Regulation of Starch Synthesis - ACS Symposium Series (ACS

Chapter 6

Regulation of Starch Synthesis Biochemical and Genetic Studies 1

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Jack Preiss , Douglas Cress , Jan Hutny , Matthew Morell , Mark Bloom , Thomas Okita , and Joseph Anderson Downloaded by CHINESE UNIV OF HONG KONG on March 4, 2016 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch006

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Department of Biochemistry, Michigan State University, East Lansing, MI 48824 Institute of Biological Chemistry, Washington State University, Pullman, WA 99164

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A key site in the regulation of starch synthesis is the enzymatic step where ADPglucose (ADPGlc) is synthesized. 3-P-Glycerate activates and orthophosphate (P ) inhibits ADPGlc synthetase (EC 2.7.7.27) activity. Spinach leaf ADPGlc synthetase is composed of two subunits, of 51 and 54 kilodaltons (kd) mass. Their N-terminal amino acid sequences and tryptic peptide maps are different and they are antigenically dissimilar. Pyridoxal-P (PLP), an activator analog, when reduced onto ADPGlc synthetase causes the enzyme to be more active in the absence of activator. The covalently modified enzyme is very resistant to P inhibition suggesting that PLP binds at or close to the activator site. The activator binding site sequence is close to the carboxyl terminus of the enzyme. Starchless mutants of Arabidopsis thaliana have been isolated that lack or contain low ADPGlc synthetase activity. Genetic analysis showed that the deficiency of both starch and ADPGlc synthetase activity in the mutants were attributable to a single, nuclear, recessive gene demonstrating that in vivo leaf starch synthesis is entirely dependent on a pathway involving ADPGlc synthesis. Recent results show that the photo affinity substrate analogue, 8-azidoADPglucose (8-N ADPGlc) inactivates the spinach leaf enzyme when irradiated with UV light. ADPGlc can protect the enzyme from inactivation. When labeled 8-N ADPGlc is used, incorporation is seen mainly if not solely in the 54 i

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Current address: Plant Environmental Biology Group, Research School of Biological Sciences, Australian National University, Canberra, A.C.T. 2601, Australia Current address: Public Affairs Department, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724

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0097-6156/89/0389-0084$06.00/0 1989 American Chemical Society

Whitaker and Sonnet; Biocatalysis in Agricultural Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Downloaded by CHINESE UNIV OF HONG KONG on March 4, 2016 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch006

kd subunit. A p a r t i a l deduced amino a c i d sequence of the spinach l e a f ADPGlc synthetase 51 kd subunit has been obtained from two cDNA clones and compared with the known deduced amino a c i d sequence of the E s c h e r i c h i a c o l i and r i c e endosperm ADPGlc synthetases.

ADPGlc synthetase (EC 2.7.7.27) i s a key enzyme involved i n the synthesis of starch (1_). Maize endosperm mutants, shrunken-2 and b r i t t l e - 2 a r e d e f i c i e n t i n ADPGlc synthetase a c t i v i t y . The enzyme a c t i v i t y i s less than 10? of wild type and the mutants accumulate 25% of the s t a r c h observed i n the normal endosperm (2,3). Recent r e s u l t s from our l a b o r a t o r y have d e s c r i b e d the i s o l a t i o n o f a mutant, TL25, of A r a b i d o p s i s t h a l i a n a l a c k i n g ADPGlc synthetase a c t i v i t y ( l e s s than 2% of normal a c t i v i t y ) and c o n t a i n i n g no d e t e c t a b l e l e v e l s of starch . Thus at least i n Arabidopsis the ADPGlc pathway accounts f o r 98Ï or more of the s t a r c h s y n t h e s i s o c c u r r i n g both i n l e a f and root cap. These r e s u l t s also strongly indicate that both UDPglucose synthetase and s t a r c h phosphorylase play a n e g l i g i b l e , i f any, r o l e i n s t a r c h synthesis. The mutant a l s o was not able to grow w e l l i n a 12 hour l i g h t / 1 2 hour dark photoperiod c y c l e but could grow at r a t e s s i m i l a r to the normal plant under continuous l i g h t (^0. T h i s suggested that s t a r c h may be n e c e s s a r y f o r o p t i m a l growth under normal p h y s i o l o g i c a l conditions. I t i s quite possible that under growth c o n d i t i o n s i n the absence o f l i g h t that the presence o f c h l o r o p l a s t starch i s required for i t s metabolism to provide energy and carbon f o r export to other parts of the plant. Regulation o f s t a r c h synthesis occurs at the ADPGlc synthetic s t e p (1_). A c t i v a t i o n o f ADPGlc s y n t h e s i s i s a f f e c t e d by 3-P-glycerate (3-PGA) and i n h i b i t i o n i s caused by orthophosphate (P^). These effector metabolites i n t e r a c t i n that P^ can reverse the a c t i v a t i o n of ADPGlc synthesis caused by 3-PGA and increasing concentrations of 3-PGA can overcome P^ i n h i b i t i o n . Because of the importance of ADPGlc s y n t h e t a s e f o r p l a n t s t a r c h synthesis, e f f o r t s have been made to determine the structure of the enzyme and to r e l a t e c a t a l y t i c and a l l o s t e r i c f u n c t i o n t o structure. The spinach leaf enzyme i s composed of two subunits of 51 and kd mass (5,6). The molecular mass of the n a t i v e enzyme i s 206,000 and presumably i s a tetramer composed of two of each subunit. The subunits are a n t i g e n i c a l l y d i s s i m i l a r , e x h i b i t d i f f e r e n t peptide patterns on HPLC a f t e r t r y p s i n d i g e s t i o n and their N-terminal amino acid sequences are d i f f e r e n t ( 7 ) . Thus i t i s l i k e l y that the peptide subunits are products of d i f f e r e n t genes. Pyridoxal-5-phosphate (PLP) has been shown to be an activator analogue of 3-PGA (7,8). I t activates ADPGlc synthetic r a t e s about 5- to 6-fold and can overcome P^ i n h i b i t i o n as does the activator 3-PGA (7,8). Reductive phosphopyridoxylation of the spinach l e a f enzyme with l a b e l e d PLP g i v e s incorporation into both subunits i n equimolar amounts (7,9). The PLP modified enzyme i s less dependent on t h e presence o f a c t i v a t o r (3-PGA) and i s more r e s i s t a n t t o i n h i b i t i o n by the a l l o s t e r i c i n h i b i t o r , P i (7^9). The a c t i v a t o r ,



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3-PGA, when p r e s e n t d u r i n g t h e r e d u c t i v e phosphopyridoxylation i n h i b i t s t h e i n c o r p o r a t i o n o f PLP (7,9). T h e s e d a t a s u g g e s t t h a t PLP i s b i n d i n g a t t h e a l l o s t e r i c site. T h u s t h e a l l o s t e r i c b i n d i n g s i t e may b e o n b o t h t h e 51 a n d 54 k d subunits. F o r t h e 51 k d s u b u n i t , t h e a m i n o a c i d s e q u e n c e o f t h e r e g i o n w h e r e PLP b i n d s t o an e p s i l o n a m i n o r e s i d u e o f l y s i n e has been determined ( 7 , 9 ) . Of i n t e r e s t i s t h a t t h i s s e q u e n c e i s v e r y s i m i l a r t o a d e d u c e d a m i n o a c i d s e q u e n c e o b t a i n e d from the n u c l e o t i d e s e q u e n c e o f a cDNA c l o n e o f t h e r i c e endosperm ADPGlc synthetase gene (7,9) . The a c t i v a t o r b i n d i n g s i t e i s s i t u a t e d c l o s e t o t h e c a r b o x y l t e r m i n u s o f t h e 51 k d s u b u n i t (£) . I n t h e c a s e of the b a c t e r i a l ADPGlc s y n t h e t a s e s the a c t i v a t o r b i n d i n g s i t e i s a t the amino t e r m i n a l r e g i o n of the s u b u n i t peptide (10). In o r d e r to o b t a i n f u r t h e r i n f o r m a t i o n on the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s of the p l a n t ADPGlc synthetase w i t h r e s p e c t t o t h e s u b s t r a t e s i t e we e m p l o y e d t h e u s e o f a p h o t o a f f i n i t y s u b s t r a t e a n a l o g u e , 8 - a z i d o - A D P G l c (1J_). Our preliminary r e s u l t s are reported here. We h a v e a l s o r e c e n t l y b e e n a b l e t o i s o l a t e two cDNA c l o n e s o f t h e s p i n a c h l e a f A D P G l c s y n t h e t a s e 51 kd gene and t h e s e r e s u l t s a r e a l s o r e p o r t e d . RESULTS Studies with 8-azido Nucleotide Substrates. F i g u r e 1 shows t h a t 8-azido-ATP (8-N3ATP) i s a s u b s t r a t e f o r the spinach leaf ADPGlc synthetase. The product f o r m e d was d e t e r m i n e d t o be 8 - N 3 A D P G I C by p r o c e d u r e s u s e d p r e v i o u s l y ( 1 J _ ) . The isolated 8 - N 3 A D P G I C s h o w e d a t y p i c a l s p e c t r u m o f an 8 - a z i d o a d e n i n e n u c l e o t i d e w i t h an a b s o r p t i o n maximum a t 281 nm w h i c h was l o s t u p o n p h o t o l y s i s (V2). The K v a l u e f o r 8 - N 3 A T P was 0.81 ± 0.05 mM, about 7 - f o l d higher than t h e K v a l u e o f 0.12 mM f o r t h e n a t u r a l s u b s t r a t e ATP. I n t h e a b s e n c e o f a c t i v a t o r , 3-PGA, t h e K for 8 - N 3 A T P was i n c r e a s e d t o a b o u t 3 mM. Moreover, the V of the spinach l e a f e n z y m e was o n l y 0.48 y m o l - m i n " '-mg~ o f product formed, only about 1 % of that o b s e r v e d w i t h ATP (50 umol-min"^-mg~^). This r e l a t i v e a c t i v i t y seen w i t h the azido a n a l o g u e i s s i m i l a r t o w h a t was o b s e r v e d f o r t h e E s c h e r i c h i a c o l i e n z y m e (1J_) w h e r e t h e m a x i m a l v e l o c i t y r a t e s o b s e r v e d w i t h 8-N3ATP and 8 - N 3 A D P G l c w e r e o n l y 0 . 3 and 0.9% of those observed w i t h the natural substrates, respectively. For t h e s p i n a c h l e a f enzyme h o w e v e r , 8-N3ADPGIC has a K o f 80 μΜ c o m p a r e d t o t h e K f o r ADPGlc o f 2 2 5 μΜ. The m a x i m a l v e l o c i t y f o r 8-N3ADPGIC, h o w e v e r , i s o n l y 0.25 μ Γ η ο 1 - Γ η ΐ η " " - π ^ - , 0.6% of t h a t observed w i t h ADPGlc. m

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Figure 2 shows t h a t i r r a d i a t i o n of the s p i n a c h l e a f ADPGlc synthetase i n t h e p r e s e n c e o f 8 - N 3 A D P G l c , 3-PGA a n d M g resulted i n l o s s of enzyme a c t i v i t y . No o r s l i g h t i n a c t i v a t i o n o f the e n z y m e , h o w e v e r , w a s o b s e r v e d w h e n t h e e n z y m e w a s e x p o s e d t o UV l i g h t w i t h o u t 8 - N 3 A D P G I C o r when t h e enzyme was i n c u b a t e d w i t h the a n a l o g i n the dark. T h i s may be due t o a s l i g h t i n s t a b i l i t y o f t h e enzyme a c t i v i t y d u r i n g t h e i n c u b a t i o n p e r i o d . Almost 70% i n a c t i v a t i o n w a s o b s e r v e d i n t h e p r e s e n c e o f 1.5 mM 8 - N 3 A D P G I C . E f f e c t i v e p r o t e c t i o n w a s o b s e r v e d w i t h 10 mM A D P G l c . Neither U D P g l u c o s e ( U D P G l c ) n o r A DP w e r e a s e f f e c t i v e a s A D P G l c . F i g u r e 2A 2 +




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Figure 1. 8-azido-ATP s a t u r a t i o n curves of ADPGlc-synthetase i n t h e p r e s e n c e (·) o r a b s e n c e ( o ) o f 1 mM 3-PGA. Enzyme was i n c u b a t e d 10 m i n a t 37° a t i n d i c a t e d c o n c e n t r a t i o n s o f 8 - N 3 A T P i n 100 mM H e p e s ( p H 7 . 5 ) , 5 mM M g C l , 1 mM [ C ] G l c 1-P, B S A ( 5 0 u g / r e a c t i o n ) , and p y r o p h o s p h a t a s e (0.1 pL/reaction) i n a v o l u m e o f 200 y L a s p r e v i o u s l y d e s c r i b e d ( i n r e f . 5 ) . U

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F i g u r e 2. P h o t o i n a c t i v a t i o n o f A D P G l c - s y n t h e t a s e a t 1.5 mM 8-N3ADPGIC. The m i x t u r e c o n t a i n i n g 1 μΜ ( s u b u n i t s ) e n z y m e , 10 mM HEPES (pH 7 . 5 ) , 1 mM 3 - P G A , 5 mM M g C l , 1.5 mM 8 - N 3 A D P G I C and i n d i c a t e d n u c l e o t i d e , was i r r a d i a t e d i n t h e w e l l s o f C o o r s p l a t e w i t h U V S - 5 4 l a m p , a t a d i s t a n c e o f 5 cm as p r e v i o u s l y d e s c r i b e d (1J_). The t i m e o f i r r a d i a t i o n i n e x p e r i m e n t A was 3 min and i n Β i t was v a r i e d as i n d i c a t e d . A - E f f e c t of the v a r i e d c o n c e n t r a t i o n s of ADPGlc on photoinactivation. Β - E f f e c t o f 10 mM ADPGlc ( A ) , ADP (o) and UDPGlc ( • ) on t h e r a t e o f p h o t o i n a c t i v a t i o n . (x) i s c o n t r o l sample, i r r a d i a t e d without 8-N3ADPGlc. (•) i s sample p h o t o i n a c t i v a t e d w i t h o u t p r o t e c t i n g n u c l e o t i d e . 2

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shows t h a t t h e p r o t e c t i o n by A D P G l c i s c o n c e n t r a t i o n dependent r e a c h i n g maximum p r o t e c t i o n between 10 t o 15 mM. T h u s , t h e a z i d o nucleotides are substrates a n d t h e s u b s t r a t e A D P G l c i s most e f f e c t i v e i n p r o t e c t i n g from p h o t o i n a c t i v a t i o n . T h i s would s t r o n g l y s u g g e s t t h a t 8-N^ADPGlc i s b i n d i n g at the substrate b i n d i n g s i t e and p h o t o i n a c t i v a t i o n i s o c c u r r i n g a t or c l o s e t o t h a t site. L a b e l e d 8 - N 3 A D P G I C was p r e p a r e d w i t h u n i f o r m l y l a b e l e d [ C ] glucose-1-P as p r e v i o u s l y described (1_J_) a n d u s e d to p h o t o i n a c t i v a t e the s p i n a c h l e a f ADPGlc s y n t h e t a s e . About 1.33 nmol o f a n a l o g u e was i n c o r p o r a t e d p e r n a n o m o l e o f t h e native h e t e r o t e t r a m e r enzyme. The enzyme was t h e n s u b j e c t e d to sodium dodecyl sulfate gel electrophoresis. As s e e n i n F i g u r e 3 t h e i n c o r p o r a t i o n o f r a d i o a c t i v i t y i s m a i n l y i f not s o l e l y i n the 54 kd subunit (lane 3). Lane 2 shows t h a t ADPGlc p a r t i a l l y i n h i b i t s t h e i n c o r p o r a t i o n as the a u t o r a d i o g r a p h i c spot i s l e s s i n t e n s e . The i n c o r p o r a t i o n was 67% l e s s i n the p r e s e n c e o f A D P G l c a s i n d i c a t e d by t h e amount o f l a b e l p r e c i p i t a t e d w i t h 10% t r i c h l o r o a c e t i c a c i d . T h i s would suggest t h a t the s u b s t r a t e b i n d i n g s i t e i s on t h e 54 kd subunit. There is s t i l l a p o s s i b i l i t y , however, that the c a t a l y t i c / s u b s t r a t e s i t e i s shared between the 2 s u b u n i t s b u t t h a t the r e a c t i v e n i t r e n e r a d i c a l r e a c t s w i t h a r e a c t i v e nucleophile r e s i d u e on the l a r g e r s u b u n i t c a u s i n g most o f t h e l a b e l t o r e s i d e on t h a t s u b u n i t . N e v e r t h e l e s s , the d a t a do s u g g e s t a f u n c t i o n f o r the 54 kd s u b u n i t .


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cDNA C l o n e s o f t h e S p i n a c h L e a f A D P G l c S y n t h e t a s e G e n e . Using a n t i b o d i e s p r e p a r e d w i t h t h e s u b u n i t s o f t h e s p i n a c h l e a f ADPGlc s y n t h e t a s e and w i t h t h e n a t i v e e n z y m e , two cDNA c l o n e s , SL1 and S L 5 , were i s o l a t e d from a Xgt11 s p i n a c h l e a f l i b r a r y p r e p a r e d by C l o n e t e c h L a b o r a t o r i e s , I n c . , Palo A l t o , CA. B o t h c l o n e s gave e x p r e s s i o n o f p r o t e i n s t h a t r e a c t e d w i t h a n t i b o d y made w i t h t h e 51 kd s u b u n i t and were sequenced v i a the dideoxy n u c l e o t i d e t e c h n i q u e (13). SL1 , found t o be 382 base p a i r s i n s i z e , and S L 5 , a b o u t 1176 b a s e p a i r s i n s i z e , were s e q u e n c e d . U n f o r t u n a t e l y , t h e r e i s no o v e r l a p w i t h t h e s e cDNA c l o n e s . P r e s e n t l y , a b o u t 18 n u c l e o t i d e b a s e p a i r s o f SL5 have not been sequenced a t the Hind I I I r e g i o n as fragments o f the DNA were p r e p a r e d from SL5 f o r s e q u e n c i n g a t t h a t region. F i g u r e 4 shows t h e d e d u c e d amino a c i d sequence from the n u c l e o t i d e s e q u e n c e o f t h e two c l o n e s and c o m p a r e s i t w i t h t h e c o m p l e t e r i c e s e e d enzyme d e d u c e d amino a c i d sequence ( 9 ) . There i s a l a r g e amount o f i d e n t i t y b e t w e e n t h e a m i n o a c i d s e q u e n c e s , c o r r e s p o n d i n g t o a b o u t 76%. Most n o t a b l e i s the sequence between r e s i d u e s 424-434 i n s p i n a c h l e a f where i t h a s b e e n shown t h a t L y s 431 i s t h e s i t e o f c h e m i c a l m o d i f i c a t i o n by PLP ( 7 , 9 ) . There i s complete agreement o f t h i s sequence i n the same a r e a w i t h t h e r i c e s e e d enzyme sequence 462-472. M o r e o v e r , t h e r e i s complete i d e n t i t y of the deduced amino a c i d sequences o f amino a c i d s 4 0 8 - 4 3 4 i n t h e s p i n a c h l e a f enzyme 51 kd s u b u n i t w i t h amino a c i d s 446-472 o f the r i c e endosperm enzyme s u b u n i t . P r e s e n t s t u d i e s a r e now i n v o l v e d i n d e t e r m i n i n g the s i t e o f l a b e l i n g o f the 54 kd s u b u n i t by 8 - N 3 A D P G I C and i s o l a t i o n o f t h e 54 kd s u b u n i t cDNA c l o n e s .



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Figure 3. SDS-Ε1ectrophoresis of ADPGlc-synthetase photoinactivated with [ C] 8-N3ADPGIC. 1.8 μΜ (subunits) e n z y m e i n 20 mM HEPES, 1 mM 3~PGA, 5 mM M g C l a n d 1.5 mM [ C ] 8 - N 3 A D P G I C ( s p e c i f i c a c t i v i t y , 1.8 χ 10? cpm p e r p m o l e ) , w a s i r r a d i a t e d 1 0 m i n w i t h U V S - 5 4 l a m p f r o m a d i s t a n c e o f 5 cm. Lane 1 - u n i r r a d i a t e d c o n t r o l ; Lane 2 enzyme p h o t o i n a c t i v a t e d i n t h e p r e s e n c e o f 1 0 mM A D P G l c ; L a n e 3 photoinactivated enzyme. A-Coomassie stained g e l ; B - a u t o r a d i o g r a p h o f t h e same g e l . 1 l |

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Figure 4. Comparison of the primary sequence of the spinach leaf ADPGlc synthetase with the r i c e endosperm enzyme deduced amino a c i d s e q u e n c e . The n o n - i d e n t i t i e s of amino a c i d s between the two enzymes are enclosed i n boxes. The s t a r t and beginning of the SL1 and SL5 clones are indicated with respect to open r e a d i n g frame as w e l l as the Hind I I I r e s t r i c t i o n nuclease r e g i o n where the n u c l e o t i d e sequence has not been determined.


92


Acknowledgment T h i s r e s e a r c h was s u p p o r t e d i n p a r t by NSF r e s e a r c h g r a n t DMB 86-10319. Literature Cited


1. 2. 3.

Preiss, J. In The Biochemistry of Plants; Preiss, J., Ed.; Academic Press, New York, 1988; Vol. 14, pp 182-254. Tsai, C.Y.; Nelson, O.E. Science 1966, 151, 341-43. Dickinson, D.B.; Preiss, J. Plant Physiol. 1969, 44,

4.

Lin, T.-P.; Caspar, T.; Somerville, C.; Preiss, J. Plant

1058-62.

Physiol. 1988, 86, 1131-35.

5. Copeland, L.; Preiss, J. Plant Physiol. 1981, 68, 996-1001. 6. Morell, M.; Bloom, M.; Knowles, V.; Preiss, J. Plant Physiol. 1987, 85,

7.

182-7.

Morell, M.K.; Bloom,M.;Preiss, J. J. Biol. Chem. 1988, 263, 633-7.

8.

9.

Preiss, J.; Morell, M.K.; Bloom, M.; Knowles, V.L.; Lin, T.-P. Proceedings of the VII International Congress on Photosynthesis Vol. III, 1987, Biggins, J., Ed.; Martinus Nijhoff, Dorchecht, The Netherlands, pp 693-700. Morell, M.K.; Bloom, M.; Larsen, R.; Okita, T.W.; Preiss, J. In Plant Gene Systems and their Biology. UCLA Symposia on Molecular and Cellular Biology, New Series Vol. 62. Key, J.L.; McIntosh, L., Eds.; A.R. Liss, Inc. New York 1987; pp 227-242.

10. 11.

Parsons, T.F.; Preiss, J. J. Biol. Chem. 1978, 253, 7638-45. Lee, Y.M.; Mukherjee, S.; Preiss, J. Arch. Biochem. Biophys.

12.

Muneyama, K.; Baur, R.F.; Shuman, D.A.; Robbins, R.D.; Simon,

13.

Sanger, F.; Nicklen, S.; Coulson, A.R. Proc. Natl. Acad. Sci.

1986, 244,

L.N.

585-95.

Biochemistry 1971, 10, 2390-5.

USA 1977, 74, RECEIVED

5463-67.

October 26, 1988


Regulation of Starch Synthesis - ACS Symposium Series (ACS

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