THE METABOLISM, STRUCTURE, AND

FUNCTION OF P L A N T LIPIDS

Edited by

Paul K. Stumpf U n i v e r s i t y o f C a l i f o r n i a , D a v i s

D a v i s , C a l i f o r n i a

J . Brian Mudd A R C O P l a n t C e l l R e s e a r c h I n s t i t u t e

D u b l i n , C a l i f o r n i a

and

W . David Nes U.S. D e p a r t m e n t o f A g r i c u l t u r e

A l b a n y , C a l i f o r n i a

P L E N U M P R E S S • N E W Y O R K A N D LONDON

Library of Congress Cataloging in Publication Data

International Symposium on Plant Lipids (7th: 1986: L'niversity of California, Davis) The metabolism, structure, and function of plant lipids.

"Proceedings of the Seventh International Symposium on Plant Lipids, held July 27-August 1, 1986, at the University of California, Davis, Davis, California"-T.p. verso. Includes bibliographies and index. 1. Plant lipids-Congresses. I. Stumpf, Paul K. (Paul Karl), 1919- . II. Mudd, J.

Brian (John Brian), 1929- . III. Nes, W. David. IV. Title. [DNLM: 1. Lipids-metab-olism-congresses. 2. Plants-metabolism-congresses. W3 IN921BU 7th 1986b / QK 898.L56 161 1986b] QK898.L56I556 1986 581.19/247 87-1749 ISBN 0-306-42492-4

No part of this book may be reproduced, stored in a retrieval System, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,

recording, or otherwise, without written permission from the Publisher

Proceedings of the Seventh International Symposium on Plant Lipids, held July 27-August 1, 1986, at the University of California, Davis,

Davis, California

c> 1987 Plenum Press, New York A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013

All rights reserved

Printed in the United States of America

CONTENTS

BIOSYNTHESIS AND FUNCTION OF STEROLS AND OTHER ISOPRENOIDS

M u l t i p l e Roles f o r P l a n t S t e r o l s 3 W.R. Nes

Stereochemistry of Monoterpene C y c l i z a t i o n 11 R. Croteau

Diterpenes - The G i b b e r e l l i n B i o s y n t h e t i c Pathway i n Zea mays . . 19 B.O. Phinney and C R . Spray

Synthesis of Plastoquinone-9, a-Tocopherol and Phylloquinone (Vitamin K i ) and I t s I n t e g r a t i o n i n C h l o r o p l a s t Carbon Metabolism of Higher P l a n t s 29

D. S c h u l z e - S i e b e r t , U. Homeyer, J . S o l l and G. Schultz

Carotenoid B i o s y n t h e s i s and Carotenogenic Enzymes i n P l a s t i d s . . 37 H. K l e i n i g

C e l l Walls and Secondary Products as Obstacles to P l a n t Enzyme I s o l a t i o n : Problems and S o l u t i o n s , I n c l u d i n g a Simple L i q u i d N i t r o g e n Homogenizer f o r Bulk T i s s u e E x t r a c t i o n . . . 45

R.P. Sandstrom and W.D. Loomis

Developmental Regulation of S t e r o l and P e n t a c y c l i c T r i t e r p e n e B i o s y n t h e s i s and Composition: A C o r r e l a t i o n with Sorghum F l o r a l I n i t i a t i o n 53

R.C. Heupel, W.D. Nes and J.A. Verbeke

E f f e c t of I n t r a c e l l u l a r S t e r o l Concentration on S t e r o l E s t e r i f i c a t i o n i n Yeast 57

L.W. Parks, T.A. Lewis, C. Low and K. Haeckler

F u n c t i o n a l O r g a n i z a t i o n of Carotenoids and Prenylquinones i n the P h o t o s y n t h e t i c Membrane 63

H.K. L i c h t e n t h a l e r

A r a c h i d o n i c and Eicosapentaenoic A c i d s , Glucans and Calcium as Regulators of Resistance to a P l a n t Disease 75

M. Zook and J . Kuc

Presence of Unusually High L e v e l s of C h o l e s t e r o l i n the Shoot-Apices of Flowering P l a n t s 83

V.K. Garg, T.J. Douglas and L.G. Paleg

ix

Changes i n S t e r o l B i o s y n t h e s i s From [ 2- l l*C]Mevalonic Acid Düring Development of C u c u r b i t a maxima Seedlings 87

V.K. Garg and W.R. Nes

I n h i b i t i o n of Growth of C e l e r y C e l l s by P a c l o b u t r a z o l and I t s Reversal by Added S t e r o l s 91

P.A. Haughan, J.R. Lenton and L . J . Goad

The S t e r y l E s t e r s of Higher P l a n t s 95 L . J . Goad, J . Zimowski, R.P. Evershed and V.L. Male

Synthesis and B i o l o g i c a l E v a l u a t i o n of Fungal B i o r e g u l a t o r s of S t e r o l B i o s y n t h e s i s 103

E.J. P a r i s h , P.K. Hanners and W.D. Nes

A Comparison of S t e r o l and Long Chain F a t t y A l c o h o l B i o s y n t h e s i s i n Sorghum b i c o l o r 107

Y. Sauvaire, B. T a l , R.C. Heupel, R. England, P.K. Hanners, W.D. Nes and J.B. Mudd

Regulation of Terpenoid B i o s y n t h e s i s i n Tapped Latex 111 G.J. P i a z z a , E.J. Saggese and M.P. Thompson

T r i t e r p e n o i d B i o s y n t h e s i s i n Euphorbia l a t h y r i s 115 C.L. Skrukrud, S.E. T a y l o r , D.R. Hawkins and M. C a l v i n

P h y t o s t e r o l S t r u c t u r e and Composition i n the Chemosystematics of the C a r y o p h y l l a l e s 119

J.H. A d l e r and T.A. S a l t

B i o s y n t h e s i s of Diterpene P h y t o a l e x i n P r e c u r s o r s i n C e l l - f r e e E x t r a c t s of Rice 123

K. Wickham and C A . West

Endogenous G i b b e r e l l i n s i n Wheat Shoots 127 J.-T. L i n and A.E. S t a f f o r d

Molecular A s s o c i a t i o n s i n L i p i d - c a r o t e n o i d Monolayers 131 M. Tomoaia-Cotis,el, J . Zsako, E. C h i f u and P.J. Quinn

Separation and I d e n t i f i c a t i o n of C a r o t e n o i d - e s t e r s i n Red Pepper /Capsicum annum/ Düring Ripening 135

P.A. B i a c s , J . Bodnär, Ä. Hoschke, A. Cs. P a v i s , H. Daood, F. Hajdu and N. Kiss-Kutz

On the B i o s y n t h e s i s of Fragrance Compounds i n Ambrette (H i b i s c u s abelmoschus) Seeds 139

M.R. P o l l a r d , S. Jamil-Panah and T.Y. Nee

STRUCTURE AND FUNCTION OF LIPIDS

S t r u c t u r a l and Dynamic Aspects of Membrane L i p i d s 145 R.A. Demel

L i p i d S a t u r a t i o n by C a t a l y t i c Hydrogenation and I t s E f f e c t on Membrane S t r u c t u r e and F u n c t i o n 153

L. Vigh

x

S p a t i a l O r g a n i z a t i o n and F u n c t i o n a l Roles of A c y l L i p i d s i n T h y l a k o i d Membranes 161

P.A. S i e g e n t h a l e r , A. Rawyler and C. Giroud

U l t r a s t r u c t u r a l Studies on P l a n t Membranes 169 W.W. Thomson, K.A. P l a t t - A l o i a and R.D. B l i s s

Evidences f o r D i f f e r e n t A c y l L i p i d Domains i n Spinach and Oat T h y l a k o i d Membranes Supporting Various Photosynthetic Functions 177

P.A. S i e g e n t h a l e r , C. Giroud and J . Smutny

Phase T r a n s i t i o n Behavior of M o n o g a l a c t o s y l d i a c y l g l y c e r o l 181 P.J. Quinn and L. J . L i s

Is Monogalactosyl D i a c y l g l y c e r o l Involved i n the Packaging of L i g h t - h a r v e s t i n g C h l o r o p h y l l P r o t e i n s i n the T h y l a k o i d Membrane? 185

P.J. Dominy and W.P. Wi l l i a m s

Mechanisms of L i p i d - p r o t e i n Binding i n Photosynthetic Membranes . . 189 D.J. Murphy

L i p i d M o l e c u l a r Species Composition of Granal and Stromal Lamellae 193

H.A. Norman, J.B. St. John, F.E. Cal l a h a n , A.K. Mattoo and W.P. Wergin

On the F u n c t i o n of Methyl-branched Chain F a t t y Acids i n P h o s p h o l i p i d s of C e l l Membranes of Higher P l a n t s 197

A. Radunz

Free z i n g R e s i s t a n c e and L i p i d Changes i n C h o l i n e - t r e a t e d Wheat Seedlings 201

W.P. W i l l i a m s , I. Horvath, P.J. Quinn, P.G. Thomas and L. Vigh

Drought S t r e s s E f f e c t s on Root C e l l Membranes 205 H. Svenningsson, M. Andersson and C. L i l j e n b e r g

M a n i p u l a t i n g Membrane F a t t y A c i d Compositions of Soybean P l a n t s . . 209 W.B. T e r z a g h i

Plasma Membrane L i p i d A l t e r a t i o n s F o l l o w i n g Cold A c c l i m a t i o n : P o s s i b l e Relevance to Freeze Tolerance 213

D.V. Lynch and P.L. Steponkus

Plasma Membrane and Tonoplast F r a c t i o n s I s o l a t e d from Spinach Leaves by P r e p a r a t i v e Free Flow E l e c t r o p h o r e s i s : E f f e c t of Photoinduction 217

C. Pen e l , G. Auderset, S. K i e f e r , A. Sandelius, A. Brightman, H. Greppin and D.J. Morre

Study of the I n t r a c e l l u l a r T r a n s f e r of L i p i d s to the Plasmalemma 221

P. Moreau, H. J u g u e l i n , R. L e s s i r e and C. Cassagne

C a 2 + and Int e r - m o l e c u l a r B r i d g i n g of Membranal Phos p h o l i p i d s and P r o t e i n s 225

Y.Y. Leshem

xi

A Membrane-located, Calcium-/Calmodulin-activated Phospholipase Stimulated by Auxin

D.J. Morre and B. Drobes 229

Regulation of Phospholipase A c t i v i t y i n Potato Leaves by P r o t e i n Phosphorylation-dephosphorylation and P r o t e o l y t i c A c t i v a t i o n 233

R.A. Moreau

BIOSYNTHESIS OF COMPLEX LIPIDS

Synthesis and Degradation of L i p i d Bodies i n the S c u t e l l a of Maize 239

A.H.C. Huang, R. Qu, S.-m. Wang, V.B. Vance, Y.-z. Cao and Y.-h. L i n

On the C o n t r o l of Fa t t y A c i d Compositions of P l a n t G l y c e r o l i p i d s 247

G. Roughan

L i p i d D i s t r i b u t i o n and Synthesis Within the P l a n t C e l l 255 R. Douce, C. Alban, R. B l i g n y , M.A. Block, J . Coves, A.-J.

Dorne, E.-P. Journet, J . Joyard, M. Neuberger and F. R e b e i l l e

Regulation of Phosp h o l i p i d Headgroup Composition i n C a s t o r Bean Endosperm 265

T.S. Moore, J r .

M o l e c u l a r Species of P h o s p h a t i d y l c h o l i n e In P l a n t s : B i o s y n t h e s i s and Role In Oleate D e s a t u r a t i o n or F r e e z i n g R e s i s t a n c e . . . . 273

C. Demandre, A.M. J u s t i n , X.V. Nguyen, M. Gawer, A. Tremolieres and P. Mazliak

F a c t o r s A f f e c t i n g the F a t t y A c i d Composition of P h o s p h a t i d y l g l y c e r o l as Related to C h i l l i n g S e n s i t i v i t y i n Higher P l a n t s 283

S.R. Thomas, J . Sänchez and J.B. Mudd

On the Synthesis of D i g a l a c t o s y l d i a c y l g l y c e r o l i n C h l o r o p l a s t s , and I t s R e l a t i o n to Mon o g a l a c t o l i p i d Synthesis 293

J.F.G.M. Wintermans and J.W.M. Heemskerk

C h a r a c t e r i z a t i o n of G a l a c t o s y l t r a n s f e r a s e s i n Spinach C h l o r o p l a s t Envelope Membranes — A p p l i c a t i o n s of an Assay f o r U D P G a l : D i a c y l g l y c e r o l G a l a c t o s y l t r a n s f e r a s e . . . . 301

J.W.M. Heemskerk, M.A.M. S c h e i j e n , F.H.H. Jacobs and J.F.G.M. Wintermans

G a l a c t o s y l t r a n s f e r a s e A c t i v i t i e s i n I n t a c t Spinach C h l o r o p l a s t s and Envelope Membranes 305

J.W.M. Heemskerk, F.H.H. Jacobs, G. Bögemann and J.F.G.M. Wintermans

B i o s y n t h e s i s of S u l f o q u i n o v o s y l d i a c y l g l y c e r o l i n Ch l o r o p l a s t s of Higher P l a n t s 309

K.F. Kleppinger-Sparace and J.B. Mudd

xii

S u l f o l i p i d S ynthesis by I s o l a t e d I n t a c t Spinach C h l o r o p l a s t s . . . . 313 J . J o y a r d , E. Blee and R. Douce

Calcium C h l o r i d e E f f e c t on G l y c e r o l i p i d s Metabolism i n O l i v e Tree Leaf 317

B. Marzouk, M. Zarrouk, A. C h e r i f and P. Mazliak

L i p i d Metabolism i n Potato Leaf D i s c s : E f f e c t of Calmodulin A n t a g o n i s t s 321

G.J. P i a z z a and R.A. Moreau

L o c a l i z a t i o n and P r o p e r t i e s of Cholinephosphate C y t i d y l y l t r a n s f e r a s e A c t i v i t y i n Castor Bean Endosperm . . . . 325

A.J. Kinney and T.S. Moore, J r .

C h o l i n e Kinase A c t i v i t y i n Castor Bean Endosperm 329 A.J. Kinney and T.S. Moore, J r .

The C o n t r o l of CTP:Cholinephosphate C y t i d y l y l t r a n s f e r a s e i n Pea Sterns 333

M.J. P r i c e - J o n e s and J.L. Harwood

Cocoa B u t t e r B i o s y n t h e s i s . Cocoa Seed D i a c y l g l y c e r o l A c y l t r a n s f e r a s e : Studies on the Microsomal Bound Enzyme 337

L. McHenry and P.J. F r i t z

S ubstrate S p e c i f i c i t y of P l a n t L i p a s e s 341 Y.H. L i n , C. Yu, J . Olsen and A.H.C. Huang

L i p i d s from Rice Anthers 345 S. Toriyama, K. Hinata, I. N i s h i d a and N. Murata

L i p i d s of Soybean Inoculated with Microsymbionts 349 R.S. Pacovsky and G. Füller

P r o p e r t i e s and I n v i t r o Synthesis of P h o s p h o l i p i d T r a n s f e r P r o t e i n s 353

F. Tchang, F. Guerbette, D. Douady, M. Grosbois, C. V e r g n o l l e , A. J o l l i o t , J.P. Dubacq and J.C. Kader

G a l a c t o l i p i d Synthesis i n I s o l a t e d Pea C h l o r o p l a s t s 357 J.P. Dubacq, R.O. Mackender and P. Mazliak

T r i a c y l g l y c e r o l B i o s y n t h e s i s i n Developing Cotyledons of S a f f l o w e r (Carthamus t i n c t o r i u s ) 361

G. G r i f f i t h s , K. Stobart and S. Stymne

Long Chain T r i a c y l g l y c e r o l A c y l Hydrolase (Lipase) A c t i v i t y i n Wheat G r a i n 365

T. G a l l i a r d , M. Lond and D.M. Gallagher

G l y c o p r o t e i n Nature of L y p o l y t i c A c y l Hydrolases i n Potato Tubers and Leaves 369

R.A. Moreau and G. Nagahashi

C o r r e l a t i o n of Metabolie Rate Changes and Membrane T r a n s i t i o n s Determined by M i c r o c a l o r i m e t r i c Methods 373

R.W. Breidenbach, R.S. C r i d d l e , E. Lewis and L. Hanson

xiii

Molecular Species Composition of P h o s p h a t i d y l g l y c e r o l i n Leaves of C a m e l l i a Species and C h i l l i n g S e n s i t i v i t y

J . Sekiya, H. Koiso, A. M o r i t a and A. Hatanaka 377

OXYGEN REQUIRING SYSTEMS - OXYGENASES AND DESATURASES

The Lipoxygenase Pathway 383 B.A. V i c k and D.C. Zimmerman

Enzymic Oxygenative-cleavage Reaction of L i n o l e n i c A c i d i n Leaves - C h l o r o p l a s t i c Lipoxygenase and F a t t y Acid Hydroperoxide Lyase i n Tea Leaves 391

A. Hatanaka, T. Kajiwara and J . Sekiya

F a t t y A c i d 3-0xidation i n Higher P l a n t s 399 B. Gerhardt

D e s a t u r a t i o n of F a t t y A c i d s on Complex-lipid Substrates 405 S. Stymne, G. G r i f f i t h s and K. Stobart

Rapid Enzymic P e r o x i d a t i o n of Polyunsaturated F a t t y A c i d s on Hydration of Wheat M i l l i n g Products 413

T. G a l l i a r d , S.P.C. T a i t and D.M. Gallagher

A c t i o n of Boron on Ethylene Production and Lipoxygenase A c t i v i t y i n Microsomes From Sunflower Cotyledons 417

A. B e l v e r , P. Rodriguez, M. Roldän and J.P. Donaire

P r e l i m i n a r y C h a r a c t e r i z a t i o n of Lipoxygenase From the Entomopathogenic Fungus Lagenidium giganteum 421

C A . Simmons, J.L. Kerwin and R.K. Washino

C h a r a c t e r i z a t i o n of Tomato Lipoxygenase 425 P.A. B i a c s and H. Daood

M u l t i p l e Pathways of L i n o l e n i c A c i d Synthesis Operate and I n t e r a c t i n Leaf T i s s u e 429

H.A. Norman and J.B. St. John

The E f f e c t of Temperature on D e s a t u r a t i o n of G a l a c t o l i p i d F a t t y Acids i n B r a s s i c a napus 433

J.P. W i l l i a m s , K. M i t c h e l l and M. Khan

A c y l L i p i d Metabolism i n Rhodotorula g r a c i l i s (CBS 3043) and the E f f e c t s of Methyl S t e r c u l a t e on F a t t y A c i d D e s a t u r a t i o n 437

C E . Rolph, R.S. Moreton, I.S. Small and J.L. Harwood

Metabolism of E i c o s a p o l y e n o i c A c i d L i p i d s i n Race S p e c i f i c I n t e r a c t i o n s Between Phytophthora i n f e s t a n s and Potato . . . . 441

R.M. Bostock

MEDIUM AND LONG CHAIN BIOSYNTHESIS

Modulation of F a t t y A c i d Synthesis i n P l a n t s by T h i o l a c t o m y c i n . . . 447 M. Yamada, M. Kato, I. N i s h i d a , K. Kawano, A. Kawaguchi

and T. Ehara

xiv

F a t t y A c i d S y n t h e s i s i n Developing O i l s e e d s 455 M.R. P o l l a r d and S.S. Singh

Medium and Long-chain F a t t y A c i d Synthesis 465 J.L. Harwood

C u t i c u l a r L i p i d s i n Plant-microbe I n t e r a c t i o n s 473 P.E. Kolattukudy, W.F. E t t i n g e r and J . Sebastian

P l a n t Elongases 481 C. Cassagne, R. L e s s i r e , J . - J . Bessoule and P. Moreau

Genes, Elongases and A s s o c i a t e d Enzyme Systems i n E p i c u t i c u l a r Wax Synthesis 489

P. von Wettstein-Knowles

The P u r i f i c a t i o n of A c e t y l CoA:Acyl C a r r i e r P r o t e i n T r a n s a c y l a s e from B r a s s i c a campestris Leaves 499

A. -M.A. Wolf and J.T. Perchorowicz

R e g u l a t i o n of A c e t y l Coenzyme A Synthesis i n C h l o r o p l a s t s 505 K.-P. H e i se and H.-J. Treede

L i p i d P r e c u r s o r s i n P l a n t C e l l s : The Problem of A c e t y l CoA G e n e r a t i o n f o r P l a s t i d F a t t y A c i d Synthesis 509

B. L i e d v o g e l

P a r t i a l P u r i f i c a t i o n and C h a r a c t e r i z a t i o n of Acetyl-CoA Synthetase from Mature Spinach Leaves 513

C A . Z e i h e r and D.D. Randall

Acetyl-CoA Carboxylase and B i o t i n - c o n t a i n i n g P r o t e i n s i n C a r r o t Somatic Embryogenesis 517

B.J. N i k o l a u , J . Croxdale, T.H. U l r i c h and E.S. Wurtele

Pyruvate R e v e r s a l of S-Ethyl Dipropylcarbamothioate (EPTC) I n h i b i t i o n of Pyruvate Dehydrogenase Complex 521

R.E. W i l k i n s o n and T.H. Oswald

Acyl-CoA E l o n g a t i o n Systems i n A l l i u m porrum Microsomes 525 R. L e s s i r e , J . - J . Bessoule and C. Cassagne

L i p i d B i o s y n t h e s i s i n O i l Palm P r o t o p l a s t s 529 R. Sambanthamurthi, K.-C Oo and A.S.-H. Ong

I d e n t i f i c a t i o n of P r o t e i n s A s s o c i a t e d with Changes i n the L i n o l e n a t e Content of Soybean Cotyledons 533

X. Wang, D.F. Hildebrand and G.B. C o l l i n s

A c y l t r a n s f e r a s e s i n Developing Seeds of O i l s e e d Rape 537 D. J . Murphy

P r o p e r t i e s of A c y l - ( A c y l - C a r r i e r Protein):Glycerol-3-Phosphate A c y l t r a n s f e r a s e from Greening Squash Cotyledons 541

I. N i s h i d a , M. Frentzen and N. Murata

L i p i d B i o s y n t h e s i s i n Epidermal, Guard and Mesophyll C e l l P r o t o p l a s t s from Leaves of V i c i a faba L 545

N. Sato

XV

Ontogenetic V a r i a t i o n s i n the Chemical Composition of Maize Surface L i p i d s 549

P. Avato, G. B i a n c h i and F. Salamini

Mechanism of B i o s y n t h e s i s of 3-Diketones and Alkan-2-0L E s t e r s from E p i c u t i c u l a r Waxes 553

G. B i a n c h i

E p i c u t i c u l a r Wax Formation on Needles of P i c e a abies and Pinus cembra 557

M.S. Günthardt-Goerg

INTERACTION OF FEDERAL, INDUSTRIAL AND ACADEMIC RESEARCH

I n t e r - o r g a n i z a t i o n a l C o l l a b o r a t i o n : A Key to Success i n T e c h n o l o g i c a l Innovation 563

R.L. Sampson

The Palm O i l Research I n s t i t u t e of M a l a y s i a - A Unique Research Uni t 571

A. Ong

Biotechnology of L i p i d s i n Industry 587 R.D. Schmid

ALGAL LIPIDS

L i t t l e Known Facts of Pl a n t L i p i d Metabolism 599 A.A. Benson

Unique C h a r a c t e r i s t i c s of C y a n o b a c t e r i a l G l y c e r o l i p i d s 603 N. Murata

L i p i d s of Diatoms and of H a l o p h i l i c D u n a l i e l l a Species 613 M. Kates

Metabolism of G a l a c t o l i p i d s i n D u n a l i e l l a s a l i n a 623 S.H. Cho and G.A. Thompson, J r .

S t e r o l Synthesis and D i s t r i b u t i o n and A l g a l Phylogeny 631 G.W. Pat t e r s o n

L i p i d s of A c e t a b u l a r i a mediterranea. Composition, C e l l u l a r L o c a l i z a t i o n and B i o s y n t h e s i s 637

W. Eichenberger and A. Gerber

The E f f e c t of Environmental Conditions on F a t t y A c i d Composition of the Red Alga Porphyridium cruentum 641

Z. Cohen, A. Vonshak and A. Richmond

N i l e Red: A Fluorophore U s e f u l i n As s e s s i n g the R e l a t i v e L i p i d Content of S i n g l e C e l l s 645

K.E. Cooksey, S.A. Wil l i a m s and P.R. C a l l i s

L i p i d Accumulation i n S i l i c o n - D e f i c i e n t Diatoms 649 P.G. Roessler

xvi

Composition and P o s i t i o n a l D i s t r i b u t i o n of F a t t y Acids i n L i p i d s from the Diatom Phaeodactylum tricornutum 653

A. Kawaguchi, T. Arao and M. Yamada

L i p i d Metabolism i n Two Species of Red Marine Algae as M o d i f i e d by Environmental Factors 657

T.R. P e t t i t t and J.L. Harwood

L i p i d s of C h a t t o n e l l a antiqua (Raphidophyceae) 661 N. Sato, Y. Nemoto and M. Furuya

THE FUTURE - GENETICS/BIOTECHNOLOGY

The Future - Genetics/Biotechnology 667 P.K. Stumpf

Genetic M a n i p u l a t i o n of O i l s e e d Q u a l i t y 669 R.K. Downey

Biotechnology i n the Improvement of the O i l Palm 677 L.H. Jones

Mutants of A r a b i d o p s i s D e f i c i e n t i n F a t t y A c i d D e s a t u r a t i o n . . . 683 C R . S o m e r v i l l e , P. McCourt , L. Kunst and J . Browse

A c y l C a r r i e r P r o t e i n as a Probe of the Molecular B i o l o g y of P l a n t F a t t y A c i d Synthesis 689

J.B. Ohlrogge, P.D. Beremand, D.J. Hannapel, D.J. Guerra, D.E. Elmore and D.N. Kuhn

O i l Seed Rape A c y l C a r r i e r P r o t e i n (ACP): P r o t e i n and Gene S t r u c t u r e 697

A.R. Slabas, J . Harding, P. Roberts, A. H e i i g e r , C Sidebottom, C G . Smith, R. S a f f o r d , J . d e S i l v a , C. Lucas, J . Windust, CM. James and S.G. Hughes

Complete Amino A c i d Sequence of N o n - s p e c i f i c L i p i d T r a n s f e r P r o t e i n from Castor Bean Seeds 701

M. Yamada, S. Watanabe, K. Takishima and C Mamiya

A P o s s i b l e D i f f e r e n t i a l Role f o r Plant A c y l C a r r i e r P r o t e i n Isoforms i n Higher P l a n t s 705

D.J. Guerra, J.B. Ohlrogge and M. Frentzen

A P r e l i m i n a r y C h a r a c t e r i z a t i o n of Plant Holo-Acyl C a r r i e r P r o t e i n Synthase 709

S.A. E l h u s s e i n , J.A. Miernyk and J.B. Ohlrogge

P h y s i o l o g i c a l and T r a n s f o r m a t i o n a l Analyses of Lipoxygenases . . . 715 D.F. H i l d e b r a n d , M. A l t s c h u l e r , G. Bookjans, G. Benzion,

T.R. Hamilton-Kemp, R.A. Andersen, J . C Rodriguez, J . C Polacco, M.L. Dahmer, A . C Hunt, X. Wang and G.B. C o l l i n s

INDEX 719

xvii

SYNTHES IS OF PLASTOQUINONE-9, , i-TOCOPHEROL AND PHYLLOQUINONE (VITAMIN K } )

AND ITS INTEGRATION IN CHLOROPLAST CARBON METABOLISM OF HIGHER PLANTS

D. S c h u l z e - S i e b e r t , U. Homeyer, J . S o l l and G. S c h u l t z

Botanisches I n s t i t u t T terar ztL iche Hochschule Hannover D 3000 Hannover 71, F.R.G.

INTRODfJCT ION

Plastoquinone-9, <\-tocopheroL and phy1Loquinone are known as p l a s t i d i c prenylquinones f u l f i l l i n g important f u n c t i o n s : Plastoquinone-9 a c t s as mobile e l e c t r o n and proton c a r r i e r in p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t and i s i n -volved i n b u i l d i n g up the e l e c t r o c h e m i c a l proton p o t e n t i a l a t the c h l o r o p l a s t cytochrom b^/f complex /1,2/. a-Tocopherol i s involved i n i n a c t i v a t i n g e nergized oxygen s p e c i e s , formed in the l i g h t , by scavenging r a d i c a l s and quenching s i n g l e t t oxygen /3/. Phy1loquinone i s known as o b l i g a t o r y c o n s t i t u e n t of PS I ( K ^ / c h l o r o p h y l l of PS I r a t i o about 1:100 /4/) .

T h i s r e p o r t d e a l s with the s y n t h e s i s of '»-tocopherol and plastoquinone-9 from homogentisate and of phy1loquinone from 1,4-dihydroxy-2-naphthoate i n c h l o r o p l a s t s . Furthermore, experimental data are presented to support e a r l i e r f i n d i n g s on the autonomic r o l e of c h l o r o p l a s t carbon metabolism i n forming p l a s t i d i c i s o p r e n o i d s . - The methods a p p l i e d are d e s c r i b e d in /5,6,7,8,9/.

RE SU LT S AND DISCUSSION

S y n t h e s i s of tt-Tocopherol and Plastoquinone-9 from Homogentisate a t the C h l o r o p l a s t Envelope Membrane

Homogentisate represents the aromatic intermediate in the formation of ( i T and PQ /10/ ( F i g . 1). I t i s formed from Tyr v i a 4-hydroxyphenylpyruvate by

A b b r e v i a t i o n s : DAHP, deoxy-D-arabinoheptulosonate-7-phoshate; DHAP, dihydroxyacetone phosphate; E'ase, 2-phosphoglycerate hydrolyase, enolase; rev. NADP GAPDH, r e v e r s i b l e NADP glyceraldehyde 3-phosphate dehydrogenase; GG, g e r a n y l g e r a n i o l ; GGPP, geranylgeranyl-pyrophosphate; GK, g l y c e r a t e kinase; HPP, 4-hydroxyphenylpyruvate; IPP, is o p e n t e n y l pyrophosphate; KGA, 2-oxoglutarate; Me-6-PhQ(H2) and isomers, 2-methyl-6-phytylquinone ( q u i n o l ) and isomers; 2,3-Me2~PhQ(H ), 2,3-dimethyl-5-phytylquinone ( q u i n o l ) ; MITO, mitochondrion; PDC, pyruvate dehydrogenase complex; PEP, phosphoenolpyruvate; PER, peroxysome; 3-PGA and 2-PGA, 3- and 2-phospho-D-glycerate; PK, pyruvate kinase; PGM, phosphoglycerate mutase; PQ(H ), plastoquinone-9 ( q u i n o l - 9 ) ; PS I, PS I I , photosystem I and I I ; SAM, S-adenosylmethionine; SkA, shikimate; SORase, shikimate oxidor' ductase; a, ß, y and ̂ T, <*-, ß-, y- and fS-tocopherol

The author's work was supported by the Deutsche Forschungsgemeinschaft.

29

F i g . 1 . I n c o r p o r a t i o n of /3- C / t y r o s i n e i n t o p l a s t o q u i n o l and n-tocopherol i n ^ i g h e r P l a n t / * \ Q / c h l o r o p l a s t s 75,6,8/. A s t e r i s k s i n d i c a t e C l a b e l .

oxidases at the stromal face of c h l o r o p l a s t membranes /11/. A l l the f o l l o w i n g r e a c t i o n s of prenylquinone s y n t h e s i s occur s o l e l y at the inner c h l o r o p l a s t envelope membrane /8/ ( F i g . 4); c h l o r o p l a s t stroma or t h y l a k o i d s are i n a c t i v e /6,12/ ( F i g . 4 ) . In the p r e n y l t r a n s f e r a s e r e a c t i o n , the c a r b o x y l group of homogentisate i s e l i m i n a t e d and a methylquinol i s formed which i s preny1-ated s p e c i f i c a l l y at p o s i t i o n 6 /6/. The p r e n y l s i d e chain i s always in tranis c o n f i g u r a t i o n /13/. In t h i s and the f o l l o w i n g methylation r e a c t i o n only the q u i n o l and not the quinone stage of the aromatic Compound i s a c t i v e /5/. Phytyl-PP i s the only preny1-PP s u i t e d f o r *»T s y n t h e s i s i n c h l o r o p l a s t s and nonaprenyl- ( s o l a n e s y l - ) PP i n PQ s y n t h e s i s /6/. Consequently, the i n t r o -ductory step of prenylquinone s y n t h e s i s i s s t r i c t l y s p e c i f i c in respect to Substrate and p o s i t i o n of a t t a c k , and the f o l l o w i n g r e a c t i o n s are s t r o n g l y d i r e c t e d to a homogenous p a t t e r n of products.

As can be seen from the methylation step by SAM i n F i g . 2, 2-methyl-6-p h y t y l q u i n o l i s s t r o n g l y p r e f e r r e d to i t s isomers. Thus, the main product i s 2,3-dimethyl-5-phytylquinol which undergoes r i n g c l o s u r e (only v e r i f i e d in i n t a c t c h l o r o p l a s t s /5/) to form )'T which i s methylated by SAM to f o r m a T . The chromanol stage i s p r e r e q u i s i t e f o r the second methylation r e a c t i o n ; no t r i m e t h y l p h y t y l q u i n o l was formed from d i m e t h y l p h y t y l q u i n o l s /5,14/. The

tocopherol m e t h y l t r a n s f e r a s e of Capsicum annuum was p u r i f i e d to homogeneity by d'Harlingue and Camara 1985 /15/. M has been determined to 33 kDa, K f o r }"T to 13.7 uM and f o r SAM 2.5 uM. Thus the main sequence for.»T synthesis i n spinach i s : Homogentisate -—* 2-Methyl-6-phytylquinol —*• 2,3-Dimethyl-5-p h y t y l q u i n o l * )T — * <*T ( F i g . 3). Another sequence caused by changing methylation and c y c l i z a t i o n r e a c t i o n may occur a d d i t i o n a l l y but at lower ra t e s /16/: Homogentisate * 2-Methyl-6-phytylquinol —»• ,ST * )'T — » <*T.

F i g . 2 Substrate s p e c i f i c i t y of the f i r s t (upper s e r i e s ) and second methylation r e a c t i o n (lower s e r i e s ) of cv-tocopherol s y n t h e s i s i n sginach c h l o r o ­p l a s t ^ /5/. / C/-Methyl from /Me- C/SAM was i n c o r p o r a t e d f o r l a b e l l i n g the S u b s t r a t e s . The products were p u r i f i e d by co-chromatography adding refe r e n c e substances and i d e n t i -f i e d by r a ^ i o s c a n . A s t e r i s k s i n d i c a t e C - l a b e l ; the e n c i r -c l e d f i g u r e s i n d i c a t e the r a t i o o f products formed from d i f f e r e n t q u i n o l s (upper s e r i e s ) and toco-p h e r o l s (lower s e r i e s ) .

* H 3 ^

n. d.

' j m

H O rSn ,3.yo>o

J M e 6 P h Q H 2 j M e 5 P h Q H 2 C-Methylot ion

J «* * H 3

Cyc l i sa t ion

C-Methylat ion

©

30

Phytyl-PP i s formed from GGPP by hydrogenation a t the envelope membrane /!/ ( F i g . 3 ) . Another pathway i s the stepwise hydrogenation of GG-chloro-p h y l l i d e a to form C h l o r o p h y l l a at t h y l a k o i d membranes /17/ ( F i g . 3). NADPH f u n c t i o n s as e l e c t r o n donor i n both r e a c t i o n s . A kinase which forms phytyl-PP from p h y t o i p l u s ATP i s l o c a l i z e d i n the stroma /6/. GGPP i t s e l f i s formed from IPP by a recombinated System of envelope or t h y l a k o i d membranes pl u s stroma p r o t e i n /18/.

j u i i i i i i i i l i i i n Chlorophyll a

Stroma plus membranes envelope or thylakoids)

1111111111 Geranylgeranyl-PP •

Geronylgeronylchlorophyllid a

Chlorophyllid q | T h , a k o i d s | 111111111111111 n 11111 n' »

' P P ^ CALVIN C Y C L E

l i i l l l l l i i i l l l l i i l i i i i

Stroma Geranylgeranyl- P P SHIKIMATE PATHWAY_

W0^\ p-Hydroxyphenylpyruvate

:-\ 2

H O r ^ r r ? Homogentisate

' 2 ' " '

L J ,

HH HH HH ' f ^ ^ ^ ^ l 2-Me-6-phytylquinol

^Wty l-PP V ^ M ^ ; ^ ^ e - 6 - P h Q H 2 )

C H y C O O H

CH0-0-(ßs-Cß\ —

'Prenylation S A M fY^il Methylation

2J3-Me2"5-phytylquinol 12,3-Me2 PhQH 2)

' 9 Ä . r > \

^^Methy la t ion ' T ' O ^ R

Cyclisation

F i g . 3 Synthesis of a-to c o p h e r o l /5,6,12/ from homogentisate /10/ and phytyl-PP at the inner envelope membrane of c h l o r o p l a s t s / 8 / . Plastoquinone-9 (see t e x t and F i g . 4) i s sy n t h e s i z e d from homogentisate and nonaprenyl-(solanesyl-)PP a t the same membrane /6,8/. For s y n t h e s i s of homogentisate /1 1/ and phytyl-PP /!/ i n c h l o r o p l a s t s see t e x t .

The r e a c t i o n mechanism of PQ sy n t h e s i s equals t h a t of aT s y n t h e s i s . The sy n t h e s i s a l s o occurs e x c l u s i v e l y at the inner c h l o r o p l a s t envelope membrane /8/ ( F i g . 4), however, i t can be assumed that e i t h e r prenylquinone i s formed by i t s own enzyme g a r n i t u r e . 2-Methyl-6-nonaprenyl-(solanosyl-)quinol i s formed from homogentisate p l u s nonaprenyl-(solanosyl-)PP. The q u i n o l formed i s then methylated by SAM to y i e l d PQH 2 /6/ ( F i g . 4 ) . Even i f the sequence i n PQ s y n t h e s i s i s c l a r i f i e d : Homogentisate — * 2-Methyl-6—nonaprenylquinol — * PQH 9 no data are a v a i l a b l e f o r the s y n t h e s i s of hydroxylated quinones Q and Q a c t i n g as primary e l e c t r o n acceptors i n PS II /19/.

31

A B C

F i g . 4 Synthesis of pl a s t o q u i n o l - 9 i n spinach c h l o r o p l a s t s ( c a l c u l a t e d from /6,8/). (A) P r e n y l a t i o n r e a c t i o n : Homogentisate + Nonaprenyl-(solanesyl-)PP — * 2-Methyl-6-nonaprenylquinol; (B) Methylation r e a c t i o n : The q u i n o l of (A) + SAM —*> P l a s t o q u i n o l - 9 . (C) O v e r a l l r e a c t i o n : Homogentisate + Nonaprenyl-PP + SAM —*• P l a s t o q u i n o l - 9 . The r e a c t i o n s occur at the inner membrane (C) /8/ of the c h l o r o p l a s t envelope (A, B) /6/; stroma and t h y l a k o i d membranes are i n a c t i v e .

Synthesis of Phylloquinone (Vitamin ) i n Higher P l a n t s

Feeding experiments using t o t a l p l a n t s revealed t h a t phylloquinone i s formed i n leaves from shikimate /20/ and 2-succinylbenzoate /2 1/. J u s t r e c e n t l y L e i s t n e r ' s group provided evidence from s t u d i e s on E. c o l i /22/ t h a t isochorismate and not chorismate r e a c t s with 2-oxoglutarate to form 2-succinylbenzoate. The r e s u l t s from s t u d i e s on c e l l c u l t u r e s /23,24/ and c h l o r o p l a s t s /25,26/ are summarized i n F i g . 5. The c h l o r o p l a s t envelope i s the s i t e of p r e n y l a t i o n /25/ and the t h y l a k o i d membrane of methylation r e a c t i o n /26/, however, compartmentation of the other r e a c t i o n s remains s t i l l u n c l e a r . The s y n t h e s i s i n pl a n t s resembles the m i c r o b i a l one /27/ though phytyl-PP i s p r e f e r r e d as prenyl donor i n p l a n t s /25/.

1. i. -Oihydroxy - 2-Phy tyl-1. L -naphthoquinol

F i g . 5 Phylloquinone (K^) sy n t h e s i s i n Higher P l a n t s /23-26/. Probably isochorismate as i n E. c o l i /22/ and 2-oxoglutarate (aKGA) forms 2-succinylbenzoate.

32

Role of C h l o r o p l a s t Carbon Metabolism i n P l a s t i d i c Prenylquinone S y n t h e s i s

The carbon flow from 3-phosphoglycerate, phosphoenolpyruvate, pyruvate and acetyl-CoA. Even i f the s y n t h e s i s of aromatic amino a c i d s by shikimate pathway /28,29,30,3V and a l s o preny1-PP synthesis v i a mevalonate /32,33,34/ has been e s t a b l i s h e d i n c h l o r o p l a s t s by i d e n t i f i c a t i o n of r e s p e c t i v e p l a s t i d i c enzymes, i t i s s t i l l a matter of d i s c u s s i o n from where PEP o r i g i n s t o supply DAHP s y n t h e s i s of the shikimate pathway and from where pyruvate i s d e l i v e r e d t o supply the p l a s t i d i c pyruvate dehydrogenase complex ( f o r i s o l a -t i o n see Treede and Heise, t h i s Conference). Because phosphoglycerate mutase (PGM) t o form 2-PGA from 3-PGA co u l d not be detected i n c h l o r o p l a s t s /35/ and acetyl-CoA i s p r e f e r a b l y s y n t h e s i z e d from added acetate by the actetyl-CoA synthetase /36/, p a r t i c u l a r l y i n spinach c h l o r o p l a s t s , i t was argued that c h l o r o p l a s t s are dependent on import of these Substrates from the e x t e r n a l S i t e . Evidence f o r PEP formation from 3-PGA with i n the c h l o r o p l a s t could be obt a i n e d by three d i f f e r e n t approaches (D. Sch u l z e - S i e b e r t , A. Heintze and G. S c h u l t z , i n pr e p a r a t i o n ; D. Sc h u l z e - S i e b e r t and G. S c h u l t z , i n p r e p a r a t i o n , f o r p l a s t i d i c isoenzyme of PGM i n R i c i n u s see / Z I / and i n B r a s s i c a /38/).

( i ) The only enzyme of carbon metabolism h i t h e r t o questioned to be present i n spinach c h l o r o p l a s t s , PGM, could be i d e n t i f i e d by the latency method. The enzyme thus i d e n t i f i e d e x h i b i t s an i d e n t i c a l behaviour i n comparison t o r e v e r s i b l e NADP D-glyceraldehyde 3-phosphate dehydrogenase and shikimate oxidoreductase as p l a s t i d i c marker enzymes ( F i g . 6 ) .

F i g . 6 Phosphoglycerate mutase (PGM) i n spinach c h l o r o p l a s t s i d e n t i f i e d by the l a t e n c y method. P u r i f i e d , i n t a c t c h l o r o p l a s t s were subjected to step-wise o s m o t i c a l shock by lowering the s o r b i t o l conc. of the medium. The enzyme a c t i v i t y r e l e a s e d from c h l o r o ­p l a s t was determined i n the supernatant. In the latency s t u d i e s , PGM e x h i b i t s an i d e n t i c a l behaviour compared to r e v e r s i b l e NADP-GAPDH and shikimate oxidoreductase (SORase) as marker enzymes f o r c h l o r o p l a s t s .

A A

Sofbitol,mM

( i i ) The carbon flow from 3-PGA to PEP and pyruvate was demonstrated w i t h i n c h l o r o p l a s t s by adding l a b e l l e d g l y c e r a t e which i s known to be phosphorylated by the g l y c e r a t e kinase l o c a l i z e d s o l e l y i n the c h l o r o p l a s t stroma /39/. A f t e r spunning down the c h l o r o p l a s t s ( F i g . 7), 3-PGA, 2-PGA, PEP and pyruvate were found at c o n s i d e r a b l e amounts only i n the c h l o r o p l a s t p e l l e t and thus support above f i n d i n g s on p l a s t i d i c PGM. Only 3-PGA was nea r l y e q u a l l y d i s t r i b u t e d between c h l o r o p l a s t and Suspension medium which can be a t t r i b u t e d to a c t i o n of the phosphate t r a n s l o c a t o r . To reduce the a c t i v i t y of the t r a n s l o c a t o r /40/ on l y 0.5 mM P^ was a p p l i e d to the medium.

( i i i ) The inc r e a s e of the s y n t h e s i s of amino a c i d s and prenylquinones i n c h l o r o p l a s t s by Omission of P. and deminishing the exchange of t r i o s e -phosphates cou l d be demonstrated e a r l i e r /41/.

33

F i g . 7 Experiment p r o v i n g the carbon flow from 3-PGA to pyruvate v i a 2-PGA and PEP i n spinach c h l o r o p l a s t s . P u r i f i e d , i n t a c t c h l o r o p l a s t s were incubated with /1- C / g l y c e r a t e which i s phosphorylated by the g l y c e r a t e kinase l o c a l i z e d s o l e l y i n the stroma /39/. 3-PGA thus formed served as Substrate f o r C -metabolism w i t h i n the

3 c h l o r o p l a s t . A l i q u o t s taken a t i n d i c a t e d time were c e n t r i f u g e d f o r 1 min and 3-PGA, 2-PGA, PEP and pyruvate determined by modified enzymatic a n a l y s i s (D. S c h u l z e - S i e b e r t , A. Heintze and G. S c h u l t z , i n p r e p a r a t i o n ) .

• 5 - C h l o r o p l a s t

0 5

M e d i u m

10 20 30 4 0

3 mm Light f Oork

. 1 - , 4 C D,L- Glycerate

An e x t e r n a l S i t e of s y n t h e s i s of PEP from p h o t o s y n t h e t i c a l l y formed 3-PGA can be detected by r i s i n g the P. c o n c e n t r a t i o n up to 5 mM and adding exogenously excessive amounts of r a b b i t PGM and enolase to i n t a c t c h l o r o p l a s t s (10 and 2.5 units/50 ug C h l o r o p h y l l ) s i m i l a r as i n /42/. The a c t i v i t y of the shikimate pathway (measured as nmol Phe and Tyr formed) was c o n s i d e r a b l y enhanced as a r e s u l t of supply of high amounts of PEP by t h i s enzyme r e a c t i o n and re-import by the phosphate t r a n s l o c a t o r . Consequently, t h i s way might be considered as an a d d i t i o n a l S i t e of supply optimized under i n v i t r o c o n d i t i o n s by adding enzymes i n excess. Under i n v i v o c o n d i t i o n s , the carbon flow from triosephosphates of pho t o s y n t h e t i c carbon f i x a t i o n i s preponderantly d i r e c t e d t o sucrose s y n t h e s i s /43/ and l e s s to PEP.

The o r i g i n of acetyl-CoA f o r i s o p r e n o i d s y n t h e s i s . As shown i n Table 1, high e s t amounts of PQ and ß-carotene were formed from added bicarbonate by spinac h p r o t o p l a s t s i n the l i g h t and only lower ones from added a c e t a t e . On the other hand, S a t u r a t i o n of f a t t y a c i d formation i s only achieved by adding a c e t a t e . Therefore, the hypothesis was r a i s e d t h a t a more or l e s s c h a n n e l l i n g o f pyruvate dehydrogenase complex and i s o p r g n o i d s y n t h e s i s i n c h l o r o p l a s t s may e x i s t . To prove t h i s the d i l u t i o n of / C/bicarbonate by i n c r e a s i n g amounts of added acetate^was s t u d i e d . I n s p i t e of i n c r e a s i n g amounts of added ac e t a t e no d i l u t i o n of / c/ a c t i v i t y i n ß-carotene and PQ coul d be observed. Only f a t t y a c i d s formed i n s i d e and s t e r o l s formed o u t s i d e the c h l o r o p l a s t s were d i l u t e d as expected ( F i g . 8 ) . A l s o a d d i t i o n a l v i c e versa experiments (data not shown) po i n t e d a t the same d i r e c t i o n .

T a b l e 1 F o r m a t i o n ^ f plastoquinone-9, ^g-carotene, f a t t y a c i d s (F.A.) and s t e r o l s from / c/bicarbonate, /2- C/acetate and / c/-mevalonate usin g spinach p r o t o p l a s t s a t pH 7.6. Note the preference of bicarbonate i n the i s o p r e n o i d and of acetate i n the f a t t y a c i d s y n t h e s i s . Plastoquinone-9 and ß-carotene are not formed i f mevalonate i s a p p l i e d from the e x t e r n a l s i t e .

14 nmol Acetate u n i t s formed per mg C h l o r o p h y l l x h

L i p i d s S t e r o l s F.A. Carotene PQ N a H 1 4 C 0

3 0 # 5 m 5 ' 1 ± °- 3 ° - 6 7 1 - 5 8 1 .18 1 .12

/ 2 - ^ C / A c e t a t e 0.1 mM 33 .3 + 6.0 2 .00 28.64 0.33 0 .67 /2- C/Mevalonate 3.5+0.6 1.80 0 0.03 0.03

34

F i g . 8 Demonstration of the metabolic c h a n n e l l i n g of carbon metabolism and i s o p r e n o i d s y n t h e s i s i n c h l o r o p l a s t s by^a competition experiment adding / c/bicarbonate and i n c r e a s i n g amounts of acetate to spinach proto­p l a s t s . A competition e f f e c t from added acetate c o u l d only be observed i n f a t t y a c i d and s t e r o l s y n t h e s i s . Plastoquinone-9 and ß-carotene s y n t h e s i s remained u n a f f e c t e d .

100

90

80

70

60

50

40

30

•

\

3 5

m M

Fatty acids-'

10

N a - a c e t a t e

The scheme i n F i g . 9 summarizes the above r e s u l t s .

SPINACH CHLOROPLAST

F i g . 9 Proposed scheme of carbon flow from primary photo­s y n t h e t i c products to i s o p r e n o i d Compounds, f a t t y a c i d s , amino a c i d s and other Compounds i n spinach c h l o r o p l a s t s . Based on rec e n t f i n d i n g s the scheme demonstrates the f o l l o w i n g p o i n t s : ( i ) A carbon flow from 3-PGA to 2-PGA, PEP, and pyruvate w i t h i n the c h l o r o p l a s t (and to some extent o u t s i d e the c h l o r o p l a s t ) e x i s t s to p rovide Substrates f o r the s y n t h e s i s of amino a c i d s , i s o p r e n o i d s and prenylquinones. ( i i ) P l a s t i d i c pyruvate dehydro­genase complex and p l a s t i d i c i s o p r e n o i d s y n t h e s i s are more or l e s s Channelled Systems. ( i i i ) The supply with acetate f o r f a t t y a c i d s y n t h e s i s predominantly occurs from the e x t e r n a l s i t e .

35

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