e n t l i q u i d s c i n t i l l a t o r f o r counting aqueous s o l u t i o n s i n a l i q u i d s c i n t i l l a -t i o n counter. Anal. Biochem. 1: 279-285. 3. Downton, John, Berry, J . , and Tregunna, E. Bruce. I969. Photosynthesis: Temperate and t r o p i c a l c h a r a c t e r i s t i c s w i t h i n a s i n g l e grass genus. Science 163. 78-79. 4. J e n k i n s , W. Terry , Yphantis, David A., and S i z e r , I r w i n W. 1959. Glutamic a s p a r t i c transaminase. I . Assay, p u r i -f i c a t i o n , and general p r o p e r t i e s . J . B i o l . Chem. 234: 51-57. 5. M u k e r j i , S.K., and Ti n g , I r w i n P. 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 C0 ? metabolism enzymes i n cactus p h y l l o c l a d e s . Phytochem. 7 (1968) 903-912. 6. Osmond, C.B. 1969. / 3-carboxylation, photosynthesis and p h o t o r e s p i r a t i o n i n higher p l a n t s . Biochim. Biophys. Acta 172: 144-149. 7. S l a c k , CR., and Hatch, M.D. 1967. Comparative s t u d i e s on the a c t i v i t y of carboxylases and other enzymes i n r e -l a t i o n t o the new pathway of photosynthetic carbon d i o x i d e f i x a t i o n i n t r o p i c a l grasses. Biochem. J . 103: 66O-665. 8. Tregunna, E.B., and Thomas, E. Ann. 1968. Measurement of ino r g a n i c carbon and photosynthesis i n seawater by P C0 2 and pH a n a l y s i s . Can. J . Bot. 46: 481-485. 9. Walker, D.A., and Brown, J.M.A. 1957. P h y s i o l o g i c a l s t u d i e s on a c i d metabolism. 5 Biochem. J . 67: 79-83. 50 l Appendix II Chromatography A one d i m e n s i o n a l chromatograph ic system was used i n these s t u d i e s . E x t r a c t s were a p p l e d i n a 0 .5 t o 1 cm band t o Whatman no . 1 paper s t r i p s ( I § " x 2 4 " ) . These were i r r i g a t e d by d e s c e n d i n g chromatography w i th l i q u e f i e d p h e n o l : wa te r : ( g l a c ) a c e t i c a c i d:lMH ; e t h y l e n e d iamine t e t r a c e t i c a c i d (840: 160: 10 : 1 ) . T h i s t e c h n i q u e was adapted f rom a two d i m e n s i o n a l t e c h n i q u e d e s c r i b e d by P e d e r s e n et a l . ( 4 ). I t was s e l e c t e d f rom many t e s t e d as i t a c h i e v e d a complete s e p a r a t i o n of C a l v i n c y c l e I n t e r m e d i a t e s f rom the C ^ - a c i d s . Two d i m e n s i o n a l t e c h -n i q u e s a r e s u p e r i o r t o t h i s i n r e s o l v i n g power, but were not adapted t o work w i th a chromatogram s c a n n e r . S t a n d a r d i z a t i o n C o m m e r c i a l l y a v a i l a b l e s t a n d a r d s were r u n as d e s c r i b e d a b o v e . Amino a c i d s were d e t e c t e d by s p r a y i n g w i t h n i n h y d r i n reagent ( 1 ) . P h o s p h o r y l a t e d compounds were d e t e c t e d e i t h e r w i t h the ammonium molybdate o r P e C l ^ r e a g e n t s d e s c r i b e d by B e l e s k i and Young ( 2 ) . " ^ - l a b e l l e d sugars and m a l i c a c i d were d e t e c t e d w i t h a s t r i p c h a r t s c a n n e r . Rp v a l u e s f o r some r e p r e s e n t a t i v e compounds a r e p r e s e n t e d i n T a b l e I . The Rp v a l u e s of most compounds a r e low , hence the s o l v e n t f r o n t was u s u a l l y run o f f the p a p e r . T h i s system proved adequate t o r e s o l v e the s i m p l e m i x t u r e s 14 of C - l a b e l l e d compounds d e r i v e d f rom s h o r t - t e r m f e e d i n g s . 1 D e t a i l s of t h i s chromatography system a r e i n c l u d e d i n a m a n u s c r i p t by Tregunna et a l . ( 5 ) submi t ted September \ 30 , 1969 t o the C a n . J . Botany and i n the PhD. t h e s i s of W . J . S . Downton, ( 3 ) . 51 Appendix I I TABLE I . l ip v a l u e s f o r some compounds s e p a r a t e d by one-dimen-s i o n a l paper chromatography u s i n g l i q u e f i e d p h e n o l ( c a . 90%)t a c e t i c a c i d : w a t e r : IM e t h y l e n e d i a m i n e -t e t r a a c e t i c a c i d (840 : 1 6 0 : 1 0 : 1 ) as t h e s o l v e n t . Compound R,p v a l u e a l a n i n e 0.62 a s p a r t i c a c i d 0.23 g l y c i n e 0.45 g l u t a m i c a c i d 0.67 s e r i n e 0.39 g l u c o s e 0.43 s u c r o s e 0.45 g l j f c o l i c a c i d 0.49 m a l i c a c i d 0.32 3 - p h o s p h o g l y c e r i c a c i d 0.08 g l u c o s e - 6 - p h o s p h a t e 0.11 r i b u l o s e - 1 , 5-diphosphate 0.03 52 The monophosphate e s t e r s moved as a s i n g l e band which was not r e s o l v e d i n t o i t s component compounds. T h e s e , a s p a r t i c a c i d , and m a l i c a c i d moved as d i s t i n c t bands which d i d not o v e r l a p w i th any o t h e r compounds t e s t e d . R i b u l o s e and f r u c t o s e d i -phosphates were not r e s o l v e d , and sometimes d i d not move enough f rom the o r i g i n t o be r e s o l v e d f rom i n s o l u b l e m a t e r i a l . The two d i m e n s i o n a l system d e s c r i b e d by P e d e r s e n et a l . ( 4 ) was used t o r e s o l v e the more complex m i x t u r e s of compounds d e r i v e d f rom l o n g e r term f e e d i n g s , and t o v e r i f y the c o m p o s i -t i o n of some e x t r a c t s s e p a r a t e d on the one d i m e n s i o n a l sys tem. T h e i r s t a n d a r d map was used f o r i d e n t i f i c a t i o n . A p r o m i s i n g m o d i f i c a t i o n of t h i s t e c h n i q u e i n v o l v e s the use of the same s o l v e n t systems on t h i n l a y e r s of " A v l c e l " m i c r o c r y s t a l l i n e c e l l u l o s e . The t ime r e q u i r e d f o r development c o u l d be s h o r t -ened s u b s t a n t i a l l y w i th s i m i l a r r e s o l u t i o n . The t h i n l a y e r s c o u l d not however t o l e r a t e n e a r l y as much e x t r a c t as c o u l d the p a p e r . 53 L i t e r a t u r e C i t e d 1 . Von Arx, E., and Neher, R. 1963. A m u l t i - d i m e n s i o n a l technique f o r the chromatographic i d e n t i f i c a t i o n of amino a c i d s . J . Chromatog. 1 2 : 3 2 9-341. 2 . B i e l e s k i , R.L., and Young, Roy E. 1 9 6 3 . E x t r a c t i o n and s e p a r a t i o n of phosphate e s t e r s from p l a n t t i s s u e . A n a l . Biochem. 6 : 5 ^ - 6 8 . 3 . Downton, W.J.S. I 9 6 9 . Some i n t e r r e l a t i o n s of phot 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 o n among s p e c i e s . Ph.D. T h e s i s U n i v e r -s i t y of B r i t i s h Columbia,Vancouver 8 , Canada. 4. Pedersen, P.A., K i r k , Martha, and Bassham, J.A. 1 9 6 6 . I n -h i b i t i o n of ph o t o p h o s p h o r y l a t i o n and p h o t o s y n t h e t i c carbon c y c l e r e a c t i o n s by f a t t y a c i d s and e s t e r s . Biochim. Biophys. A c t a 1 1 2 : 1 8 9 - 2 0 3 . 5 . Tregunna, E.B., Smith, B.N., Berry, J.A., and Downton, W.J.S. 1 9 7 0 . Some methods f o r s t u d y i n g the p h o t o s y n t h e t i c t a x -onomy of the angiosperms. Can. J . Bot. ( i n p r e s s ) . 50 Appendix I I I Nonaqueous 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 The normal t e c h n i q u e s f o r the I s o l a t i o n o f c h l o r o p l a s t s i n aqueous media have the d i s a d v a n t a g e t h a t some s o l u b l e com-ponents a r e l e a c h e d away f rom the c h l o r o p l a s t body d u r i n g i s o -l a t i o n . Such i s o l a t e d c h l o r o p l a s t s a r e u n s u i t a b l e f o r s t u d i e s o f the s o l u b l e c h l o r o p l a s t components, such as enzymes and m e t a b o l i c i n t e r m e d i a t e s . Independent ly T h a l l a c k e r and Behrens (8) and S t o c k i n g (7) d e v e l o p e d a t e c h n i q u e f o r i s o l a t i o n o f c h l o r o p l a s t s i n c a r b o n t e t r a c h l o r i d e - hexane m i x t u r e s f rom f r e e z e - d r i e d p l a n t m a t e r i a l . As no aqueous s o l v e n t s a r e u s e d , no l e a c h i n g o f water s o l u b l e components c a n o c c u r d u r i n g p r e p -a r a t i o n . T h e i r t e c h n i q u e was based on a t e c h n i q u e deve loped f o r i s o l a t i o n o f n u c l e i Behrens (1). T h i s t e c h n i q u e has been used by s e v e r a l workers t o s tudy the c h l o r o p l a s t complement o f enzymes (3), c e l l u l a r components (2), and t o s tudy the meta -b o l i c r e l a t i o n s h i p o f the c h l o r o p l a s t s t o the s u r r o u n d i n g c y t o -p lasm (2). S e v e r a l t i s s u e s , t e c h n i c a l m o d i f i c a t i o n s , and methods o f d a t a p r e s e n t a t i o n have been u s e d . P r e l i m i n a r y e x -per iments were per formed i n o r d e r t o f i n d methods most s u i t a b l e t o the t i s s u e and purposes we i n t e n d e d . The l e a v e s o f both Zea mays and Gomphrena g l o b o s a a r e tougher than t h o s e o f p l a n t s p r e v i o u s l y u s e d . G r i n d i n g t e c h -n i q u e s used by o t h e r workers proved inadequate t o r e l e a s e most o f the c h l o r o p l a s t s f rom the bund le s h e a t h . Except f o r m o d i f i c a t i o n i n the g r i n d i n g p r o c e d u r e , s t a n d a r d t e c h n i q u e s were employed f o r s e p a r a t i o n of the c e l l u l a r components . These a r e d e s c r i b e d i n d e t a i l e lsewhere ( p . 0). 55 C o n t r o l s The v a l i d i t y of t h i s t e c h n i q u e depends upon the assump-t i o n tha t the components b e i n g i n v e s t i g a t e d a r e not l e a c h e d f rom the c h l o r o p l a s t s d u r i n g the p r e p a r a t i o n s t e p s . The s o l v e n t s used i n c h l o r o p l a s t p r e p a r a t i o n c o n t a i n e d 1 - h% of the t o t a l r a d i o a c t i v i t y p r e s e n t i n the l e a f m a t e r i a l . Much of t h i s was a s s o c i a t e d w i t h f i n e p a r t i c u l a t e m a t e r i a l not r e -moved i n c e n t r i f u g a t i o n . Prom s i m i l a r s t u d i e s Heber c o n c l u d e d " T h e r e f o r e l e a c h i n g of water s o l u b l e compounds such as sugar phosphates o r o r g a n i c a c i d s f rom the l e a f f r a c t i o n s d u r i n g the p r o c e d u r e can be n e g l e c t e d . " (2) P r e v i o u s s t u d i e s have shown t h a t the p r o d u c t s of CO^ f i x -a t i o n by p l a n t s which have C a l v i n pathway of p h o t o s y n t h e s i s I k a p p e a r f i r s t i n the c h l o r o p l a s t s . The l o c a t i o n of c l a b e l l e d 14 compounds formed f rom co 2 d u r i n g v e r y s h o r t - t e r m f e e d i n g s t o wheat and s p i n a c h was s t u d i e d as a f u r t h e r c o n t r o l of our t e c h -n i q u e . Three week o l d wheat p l a n t s which had been grown on v e r m i c u l i t e and s p i n a c h o b t a i n e d f rom the l o c a l market were 14 f e d COg f o r 3 - 5 sec , f r o z e n , and f r e e z e d r i e d as d e s c r i b e d 14 e a r l i e r . Chromatography of the C l a b e l l e d compounds p r e s e n t 14 i n e x t r a c t s of t h e s e l e a v e s I n d i c a t e d v i r t u a l l y a l l of the C was i n P - e s t e r s . Data o b t a i n e d i n nonaqueous d e n s i t y g r a d i e n t f r a c t i o n a t i o n of these l e a v e s i s p r e s e n t e d i n T a b l e I . F o r wheat the r a t i o , dpm/ug c h l o r o p h y l l i s c o n s t a n t . The l i g h t e s t f r a c t i o n (1.29 - 1.31) c o n t a i n e d v i r t u a l l y pure c h l o r o p l a s t s w h i l e the o t h e r f r a c t i o n s c o n t a i n e d a mix tu re of c h l o r o p l a s t s and c y t o p l a s m . T h i s r e s u l t i n d i c a t e s t h a t n e a r l y a l l of the 14 C p r e s e n t i n these l e a v e s i s l o c a t e d i n the c h l o r o p l a s t s . Appendix I I I 14 TABLE I. Data from nonaqueous f r a c t i o n a t i o n of l e a v e s of wheat and s p i n a c h f e d C 0 2 f o r 6 s e c . Wheat 14 C-dpm D e n s i t y range C h l o r o p h y l l P r o t e i n dpm/ug c h l o r o p h y l l dpm/jug p r o t e i n 1 1.29 - 1.31 2 1.31 - 1 .40 3 > 1 .40 19.5 6 9 . 0 23.3 0.51 2.67 1.94 16,150 67,200 22,900 830 973 980 37,700 25,100 11 ,800 S p i n a c h 14 C-dpm D e n s i t y range C h l o r o p h y l l P r o t e i n dpm/yug c h l o r o p h y l l dpm/ug p r o t e i n 1 1.27 - 1.35 2 I . 3 5 - 1 .40 3 > 1 .40 192 191 214 3.68 4 . 5 9 9.11 269,000 603,000 1 , 0 0 0 , 0 0 0 1 ,400 3,160 4,680 73,200 129,000 110,000 57 T h i s i s c o n s i s t e n t w i t h p r e d i c t i o n s and shows t h a t no l e a c h i n g c o u l d have o c c u r r e d . The r e s u l t f o r s p i n a c h i s not as c l e a r , a l t h o u g h the p r o c e d u r e s were the same. Some o f the l a b e l l e d compounds a r e found o u t s i d e o f the c h l o r o p l a s t s , as the r a t i o dpm/jig c h l o r o p h y l l i n c r e a s e s w i t h i n c r e a s i n g p r o p o r t i o n o f c y t o -p l a s m . U s i n g the e q u a t i o n s g i v e n on page 6 i t was c a l c u l a t e d t h a t a p p r o x i m a t e l y 50% o f the "^C was l o c a t e d i n the c h l o r o -1k p l a s t s o f s p i n a c h . Some movement o f C has t h u s - o c c u r r e d . I f i t o c c u r r e d a f t e r k i l l i n g i t i s an a r t i f a c t . Johnson and B r u f f (5) have n o t e d t h a t c h l o r o p l a s t s p r e p a r e d f rom "market" s p i n a c h a r e " l e a k y " . T h i s may a l s o o c c u r i n v i v o . The pr imary d a t a f o r s e v e r a l 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 s o f Zea and Gomphrena l e a v e s f e d " ^ C 0 2 A N D - " L ^ C 0 2 a s i n d i c a t e d a r e p r e s e n t e d i n T a b l e I I I . The p e r c e n t o f t o t a l l a b e l l e d C ^ - a c i d s o r P - e s t e r s l o c a t e d i n the c h l o r o p l a s t s was c a l c u l a -t e d as d e s c r i b e d (page 5). A sample c a l c u l a t i o n o f x appears i n T a b l e IV and a sample c a l c u l a t i o n o f y appears i n T a b l e V . Data o b t a i n e d f rom two f e e d i n g exper iments a r e g r a p h i c a l l y r e p r e s e n t e d i n F i g u r e 1 and F i g u r e 2 . These d a t a s u p p o r t the c o n c l u s i o n (page 2 1 ) t h a t the C ^ - a c i d s o f Gomphrena a r e found i n bo th the c y t o p l a s m and c h l o r o p l a s t s w h i l e the P - e s t e r s a p p e a r t o be c o n f i n e d t o the c h l o r o p l a s t s . F o r Z e a . on the o t h e r hand , C ^ - a c i d s and P - e s t e r s a r e l o c a t e d i n bo th c y t o p l a s m and c h l o r o p l a s t s . Heber and W i l l e n b r i n k ( 2 ) r e p o r t t h a t - ^ C l a b e l l e d m a l i c a c i d formed d u r i n g p h o t o s y n t h e s i s i s found i n both c y t o p l a s m and c h l o r o p l a s t s o f s p i n a c h and broadbean ( p l a n t s w i t h the normal C a l v i n pathway o f p h o t o s y n t h e s i s ) . 58 Recent r e s u l t s p u b l i s h e d by S l a c k et a l . (6) c o n f l i c t w i t h our d a t a . They c o n c l u d e t h a t the C ^ - a c i d s and P - e s t e r s o f Zea a r e a s s o c i a t e d w i t h c h l o r o p l a s t s . They assumed t h a t most o f the - ^ C found a s s o c i a t e d w i t h the c y t o p l a s m was an a r t i f a c t due t o l e a c h i n g o r movement. The c o n t r o l exper iments done i n t h i s s tudy do no t c o m p l e t e l y e l i m i n a t e t h i s p o s s i b i l i -t y . Appendix I I I TABLE I I . Data from nonaqueous d e n s i t y g r a d i e n t f r a c t i o n a t i o n s of l e a v e s f e d i n t h e manner i n d i c a t e d . Gomphrena g l o b o s a 6 sec. C0 2» 1 2 0 sec. C 0 2 D e n s i t y range C h l o r o p h y l l ^ ; ) P r o t e i n •(mg) 14 C-dpm C ^ - a c i d s P - e s t e r s O r i g i n 1 1 . 2 9 - 1 . 3 4 2 1 . 3 4 - 1 . 3 7 3 1 . 3 7 - 1 . 4 5 4 1 . 4 5 2 3 5 7 9 . 3 8 8 . 2 1 3 7 . 6 1 3.1 1 5 . 6 2 9 . 4 6 6 . 6 3 1 6,000 1 6 0,000 448,000 1 , 5 ^ 2,400 7 9 . 1 $ 9 7 . 1 9 2 . 2 9 5 - 9 1 5 . 4 * 2 . 9 6 . 9 3.8 5.5% 1.0 0 . 4 Crude sample 404 2 1.1 1 , 5 2 5 , 0 0 0 9 3 . 2 5 . 5 1 . 3 Gomphrena g l o b o s a 6 sec. C 0 2 , 1 5 sec. CO D e n s i t y range C h l o r o p h y l l ^ P r o t ein( mg) l 2 ^ a C-dpm C ^ - a c i d s P - e s t e r s O r i g i n 1 1 . 2 9 - 1 . 3 4 2 1 . 3 4 - 1 . 3 7 3 1 . 3 7 - 1 . 4 5 4 ^ - 1 . 4 5 7 6 . 3 1 5 1 7 2.0 2 7 . 9 1 . 7 3 3 . 6 3 4.80 3 . 5 9 1 0 5 , 7 0 0 3 0 3,000 5 5 6,000 3 7 7 , 2 0 0 7 4 . 4 $ 7 3 . 6 8 8 . 4 9 1 . 4 2 1 . 0 $ 2 2 . 6 9 . 3 7.8 4 . 6 $ 4.8 2 . 3 1 . 7 Crude sample 3 6 3 1 5 . 1 1 , 5 8 9 , 0 0 0 9 1.0 7 . 4 1 . 6 TABLE I I . cont'd. Gomphrena globosa 6 14 sec. C 0 2 * ™ s e c * 1 2 c o 2 D e n s i t y range C h l o r o p h y l l (ug) P r o t e i n (mg) C-dpm C^-acids P - e s t e r s O r i g i n 1 1 .29 - 1.34 2 1 . 3 4 - 1 . 4 0 3 1.40 - 1.45 4 ^ 1.45 108 64.0 45.6 149.6 2.11 2.40 2 .99 11.18 390,000 630 ,000 653,000 2,440,000 46 .9* 59.0 6 7 . 4 74.4 4 4 . 8 £ 34.6 21.3 20.1 8.2# 6.4 3 .9 5.5 Crude sample 356 9 .84 2 ,065 , 000 6 8 . 5 21.4 7.0 Gomphrena globosa 6 !0 sec. C ° 2 ' 8 6 0 ,1 2 c o 2 D e n s i t y range C h l o r o p h y l l (ug) P r o t e i n (mg) 14 C-dpm C^-acids. P - e s t e r s O r i g i n 1 <=• 1 .30 2 1.34 - 1.37 3 1.37 - 1.00 4 1.40 - 1.45 5 1.45 - 1 .59 6 -> 1 .59 5.06 3.32 1.49 1.51 4.8 1.20 88,400 77,800 52,300 78,000 392 ,000 50,800 57.9 % 48.4 48.3 53.2 66.0 67 .O 42 .1 % 31.8 46.6 40.9 31.8 3 2 . 6 2.2 0.9 5 .9 2.2 Crude f r a c t i o n 9.00 482,000 66.5 2 9 . 6 3 .9 TABLE I I . cont'd. Gomphrena globosa 6 10 sec. C ° 2 ' s e c 12 . c o 2 D e n s i t y range C h l o r o p h y l l (ug) P r o t e i n (mg) 10 ^ C-dpm C^-acids P - e s t e r s O r i g i n 1 1.29 - 1 .30 2 1.34 - 1.37 3 1.37 - 1 .05 0 •> 1.05 107 229 9 0 . 5 536 1.29 0.29 5.39 16.38 10,970 126,300 158,200 397,000 37.1* 28 .9 3 5 . 9 37.1 55.5% 71.1 60.1 52.6 7 . 0 * 8 . 0 Crude sample Zea mays 6 sec. CO 12 _., 0 sec. CO 2 2 D e n s i t y range C h l o r o p h y l l (/ig) P r o t e i n (mg) 10 C-dpm C^-acids P - e s t e r s O r i g i n 1 1.29 - 1.34 2 1.34 - 1.45 3 •> 1.05 3 3 . 0 22.3 13.8 0.80 1.01 1.02 33,150 50 ,000 6 3 , 8 0 0 89.8# 72.0 82.1 10.2% 28.1 17.9 Crude sample 208 9.50 385,500 92.6 7 . 0 _ _ _ TABLE I I . c o n t ' d . Zea mays 6 sec. GO , 30 sec. CO 14 D e n s i t y range C h l o r o p h y l l (ug) P r o t e i n (mg ) C-dpm C ^ - a c i d s P - e s t e r s O r i g i n 1 1 . 2 9 - 1.34 85 2 1 . 3 4 - 1 . 4 0 49 3 J S * 1.40 33 1 .32 4 .03 6.06 167,700 299,000 614,000 5 0 . 0 $ 42.1 5 6 . 9 50.2# 51.0 3 3 . 7 6 . 9 $ 2 . 5 Crude sample 290 P i n e p a r t i c l e s s e p a r a t e d from c h l o r o p l a s t s 18.2 9.57 0 .47 958,000 48,400 50.3 48 . 7 4 3 . 9 48.8 2 . 5 Zea mays 12 sec. 1^C0„, 0 sec. 1 2 C 0 . 2 2 D e n s i t y range C h l o r o p h y l l {jig) P r o t e i n (mg) 14 C-dpm C ^ - a c i d s P - e s t e r s O r i g i n 1 1 . 2 9 - 1 . 3 4 494; 2 1 .34 - 1.40 177 3 1.40 220 7.06 5.45 13.42 188 , 8 5 0 165,500 566,000 93.7# 84 . 4 9 3 . 3 6.3# 15.6 6 . 7 Crude sample 591 2 3 . 8 5 931,500 9 2 . 5 7.5 T A B L E I I . c o n t ' d . 2 Z e a mays 2 s e c . l i | ' C 0 , 0 s e c . 1 2 C 0 *~ 2 D e n s i t y r a n g e C h l o r o p h y l l (^ag) P r o t e i n (mg) 10 C - d p m C ^ - a c i d s P - e s t e r s O r i g i n 1 1.27 - 1.30 2 1.30 - 1.37 3 1.37 - 1.05 0 > 1.05 861 O i l 325 221 13.3 13.1 16.8 12.0 369,000 029,000 806,000 021,000 93. 91.3 91.6 9 2 . 8 7 . 0 £ 8.7 7 . 0 7.2 C r u d e s a m p l e 787 2 0 . 0 876,000 92.2 7.8 2 Z e a mays 2 s e c . 10 12 CO , 5 s e c . CO 2 2 D e n s i t y r a n g e C h l o r o p h y l l (jag) P r o t e i n (mg) C - d p m C ^ - a c i d s P - e s t e r s O r i g i n 1 1.29 - 1.30 2 1.30 - 1 .00 3 -> 1.00 310 320 276 5.2 9.85 17.70 160,000 056,000 108,500 70.8# 69.3 2 5 . 2 ^ 30.7' C r u d e s a m p l e 850 28.9 1 , 2 9 0 , 0 0 0 79.6 20 .0 TABLE I I . c o n t ' d Zea mays ^ 2 sec. 14 C 0 2 , 20 sec. 12 D e n s i t y range C h l o r o p h y l l (ug) P r o t e i n (mg) 14 C-dpm C^ - a c l d s P - e s t e r s O r i g i n 1 1.29 - 1 . 3 4 2 1 . 3 4 - 1 .37 3 1 .37 - 1.45 4 •> 1 . 4 5 395 289 181 128 7-9 9.8 11.8 12.4 384,200 510,000 880,000 765,000 53.3^ 37.3 52.1 57.3 44.7^ 59.6 43.2 40 . 7 3.1$ 4 . 7 3.0 Crude sample 936 32.4 2 ,395,000 47.2 46 . 7 5.3 Zea mays ( d a r k 60 sec) 14 C-dpm D e n s i t y range C h l o r o p h y l l (^g) P r o t e i n C - a c i d s 4 P - e s t e r s O r i g i n 1 1 . 2 7 - 1 . 3 5 2 1 . 3 5 - 1.40 3 =^> 1.40 322 163 156 5.15 6.34 8.56 1 , 0 0 3,000 1 ,360,000 3,630,000 47.3$ 10.3$ Crude sample 522 16.9 5,504,000 95-5 4 . 5 1 These l e a v e s f e d November 2 5 , 1968. 2 These l e a v e s f e d December 13, 1968. 65 Appendix I I I F i g u r e 1. Nonaqueous 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 a o f f r e e z e 10 d r i e d l e a v e s . P e r c e n t o f t o t a l C , p r o t e i n , and c h l o r o p h y l l i n each f r a c t i o n , and p e r c e n t o f o*f each f r a c t i o n i n C /^ -ac ids and phosphate e s t e r s . Gomphrena g l o b o s a l e a v e s 6 sec 1^C02t 15 sec 1 2 C 0 2 . % OF F R A C T I O N % OF TOTAL 8 0 6 0 40 20 4 0 30 20 1 0 FRACTION D E N S I T Y O Crude A u u ~ o o ° o ° ° o ° 3 o 0 0 o 0 . o o o o O Q--,0-f-0 O £.5' 33 ° 1.27 1.3 A C HL 0 R O P H Y L L A \ A • 1.3 7 1.4 5 A P H O S P H A T E S • A P R O T El N T O T A L U C C 4 - A CI D. 66 Appendix I I I F i g u r e 2. Nonaqueous 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 o f f r e e z e 14 d r i e d l e a v e s . P e r c e n t o f t o t a l C , p r o t e i n , and c h l o r o p h y l l i n each f r a c t i o n , and p e r c e n t o f - ^ C o f each f r a c t i o n i n Ik C ^ - a c i d s and phosphate e s t e r s . Zea mays l e a v e s 2 sec C0 2, 20 sec 1 2 G 0 2 . 80 > 'o OFL 40 F R A C T I O N • A 20 m AO m 0/ 'o OF 30 T O T A L 20 10 - O Crude 1 2 3 FRACTION 0 " " O " " o o o o o o 1 ° X ° ° « « o o 0 °„ _° o °. _° ° P A o 7 i ^ ° o — * -> D E N S I T Y 0 • 1.29 1.34 C HLO R 0 P H Y L L P R O T E I N TOT A L U C 1.37 1.45 A P H O S P H A T E S C ^ - A C I D S 67 Appendix I I I TABLE I I I . C a l c u l a t i o n of the f r a c t i o n s of l e a f p r o t e i n a s s o c i a t e d with the c h l o r o p l a s t s . E q u a t i o n from r e f e r e n c e 6 . x = :.. 1 0 0 Where x = % c h l o r o p l a s t p r o t e i n of t o t a l p r o t e i n a = t o t a l p r o t e i n c h l o r o p l a s t sample b = t o t a l p r o t e i n crude sample c = t o t a l c h l o r o p h y l l crude sample d = t o t a l c h l o r o p l y l l c h l o r o p l a s t sample Gomphrena globosa Zea mays Feeding x Feeding x 6 + 0 5 8 ^ 62% 6 + 1 5 59 07 6 + 3 0 70 3 5 6 + 6 0 0 1 08 08 x 5 7 ± 1 1 6 0 58 ~ = 5 1 - 9 68 Appendix I I I TABLE IV. Sample c a l c u l a t i o n of the f r a c t i o n s of a m e t a b o l i t e l o c a t e d i n the c h l o r o p l a s t s . E q u a t i o n a l b 2 . 7 y _ X a 2 b l Where a n = t o t a l p r o t e i n , crude sample C a l c u l a t i o n s f o r Gomphrena globosa (6 + 0) from Ta b l e I I I . a 2 t o t a l p r o t e i n , c h l o r o p l a s t sample 14 t o t a l c dpm/compound, crude sample 14 t o t a l C dpm/compound, c h l o r o p l a s t sample a^ = 8.52 mg. &2 = 3 . 0 7 mg. h1 - ( 1 , 5 2 5 , 0 0 0 ) (5.5/IOO) = 83,700 dpm b 2 = (316,000) (15.4/100) = 48 ,600 dpm x = 57% 69 L i t e r a t u r e C i t e d 1. Behrens, M. 1932. Unter-^uchungen an i s o l i e r t e n Z e l l u n d G e w e b s b i s t a n d t e i l e n . I. M i t t e i l u n g : I s o l i e r u n g von Z e l l k u n e n des Kalbsherzmuskels (Hoppe-Seyler). Z u t s . p h y s i o l . Chem. 2 0 9 : 59-70. 2. Heber, U., and W i l l e n b r i n k , J . I960, S i t e s of s y n t h e s i s and t r a n s p o r t of p h o t o s y n t h e t i c products w i t h i n the l e a f c e l l . Biochim. Biophys. A c t a 8 3 : 3 1 3 - 3 2 0 . 3 . Heber, U., S a n t a r i u s , K.A., Hudson, M.A., and H a l l i e r , U.W, 1967. Untersuchungen zur i n t r a z e l l u l a r e n V e r t e i l u n g von Enzymen und S u b s t r a t e n i n de B l a t t z e l l e I. Z. Natur-f o r s c h g . 22b: 1189-1199. 0 . Heber, U., H a l l i e r , U.W., and Hudson, M.A. 1 9 6 7 . Untersuchun-gen z u r i n t r a z e l l u r & r e n V e r t e i l u n g von Enzymen und Sub-s t r a t e n i n der B l a t t z e l l e . I I . Z. N a t u r f o r s c h g . 2 2 b : 1 2 0 0 - 1 2 1 5 . 5 . Johnson, Emmett J . , and B r u f f , Barbara S. 1 9 6 7 . C h l o r o p l a s t i n t e g r i t y and ATP-dependent CO? f i x a t i o n i n S p i n a c i a o l e r a c e a . P l a n t p h y s i o l . 02: 1 3 2 1 - 1 3 2 8 . 6 . Latzko, E., and Gibbs, M a r t i n . I 9 6 8 . D i s t r i b u t i o n and ac-t i v i t y of enzymes of the r e d u c t i v e pentose phosphate c y c l e i n spinach l e a v e s and i n c h l o r o p l a s t s i s o l a t e d by d i f f e r e n t methods. Z. P f l a n z e n p h y s i o l . 5 9 : 180-190. 7 . S l a c k , CR., Hatch, M.D., and Goodchild, D.J. 1 9 6 9 . D i s t r i -b u t i o n of enzymes i n mesophyll and parenchymal-sheath c h l o r o p l a s t s of maize l e a v e s i n r e l a t i o n t o the C^-d i c a r b o x y l i c a c i d pathway of p h o t o s y n t h e s i s . Biochem. J . 110: 089-098. 8. S t o c k i n g , C R . 1959- C h l o r o p l a s t i s o l a t i o n i n nonaqueous media. P l a n t p h y s i o l . 30: 5 6 - 6 1 . 9 . T h a l a c k e r , R., und Behrens, M. 1 9 5 9 . Uber den R e i n h e i t s g r a d der i n einem n i c h t w a s s r i g e n s p e z i f i s c h e n Gewichtsgradien-t e n gewonnenen C h l o r o p l a s t e n . Z. N a t u r f o r s c h . 106: 0 0 3 . 70 Appendix IV B i o c h e m i c a l s , A b b r e v i a t i o n s and Sources o f S u p p l y C h e m i c a l A b b r e v i a t i o n L - a l a n i n e ammonium s u l f a t e (enzyme grade) (NH^JgSO^ D - a r a b a n o a s c o r b i c a c i d d i t h i o t h r e i t o l DTT e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d EDTA Source o f S u p p l y C a l b i o c h e m 1 2 3 NBC Eastman C a l b i o c h e m 4 f r u c t o s e - 1 , 6 - d i p h o s p h a t e g l u c o s e - U L - ^ C g l u c o s e - 6 - p h o s p h a t e L - g l u t a m i c a c i d L - g l y c i n e g l y c o l i c a c i d - l - ^ C N - 2 - h y d r o x y e t h y l p y s e r a z i n e -N - 2 - e t h a n e s u l f o n i c a c i d L - m a l i c a c i d L - m a l i c a c i d - O - ^ C F i s h e r F r u c t o s e - D - P C a l b i o c h e m New Eng land N u c C a l b i o c h e m C a l b i o c h e m C a l b i o c h e m T6 HEPES ICN C a l b i o c h e m NBC C a l b i o c h e m 1 C a l b i o c h e m , Box 5^282, Los A n g e l e s , C a l i f o r n i a . 2 N u t r i t i o n a l B i o c h e m i c a l s , 26201 M i l e s Road, C l e v e l a n d , O h i o . 3 Eastman O r g a n i c C h e m i c a l s , R o c h e s t e r 3 . New Y o r k . 0 F i s h e r S c i e n t i f i c Company L i m i t e d , 196 W. 3rd Avenue, Vancouver 3 , B . C . 5 New Eng land N u c l e a r , 575 A lbany S t r e e t , B o s t o n , Mass. 6 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 . , 801 Nor th Lake S t r e e t , Burbank, C a l i f o r n i a . 71 C h e m i c a l 2 - m e r c a p t o e t h a n o l n i c o t i n e - a d e n i n e d i n u c l e o t i d e ( r e d u c e d , d isod lum) n i c o t i n e - a d e n i n e d i n u c l e o t i d e phosphate (monosodium) n i c o t i n e - a d e n i n e d i n u c l e o t i d e phosphate ( r e d u c e d , t e t r a -sodium) o x a l o a c e t i c a c i d l i q u e f i e d pheno l P o l i n Pheno l reagent 2 - p h o s p h o e n o l p y r u v i c a c i d ( t r i s o d i u m s a l t , p e n t -h y d r a t e ) o r ( t r i c y c l o h e x y l a m m o n i u m s a l t ) 3 - p h o s p h o g l y c e r i c a c i d (sodium s a l t ) p o l y v i n y l p y r r o l i d i n o n e (MW 40,000) r i b u l o s e - 1 , 5 - d i p h o s p h a t e L - s e r i n e s u c r o s e - U L - C t r i c h l o r o a c e t i c a c i d t r i s (hydroxymethyl ) Amino methane A b b r e v i a t i o n NADH NADP NADPH p - e n o l p y r u v i c a c i d 3-PGA PVP RuDP TCA T r i s Source o f S u p p l y Eastman Ca lb iochem C a l b i o c h e m C a l b i o c h e m C a l b i o c h e m F i s h e r F i s h e r C a l b i o c h e m C a l b i o c h e m 7 Matheson' Sigma^ Ca lb iochem New Eng land N u c l e a r F i s h e r F i s h e r 7 Matheson Coleman and B e l l , Norwood ( C i n c i n n a t i ) , O h i o . 8 Sigma C h e m i c a l Company, 3500 de K a l b S t r e e t , S t . L o u i s , Mo. 72 E p i l o g u e In the p r e c e d i n g d i s c u s s i o n s i n t h i s t h e s i s d e t a i l e d a t t e n t i o n has been f o c u s e d on p a r t i c u l a r a s p e c t s o f t h i s p a t h -way o f p h o t o s y n t h e s i s . In t h i s s e c t i o n I w i l l a t tempt t o draw t o g e t h e r i n f o r m a t i o n f rom t h i s t h e s i s , f rom s e v e r a l papers which have become a v a i l a b l e d u r i n g the l a t t e r s t a g e s o f p r e p a r a t i o n o f t h i s t h e s i s , and f rom i n f o r m a l s o u r c e s t o p r o v i d e an e x p l a n a t i o n o f how I b e l i e v e t h i s pathway o f p h o t o s y n t h e s i s w o r k s . T h i s i s a t e n t a t i v e v e n t u r e which must be based on i n c o m p l e t e e v i d -e n c e . I hope t h a t i t w i l l be v a l u a b l e as a framework f o r f u r -t h e r c r i t i c a l e x p e r i m e n t s . The l o c a t i o n o f enzymes In g e n e r a l terms perhaps the most impor tan t f i n d i n g t o d a t e i s t h a t the o p e r a t i o n o f the p - c a r b o x y l a t i o n pathway o f p h o t o s y n t h e s i s r e q u i r e s the p a r t i c i p a t i o n of two s e p a r a t e l e a f t i s s u e s . Whi le a l l o f the enzymes a p p a r e n t l y r e q u i r e d a r e p r e s e n t i n t h e l e a v e s o f p l a n t s w i t h t h e ^ - c a r b o x y l a t i o n p a t h -way, n e i t h e r the bund le shea th n o r the m e s o p h y l l c e l l s c o n t a i n a l l o f t h e s e . T h i s i n d i c a t e s t h a t c o o p e r a t i o n i n p h o t o s y n t h e t i c metabo l ism must o c c u r between t h e s e two t i s s u e s . The i n t a c t l e a f thus appears t o be r e q u i r e d f o r t h e o p e r a t i o n o f t h i s p a t h -way. In c o n t r a s t t h e normal C a l v i n c y c l e c a n a p p a r e n t l y o c c u r i n a s i n g l e i n t a c t c h l o r o p l a s t . T h i s may e x p l a i n the i n a b i l i t y t o o b t a i n c e l l - f r e e p r e p a r a t i o n s c a p a b l e o f s u s t a i n i n g t h i s pathway. The l o c a t i o n o f some enzymes which have been s t u d i e d i s summarized i n T a b l e I. Some enzymes such as P - e n o l p y r u v a t e c a r -b o x y l a s e and " m a l i c " enzyme a r e a p p a r e n t l y l o c a t e d i n o n l y one 73 o f the two t i s s u e s , w h i l e o t h e r s such as g l y c e r a l d e h y d e - 3 -phosphate dehydrogenase a r e n o t . I t Is a l s o noteworthy t h a t s p e c i e s d i f f e r e n c e s o c c u r i n the d i s t r i b u t i o n o f NADP m a l i c dehydrogenase and perhaps o t h e r enzymes. D i f f e r e n t t e c h n i -ques have been employed i n t h e s e s t u d i e s . I t i s e n c o u r a g i n g t h a t the s t u d i e s o f S l a c k et a l . (15) u s i n g a nonaqueous d e n -s i t y g r a d i e n t t e c h n i q u e a g r e e , where c r o s s checks a r e a v a i l a b l e , w i t h the s t u d i e s r e p o r t e d i n C h a p t e r I u s i n g the d i f f e r e n t i a l g r i n d i n g t e c h n i q u e . The s t u d i e s o f S l a c k e t a l . (15) a l s o i n d i c a t e t h a t many o f t h e s e enzymes a r e l o c a t e d i n c h l o r o p l a s t s . However i t appears t h a t no s i n g l e c h l o r o p l a s t c o n t a i n s a l l o f the enzymes r e q u i r e d t o comple te the pathway. F i g u r e 1 p r o v i d e s a summary o f how I b e l i e v e the r e a c t i o n s a r e o r g a n i z e d w i t h i n the l e a f . The c o n -c e r t e d o p e r a t i o n o f two d i f f e r e n t o h l o r o p l a s t t y p e s i n s e p a r a t e c e l l s r e q u i r e s t h a t some m e t a b o l i t e s move f rom one t o the o t h e r . 10 E v i d e n c e was p r e s e n t e d i n C h a p t e r I and Appendix I I I t h a t C -l a b e l l e d C ^ - a c i d s a r e p r e s e n t i n the c y t o p l a s m i c p o r t i o n o f the l e a f and c o u l d be t r a n s p o r t e d as p o s t u l a t e d from the m e s o p h y l l t o the bund le shea th c h l o r o p l a s t s . A r e t u r n t r a n s p o r t o f p y -r u v a t e , o r a compound r e l a t e d to i t must a l s o o c c u r t o s u s t a i n CC>2 f i x a t i o n i n the m e s o p h y l l c e l l s . T h i s r e t u r n t r a n s p o r t has not been i n v e s t i g a t e d a t a l l . L a b e l l e d p y r u v a t e was no t d e t e c t e d i n our exper iments however i t i s p o s s i b l e t h a t much o f the p y r u v a t e was t r a n s a m i n a t e d t o a l a n i n e which was d e t e c t e d i n some f e e d i n g s . The f i g u r e a l s o i n d i c a t e s t h a t 3-PGA and t r i o s e phosphates may a l s o be t r a n s p o r t e d between the two c h l o r o p l a s t t y p e s . I n c l u s i o n o f t h i s s h u t t l e w i l l be d i s c u s s e d l a t e r as a p o s s i b l e mechanism f o r b a l a n c i n g energy consumpt ion t o energy E p i l o g u e TABLE I . The l o c a t i o n of some p h o t o s y n t h e t i c enzymes i n l e a v e s of p l a n t s w i t h t h e ^B-car b o x y l a t i o n pathway of p h o t o s y n t h e s i s . Enzyme P l a n t Location"*" R e f e r e n c e s P - e n o l p y r u v a t e c a r b o x y l a s e Zea mays Gomphrena g l o b o s a A t r i p l e x r o s e a M e s o p h y l l M e s o p h y l l M e s o p h y l l 2 2 3 p y r u v a t e , P i d i k i n a s e Zea mays Amaranthus e d u l i s M e s o p h y l l M e s o p h y l l 14 , 1 5 14 NADP-malate dehydrogenase Zea mays Gomphrena g l o b o s a M e s o p h y l l Both 2 , 15 2 NADP-glyceraldehyde dehydrogenase Zea mays Both 14 , 1 5 " m a l i c " enzyme Zea mays Gomphrena g l o b o s a Bundle Bundle Sheath Sheath 2 , 15 2 RuDP c a r b o x y l a s e Zea mays Gomphrena g l o b o s a Amaranthus e d u l i s A t r i p l e x r o s e a Bundle Bundle Bundle Bundle Sheath Sheath Sheath Sheath 2,14 ,15 2 14 3 p h o s p h o r i b u l o - k i n a s e Zea mays Bundle Sheath 15 1 L o c a t i o n s a r e a s s i g n e d e i t h e r t o the m e s o p h y l l o r t o t h e bund l e s h e a t h t i s s u e s when t h e r e i s a l a r g e d i f f e r e n c e between the s p e c i f i c : a c t i v i t i e s of t h e enzyme i n the two e x t r a c t s . The l o w e r s p e c i f i c a c t i v i t i e s of the s e enzymes i n t h e e x t r a c t of the o t h e r t i s s u e may be due t o l o w e r endogenous a c t i v i t i e s of t h e s e enzymes i n t h a t t i s s u e , o r t o c r o s s c o n t a m i n a t i o n from the o t h e r t i s s u e . TABLE I. cont'd. Enzyme P l a n t L o c a t i o n References r i b o s e phosphate isomerase Zea mays Bundle Sheath 15 f r u c t o s e diphosphate a l d o l a s e Zea mays Bundle Sheath 15 f r u c t o s e diphosphatase Zea mays Bundle Sheath 15 ADP glucose and UDP glucose p y r o p h o s p h o r y l a s e 1 s Zea mays Bundle Sheath 10 ADP glucose and UDP glucose transphosphorylase Zea mays Bundle Sheath 10 s t a r c h phosphorylase Zea mays Bundle Sheath 10 g l y c o l i c a c i d oxidase Zea mays A t r i p l e x rosea Bundle Bundle Sheath Sheath 17 3 Epilogue Figure 1. The d i v i s i o n of reactions between bundle sheath and mesophyll chloroplasts. The background electron-micrograph showing adjcent bundle sheath and mesophyll c e l l s of Sorghum  sudanense was provided courtesy of Dr. Thana Bisalputra. Note the plasmodesmata penetrating the c e l l wall which separates %% the two c e l l s . NADPH oxa 4 - ^ y iloacetaie* 1 K v malate P-enolpyruvate ^ -pyruvate " ATP 3-PQA-NADgH 1 M e s o p h y l l C h l e r o p l a s t 77 a v a i l a b i l i t y i n the bundle sheath and mesophyll c h l o r o p l a s t s . S l a c k _et a l . (15 ) a l s o present a scheme s i m i l a r t o F i g u r e 1 . They i n d i c a t e however t h a t most of the 3-PGA i s formed i n the l e a f mesophyll. T h i s i s suggested by t h e i r f i n d i n g that both C^-acids and 3-PGA occur i n the mesophyll c h l o r o p l a s t s . I t however should be p o i n t e d out that the t r a n s c a r b o x y l a s e enzyme p o s t u l a t e d to c a t a l y z e t h i s r e a c t i o n has not yet been d e s c r i b e d . In t h i s t h e s i s arguments are presented t h a t " m a l i c " enzyme and RuDP carboxylase c o u l d b r i n g about t h i s s y n t h e s i s of 3-PGA. These enzymes are found i n adequate a c t i v i t i e s i n the bundle sheath c e l l s . Johnson and Hatch (11) r e p o r t t h a t l a b e l l e d RuDP accumulated d r a m a t i c a l l y i n l e a v e s of Zea mays su b j e c t e d to c o n d i t i o n s (no C 0 2 ) which should l e a d t o d e p l e t i o n of the C^-acids (the c a r b o x y l donors). T h i s dramatic accumulation suggests t h a t RuDP may serve as a s u b s t r a t e f o r the r e a c t i o n s l e a d i n g t o the s y n t h e s i s of 3-PGA from the C ^ - a c i d s . S l a c k et a l . ( 15 ) found t h a t l a b e l l e d RuDP occurred i n the bundle sheath c h l o r o p l a s t s , as does RuDP car b o x y l a s e . A m i c r o r a d i o -a u t o g r a p h i c study of Gomphrena showed t h a t " ^ C - l a b e l l e d Ox-a c i d s move t o the c e l l s of the bundle sheath. T h i s movement ap-1k p a r e n t l y precedes the t r a n s f e r of c t o 3-PGA. I t t h e r e f o r e seems to me t h a t the bulk of the evidence a v a i l a b l e a t t h i s time supports the p o s t u l a t e t h a t RuDP carboxylase and " m a l i c " enzyme mediate the c a r b o x y l t r a n s f e r r e a c t i o n . S e v e r a l r e a c t i o n s i n d i c a t e d i n F i g u r e 1 consume energy which i s d e r i v e d from ATP and NADPH produced i n the l i g h t r e a c -t i o n s of the c h l o r o p l a s t s . The f o r m a t i o n of P-enolpyruvate from pyruvate consumes two e q u i v a l e n t s of ATP (1., 6). Net f o r m a t i o n 78 of m a l i c a c i d from o x a l o a c e t i c a c i d consumes one e q u i v a l e n t of NADPH ( 9 ). The o p e r a t i o n of the C a l v i n c y c l e consumes t h r e e e q u i v a l e n t s of ATP and two of NADPH f o r each molecule of C0 2 reduced t o the l e v e l of carbohydrate. These r e a c t i o n s a l l operate i n a concert e d f a s h i o n . The r a t e of the process may be l i m i t e d i f i n s u f f i c i e n t energy i s a v a i l a b l e a t the s i t e of any of the above r e a c t i o n s . The f a c t t h a t these r e a c t i o n s oc-cur i n separate s i t e s makes i t necessary that some mechanism e x i s t t o balance the energy requirement i n one t i s s u e t o the energy a v a i l a b l e or v i c e v e r s a . Some p l a n t s such as Gomphrena globosa and a number of other d i c o t y l e d o n s seem t o have most of t h e i r c h l o r o p h y l l i n the bundle sheath c e l l s . These are the s i t e of the C a l v i n cy-c l e r e a c t i o n s thus they would a l s o seem t o have the g r e a t e s t energy requirement. Other p l a n t s such as Zea mays, Sorghum sp and Saccharum sp a r e not c o n s t r u c t e d a c c o r d i n g t o t h i s p a t t e r n . The bundle sheath c h l o r o p l a s t s c o n t a i n a r e l a t i v e l y s m a l l e r p r o p o r t i o n of the t o t a l c h l o r o p h y l l - hence l e s s a v a i l a b l e en-ergy. I t seems imperative t h a t the r e a c t i o n be arranged t o compensate f o r the l e s s e r a v a i l a b i l i t y of energy i n the bundle sheath. Two mechanisms seem f e a s i b l e f o r s h i f t i n g consumption of NADPH from the bundle sheath t o the mesophyll c h l o r o p l a s t s . A s h u t t l e system may be arranged i n v o l v i n g : C0 2 f i x a t i o n by P-enolpyruvate c a r b o x y l a s e ; r e d u c t i o n of ox a l o a c e t a t e to malate; o x i d a t i v e d e c a r b o x y l a t i o n of malate t o CO2 and pyruvate; and r e -g e n e r a t i o n of P-enolpyruvate from pyruvate. T h i s s h u t t l e i s important i n t r a n s p o r t i n g f i x e d carbon from the mesophyll t o the bundle sheath c h l o r o p l a s t s . .Two r e a c t i o n s i n t h i s s h u t t l e i n -79 v o l v e NADPH; i t i s o x i d i z e d i n the r e d u c t i o n of o x a l o a c e t a t e , and reduced i n the o x i d a t i v e d e c a r b o x y l a t i o n of malate. No net change i n the l e v e l of NADPH should occur as these r e a c -t i o n s should occur a t the same r a t e . However the s i t e s of these two r e a c t i o n s may d i f f e r . T h i s may a l t e r the a v a i l a b i l i t y of NADPH i n one l o c a t i o n . The NADP-malate dehydrogenase of Zea mays i s l o c a t e d i n the mesophyll c h l o r o p l a s t s and the "ma l i c " enzyme i s l o c a t e d i n the bundle sheath c h l o r o p l a s t s . Opera-t i o n of t h i s s h u t t l e a p p a r e n t l y r e s u l t s i n the consumption of one NADPH i n the mesophyll and the p r o d u c t i o n of one i n the bundle sheath. T h i s NADPH produced by o x i d a t i v e d e c a r b o x y l a -t i o n can p a r t i a l l y s a t i s f y the NADPH requirement of the C a l v i n c y c l e . The NADP-specific malate dehydrogenase of Gomphrena  globosa i s l o c a t e d i n both the mesophyll and bundle sheath. T h i s may suggest t h a t the s h u t t l e i s not as important i n t h i s p l a n t as i t i s i n Zea mays. Another s h u t t l e , i n v o l v i n g 3-PGA and t r i o s e phosphates may a l s o occur. I n Zea mays the enzyme NADP-glyceraldehyde - 3-phos-phate dehydrogenase occurs i n both bundle sheath and mesophyll c h l o r o p l a s t s . The r e d u c t i o n of 3-PGA t o t r i o s e phosphate can thus occur i n e i t h e r c h l o r o p l a s t with the consumption of one ATP and one NADPH. I f t r a n s p o r t of 3-PGA and t r i o s e phosphates occurs between the c h l o r o p l a s t s then the s i t e of 3-PGA r e d u c t i o n may be separate from the s i t e of 3-PGA f o r m a t i o n ( c a r b o x y l trans< f e r ) . Return t r a n s p o r t of t r i o s e phosphate would s u b s t i t u t e f o r NADPH i n the bundle. T h i s s h u t t l e i s q u i t e s i m i l a r t o one pro-posed by S t o c k i n g and L a r s o n (1'6 ). I t would however operate between c e l l s r a t h e r than between the c h l o r o p l a s t s and 80 the cytoplasm. The presence of t h i s s h u t t l e may e x p l a i n the f i n d i n g by S l a c k et a l . (.15 ) t h a t 3-PGA occurs i n the meso-p h y l l c h l o r o p l a s t s of Zea mays. Tr a n s p o r t of 3-PGA and t r i o s e phosphates between c h l o r o p l a s t s v i a the cytoplasm may e x p l a i n the r e s u l t , d i s c u s s e d i n Chapter I and Appendix I I I , t h a t a l a r g e p o r t i o n of the phosphate e s t e r s of Zea mays are l o c a t e d i n the cytoplasm. Such mechanism may be p a r t i c u l a r l y important i n view of the f i n d i n g by Downton et a l . ( 5 ) t h a t bundle sheath c h l o r o p l a s t s of Zea mays and Sorghum sudanense may not.be capable of producing NADPH i n the l i g h t r e a c t i o n . The r e a c t i o n s of t h i s pathway of p h o t o s y n t h e s i s m u s t , l i k e other b i o c h e m i c a l r e a c t i o n sequences,be s u b j e c t to some c o n t r o l mechanism. S t u d i e s have i m p l i c a t e d pyruvate (, Pi., d i k i n a s e as a r e g u l a t o r y enzyme i n t h i s pathway ( 1 , 8 , 13). I t s a c t i v i t y r e -sponds f a i r l y q u i c k l y t o changes i n the i n c i d e n t i l l u m i n a t i o n . Other enzymes as P-enolpyruvate carboxylase a l s o respond 1, but (7) much more s l o w l y t o changes i n i l l u m i n a t i o n . The measured ac-t i v a t i o n of some enzymes p a r t i c u l a r l y " m a l i c " enzyme and P-enol-pyruvate carboxylase f a r exceed the maximum p h o t o s y n t h e t i c r a t e s , thus i t seems l i k e l y t h a t o t h e r steps l i m i t the r a t e of the e n t i r e p r o c e s s . The t r a n s i e n t b u r s t of CO2 p r o d u c t i o n r e p o r t e d In Chapter I which occ u r r e d upon i l l u m i n a t i o n of Zea mays l e a v e s i m p l i e s some form of l i g h t c o n t r o l . A survey of many other p l a n t s with t h i s pathway i n d i c a t e s t h a t s i m i l a r l i g h t - c o n t r o l l e d , C0 2-producing processes occur i n many p l a n t s , and are a p p a r e n t l y a p r o p e r t y of t h i s pathway. I t seems reasonable t o a t t r i b u t e t h i s C0 2 p r o d u c t i o n t o o x i d a t i v e d e c a r b o x y l a t i o n of m a l i c a c i d . As r e -ported i n Chapter I I no evidence was found which i n d i c a t e s l i g h t 81 c o n t r o l o f the k i n e t i c p r o p e r t i e s o f " m a l i c " enzyme. D e s p i t e the f a c t t h a t no mechanism o f c o n t r o l c a n be advanced a t t h i s t ime i t seems p r o b a b l e t h a t some form o f l i g h t c o n t r o l p r e v e n t s c o n t i n u e d d e c a r b o x y l a t i o n o f m a l i c a c i d i n the d a r k . C l e a r l y much more i s t o be l e a r n e d c o n c e r n i n g mechanisms c o n t r o l l i n g t h i s pathway. The development and s u r v i v a l o f t h i s pathway w i t h i n the p l a n t kingdom must i n d i c a t e t h a t a t l e a s t under some c o n d i t i o n s i t c o n f e r s a s e l e c t i v e advantage t o p l a n t s p o s s e s s i n g i t . In g e n e r a l p l a n t s w i t h t h i s pathway do p a r t i c u l a r l y w e l l under c o n d i t i o n s o f h i g h temperature and h i g h l i g h t i n t e n s i t y , and they a r e perhaps more r e s i s t a n t t o water s t r e s s than p l a n t s w i t h the normal C a l v i n pathway• I t i s t empt ing t o a t t r i b u t e t h e s e p r o p e r t i e s t o the absence o f d e t e c t a b l e p h o t o r e s p i r a t i o n In p l a n t s w i t h t h i s pathway. P h o t o r e s p i r a t o r y i n h i b i t i o n o f n e t COg f i x a t i o n i n c r e a s e s d r a m a t i c a l l y a t h i g h tempera ture i n p l a n t s s u c h as wheat (12, 18) which l a c k B - c a r b o x y l a t i o n . P l a n t s a b l e t o a v o i d t h i s i n h i b i t i o n a t h i g h tempera ture would be expected t o show h i g h e r growth r a t e s (3), and b e t t e r r e l a t i v e compar ison between COg uptake and water l o s s (3. 0) as do p l a n t s w i t h t h i s pathway. T h i s absence o f d e t e c t a b l e p h o t o r e s p i r a t i o n may perhaps be r e l a t e d t o the s e p a r a t i o n o f r e a c t i o n s w i t h i n the l e a f . A t l e a s t a t h i g h l i g h t i n t e n s i t y the i n h i b i t o r y e f f e c t o f p h o t o -r e s p i r a t i o n i s p r i m a r i l y due t o l o s s o f C 0 2 f rom t h e l e a f . I f p h o t o r e s p i r a t o r y COg c o u l d be r e c y c l e d r a t h e r t h a n l o s t f rom the l e a f i n h i b i t i o n would be r e d u c e d . R e c e n t l y i t has been r e p o r t e d t h a t g l y c o l i c a c i d o x i d a s e o f Zea mays (17) and A t r l p l e x r o s e a 8 2 (3) i s l o c a t e d i n the c e l l s o f the bundle s h e a t h . T h i s enzyme i s thought t o be r e s p o n s i b l e f o r p r o d u c t i o n o f p h o t o r e s p i r a -t o r y C 0 2 . The bund le s h e a t h c e l l s a r e sur rounded by the meso-p h y l l c e l l s which a r e a p p a r e n t l y v e r y e f f i c i e n t f i x e r s o f C 0 2 , and a l s o l a c k p h o t o r e s p i r a t o r y mechanisms. T h i s c o n f i g u r a t i o n may i n s u r e t h a t p h o t o r e s p i r a t o r y CC* 2 i s r e c y c l e d r a t h e r than e s c a p i n g f rom the l e a f . Thus i t may be t h a t t h i s pathway has d e v e l o p e d as a means o f masking the i n h i b i t o r y e f f e c t o f p h o t o -r e s p i r a t i o n . The pathway even w i t h o u t c o n s i d e r i n g the p o s s i b i l i t y o f r e c y c l i n g appears t o be e n e r g e t i c a l l y l e s s e f f i c i e n t than the C a l v i n pathway. A t l e a s t two a d d i t i o n a l e q u i v a l e n t s o f ATP a r e r e q u i r e d f o r each C 0 2 f i x e d . Measurement o f quantum e f f i -c i e n c y f o r A t r i p l e x r o s e a which has J 3 - c a r b o x y l a t i o n and A t r i p l e x  p a t u l a which l a c k s i t were r e c e n t l y r e p o r t e d by Bjorkman ( 3 ) » The maximum e f f i c i e n c y o f A t r i p l e x r o s e a was 35% l ower than t h a t o f A t r i p l e x p a t u l a . T h i s lower e f f i c i e n c y was o f f s e t by p h o t o -r e s p i r a t o r y i n h i b i t i o n o f A t r i p l e x p a t u l a i f the measurements were made i n normal a i r ( 2 1 # 0 2 ) r a t h e r than low 0 2 a i r (Z% 0^). Presumably the d i f f e r e n c e would be more t h a n o f f s e t i n c o n d i -t i o n s where l i g h t i s n o t l i m i t i n g . On the o t h e r hand the lower e f f i c i e n c y o f the / J - c a r b o x y l a t i o n pathway c o u l d become q u i t e impor tan t under l i g h t l i m i t i n g c o n d i t i o n s p a r t i c u l a r l y a t low tempera tures where - p h o t o r e s p i r a t o r y i n h i b i t i o n o f p h o t o s y n t h e s i s becomes l e s s i m p o r t a n t . These c o n s i d e r a t i o n s may suggest e c o l o -g i c a l and p h y s i o l o g i c a l r a m i f i c a t i o n s o f t h i s pathway. 83 L i t e r a t u r e C i t e d 1. Andrews, T. J . , and Hatch, M.D. 1969. P r o p e r t i e s and mech-anism of a c t i v a t i o n of pyruvate, phosphate d i k i n a s e from l e a v e s . Biochem. J . 110: 117-125. 2. Berry, J.A., Downton, W.J.S., and Tregunna, E.B. 1970. The p h o t o s y n t h e t i c carbon metabolism of Zea mays and Gomphrena gl o b o s a : the l o c a t i o n of the CO2 f i x a t i o n and the c a r b o x y l t r a n s f e r r e a c t i o n s . Can. J . Botany ( i n p r e s s ) . 3. Bjorkman, O l l e , Gauhl, Eckard, and Nobs, Malcolm A. I 9 6 8 -1969. Comparative s t u d i e s on A t r i p l e x s p e c i e s with and without B - c a r b o x y l a t i o n p h o t o s y n t h e s i s and t h e i r f i r s t -g e n e r a t i o n h y b r i d . C u r n i g i e I n s t , of Washington Year Book 68 ( i n p r e s s ) . 0 . Downes, R.W. 1969. D i f f e r e n c e s i n t r a n s p i r a t i o n r a t e s be-tween t r o p i c a l and temperate grasses under c o n t r o l l e d c o n d i t i o n s . P l a n t a 8 8 : 261-273. 5. Downton, J . , Ber r y , J . , and Tregunna, E.B. 1970. C^-Photo-s y n t h e s i s : N o n c y c l i c e l e c t r o n f l o w and grana develop-ment i n bundle sheath c h l o r o p l a s t s . ( I n p r e p a r a t i o n . ) 6. Hatch, M.D.,and Sl a c k , C R . I 9 6 7 . The p a r t i c i p a t i o n of phosphoenolpyruvate synthetase i n p h o t o s y n t h e t i c COg f i x a t i o n of t r o p i c a l g r a s s e s . Arch. Biochem. Biophys. 101: 2 2 0 - 2 2 5 . 7. Hatch, M.D., S l a c k , C.R., and B u l l , T.A. 1969. L i g h t - i n -duced changes i n the content of some enzymes of the C^-d i c a r b o x y l i c a c i d pathway of pho t o s y n t h e s i s and i t s e f f e c t on other c h a r a c t e r i s t i c s of ph o t o s y n t h e s i s . Phytochem. 8: 6 9 7 - 7 0 6 . 8. Hatch, M.D., and S l a c k , C R . 1969. S t u d i e s on the mechanism of a c t i v a t i o n and i n a c t l v a t i o n of pyruvate, phosphate d i k i n a s e . Biochem. J . 112: 5O9-558. 9. Hatch, M.D., and S l a c k , C R . 1969. NADP-Specific malate de-hydrogenase and g l y c e r a t e k i n a s e i n le a v e s and evidence f o r t h e i r l o c a t i o n i n c h l o r o p l a s t s . Biochem. Biophys. Res. Comm. 30: 589-593. 10. Huber, W.,^DePekete, M.A.R., and Z i e g l e r , H. 1969. Enzyme des Starkeumsatzes i n Biindelscheide - und P a l i s a d e n -c h l o r o p l a s t e n von Zea mays. P l a n t a 87: 36O-36O. 11. Johnson, H i l a r y S., and Hatch, M.D. 1969. The C ^ - d i c a r b o x y l l c a c i d pathway of ph o t o s y n t h e s i s . I d e n t i f i c a t i o n of i n t e r -mediates and products and q u a n t i t a t i v e evidence f o r the route of carbon flow. Biochem. J . 110: 127-130. 84 12. J o l l i f f e , P.A., and Tregunna, E.B. 1968. E f f e c t of tempera-t u r e , COp c o n c e n t r a t i o n , and l i g h t i n t e n s i t y on oxygen i n h i b i t i o n of photosynthesis i n wheat leaves. P l a n t P h y s i o l . 4 3 : 9 0 2 - 9 0 6 . 13. Slack, CR. 1968. The p h o t o a c t i v a t i o n of a phosphopyruvate synthase i n leaves of Amaranthus palmerl. Biochem. Biophys. Res. Comm. 3 0 : 483-488. 14. Slack, CR. I 9 6 9 . L o c a l i z a t i o n of c e r t a i n photosynthetic enzymes i n mesophyll and parenchyma sheath c h l o r o p l a s t s of maize and Amaranthus palmeri. Phytochem. 8 : 1387-1391. 15. Slack, C.R., Hatch, M . D . , and Goodchild, D.J. 1969. D i s t r i -b u t i o n of enzymes i n mesophyll and parenchyma-sheath c h l o r o p l a s t s of maize leaves i n r e l a t i o n to the C^-di-c a r b o x y l i c a c i d pathway of photosynthesis. Biochem. J . 114: 4 8 9 - 4 9 8 . 16. S t o c k i n g , CR., and Larson, Susan. 19^9. A; c h l o r o p l a s t cytoplasmic s h u t t l e and the r e d u c t i o n of e x t r a p l a s t i d NAD. Biochem. Biophys. Res. Com. 37: 278-282. 17. T o l b e r t , N.E. Communicated at the XI I n t e r n a t i o n a l B o t a n i c a l Congress Meetings August 28 - September 4, 1969. S e a t t l e , Washington. 18. Tregunna, E.B., Krotkov, G., and Nelson, CD. 1966. E f f e c t of oxygen on the r a t e of p h o t o r e s p i r a t i o n i n detached tobacco leaves. P h y s i o l . Plantarum 19: 723-733. 

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