PHOTOSYNTHESIS Volume V Chloroplast Development

Proceedings of the Fifth International Congress on Photosynthesis September 7-13, 1980, Halkidiki, Greece

Editor

GEORGE AKOYUNOGLOU

1981 Balaban International Science Services Philadelphia

CONTENTS v i i

VOLUME V- - CHLOROPLAST DEVELOPMENT

P ROTOCHLOROPHYLLIDE PHOTOREDUCTION 1

The p h o t o C h l o r o p h y l l i d e - c h l o r o p h y l l i d e c y c l e as a source of p h o t o s y n t h e t i c a l l y a c t i v e C h l o r o p h y l l s C. S i r o n v a l 3

D i f f e r e n c e a b s o r p t i o n changes i n the v i s i b l e region during and a f t e r photoreduction of p r o t o c h l o r o p h y l l i d e B. Bereza and E. Dujardin 1 5

Fluorescence quenching of p r o t o c h l o r o p h y l l i d e , c h l o r o p h y l l i d e and C h l o r o p h y l l i n e t i o l a t e d , greening and green leaves E. D u j a r d i n 7 M. C o r r e i a and C. S i r o n v a l 21

Long-wavelength-absorbing intermediate pigments of the proto-c h l o r o p h y l l i d e - p r o t e i n System as a c t i v e centres E. Dujardin 31

P r o t o c h l o r o p h y l l ( i d e ) and C h l o r o p h y l l ( i d e ) fluorescence l i f e -time and other p r o p e r t i e s i n e t i o l a t e d and greening leaves J.C. Goedheer and J.C.J.M. van der Cammen 39

Quenching of the c h l o r o p h y l l i d e fluorescence by a s h o r t - l i v e d intermediate i n the photoreduction of p r o t o c h l o r o p h y l l i d e F. Franck 45

Laser induced photoconversion of p r o t o c h l o r o p h y l l i d e to Chloro­p h y l l a Y. Inoue, T. Kobayashi, T. Ogawa and K. Shibata 55

The r o l e of NADPH i n C h l o r o p h y l l s y n t h e s i s by p r o t o c h l o r o p h y l ­l i d e reductase W.T. G r i f f i t h s 65

I d e n t i f i c a t i o n of the Polypeptides of NADPH-protochlorophyllide oxidoreductase R.P. O l i v e r and W.T. G r i f f i t h s 7 3

The i s o l a t i o n and c h a r a c t e r i z a t i o n of the NADPH-protochlorophyl­l i d e oxidoreductase of b a r l e y (Hordeum vulgare L.) H.J. Santel/ T.E. Redlinger, C. Spr i n g w e i l e r and K. Apel 83

A new method f o r the p u r i f i c a t i o n o f p r o t o c h l o r o p h y l l i d e P. Bombart, E. Dujardin and M. Brouers 87

VÜi PIGMENT AND L I P I D BIOSYNTHESIS 93

A l t e r n a t i v e routes f o r the synthesis of 5-aminolevulinic a c i d i n greening leaves - Double l a b e l l i n g w i t h 15n- a n d 14c-gluta-mate E. H a r e l , P.J. Lea, E. M e i l e r and E. Ne'eman 95

A-Aminolevulinate synthesis i n greening b a r l e y . 1. Regulation S.P. Gough, C. G i r n t h and C. G. Kannangara 107

A-Aminolevulinate s y n t h e s i s i n greening b a r l e y . 2. P u r i f i c a t i o n of enzymes C G . Kannangara, S.P. Gough and C. G i r n t h 117

A-Aminolevulinate s y n t h e s i s i n greening b a r l e y . 3. C h a r a c t e r i -z a t i o n of r e g u l a t o r y mutants C. G i r n t h , S.P. Gough and C. G. Kannangara 129

The i n f l u e n c e of l i g h t and l e v u l i n i c a c i d on the r e g u l a t i o n of enzymes f o r ALA-biosynthesis i n two pigment mutants of Scenedesmus obliquus A. Kah, D. Dörnemann, D. Rühl and H. Senger 137

The e f f e c t s of l e v u l i n i c a c i d and 4,6-dioxoheptanoic a c i d on 5-aminolevulinic a c i d metabolism i n e t i o l a t e d b a r l e y leaves E. M e i l e r and M.L. Gassman 145

C h l o r o p h y l l formation and ALA b i o s y n t h e s i s v i a two d i f f e r e n t pathways during the c e l l c y c l e of w i l d type c e l l s of Scenedesmus K. Humbeck and H. Senger 161

C h l o r o p h y l l b i o s y n t h e s i s : Mg i n s e r t i o n ; O2 and Fe requirements P.A. C a s t e l f r a n c o 171

C h l o r o p h y l l precursors and p l a s t i d u l t r a s t r u c t u r e i n dark-grown wheat leaves t r e a t e d w i t h 8-hydroxyquinoline and 6 - a m i n o l e v u l i n i c a c i d M. Ryberg and H. Ryberg 177

B i o s y n t h e s i s of c h l o r o p h y l l i d e from A L A - p r o t o c h l o r o p h y l l i d e in v i t r o ; r o l e of NADPH and NADP M. Brouers and M.R. Wolwertz 185

B i o s y n t h e s i s and accumulation of novel C h l o r o p h y l l a. and b. chromo-p h o r i c species i n green p l a n t s C A . Rebeiz, F.C. Belanger, S.A. McCarthy, G. F r e v s s i n e t ,

J.X. Duggan, S-M. Wu and J.R. Mattheis 197

C h l o r o p h y l l s y n t h e s i s i n the dark i n angiosperms H. Adamson and R.G. H i l l e r 213

E x t r a c t i o n and c h a r a c t e r i z a t i o n of a chemically new C h l o r o p h y l l , absorbing at longer wavelengths and a t t r i b u t e d to P-700 D. Dörnemann and H. Senger 223

C h l o r o p h y l l b accumulation i n normal and SAN-9789 grown wheat leaves - L i g h t independency? C. M e l i n , L. Axelsson, H. Ryberg and H. V i r g i n 233

A f f i n i t y Chromatographie p u r i f i c a t i o n of an enzyme of C h l o r o p h y l l s y n t h e s i s W.R. Richards, J.C.-S. Chan and S.B. H i n c h i g e r i

P h o t o a c t i v i t y of the P695-682 c h l o r o p h y l l i d e form M. Jouy

Studies on c a r o t e n o i d b i o s y n t h e s i s i n r e l a t i o n to development of the p h o t o s y n t h e t i c apparatus: use of a d e u t e r i u m - l a b e l l i n g method G. B r i t t o n

L i p i d and c a r o t e n o i d metabolism during p l a s t i d development i n C a p s i c u m annuum f r u i t B. Camara and J . Brangeon

The f u n e t i o n of carotenoids during c h l o r o p l a s t development. I . E f f e c t s o f the h e r b i c i d e SAN-9789 on C h l o r o p h y l l s y n t h e s i s and p l a s t i d u l t r a s t r u c t u r e B. Klockare, L. Axelsson, H. Ryberg, A.S. Sandelius and K-0. W i d e l l

The f u n e t i o n of carotenoids during c h l o r o p l a s t development. I I . P h o t o s t a b i l i t y and Organization of e a r l y forms of C h l o r o p h y l l (ide) L. Axelsson, B. Klockare, H. Ryberg and A.S. Sandelius

The f u n e t i o n o f carotenoids during c h l o r o p l a s t development. I I I . P r o t e c t i o n of the p r o l a m e l l a r body and the enzymes f o r C h l o r o p h y l l s y n t h e s i s from photodestruetion, s e n s i t i z e d by e a r l y forms of C h l o r o p h y l l H. Ryberg, L. Axelsson, B. Klockare and A.S. Sandelius

The f u n e t i o n o f carotenoids during c h l o r o p l a s t development. IV. P r o t e c t i o n of g a l a c t o l i p i d s from photodecomposition, s e n s i t i z e d by e a r l y forms of C h l o r o p h y l l A.S. Sandelius, L. Axelsson, B. Klockare, H. Ryberg and K-0. W i d e l l

The p l a s t i d i c shikimate pathway and i t s r o l e i n the sy n t h e s i s of plastoquinone-9,a-tocopherol and phylloquinone i n spinach c h l o r o p l a s t s G. S c h u l t z , H. B i c k e l , B. Buchholz and J . S o l l A r a p i d Separation method of l e a f pigments by t h i n l a y e r chromatography on s i l i c a g e l GjlP* Evangelatos and G.A. Akoyunoglou

DEVELOPMENT OF THE PHOTOSYNTHETIC ACTIVITY-BIOGENESIS OF THE THYLAKOID STRUCTURE

Thylakoid membrane formation i n the higher p l a n t c h l o r o p l a s t N.K. Boardman

Ch l o r o p l a s t assembly i n development. Chairman's opening address to Symposium X I I I I . S S t l i k

Assembly of f u n c t i o n a l components i n c h l o r o p l a s t p h o t o s y n t h e t i c membranes G.A. Akoyunoglou

Development of primary photosynthetic processes i n leaves grown under a d i u r n a l l i g h t regime N.R. Baker and.V. Miranda

C o n t r i b u t i o n of manganese to the mechanism of p h o t o a c t i v a t i o n of the oxygen-evolving System i n dark-grown spruce s e e d l i n g s Y. Lemoine

The development of the photosynthetic apparatus of Gnetum : angiosperm or gymnosperm type? C. Jeske and H. Senger

The development of the photosynthetic apparatus during l e a f opening i n s i l v e r b i r c h (Betula p e n d u l a R o t h ) N. Valanne, T. Valanne, H. Niemi and E.-M. Aro

S p e c t r a l d i s t r i b u t i o n of fluorescence a t 77°K of h e a t - t r e a t e d and developing water-stressed c h l o r o p l a s t s R. Bhardwaj and G.S. Singhai

The c h a r a c t e r i z a t i o n of the developing photosynthetic apparatus i n greening b a r l e y leaves by means of (slow) fluorescence k i n e t i c measurements C. Buschmann

E v o l u t i o n of photosynthetic c a r b o x y l a t i o n pathways during wheat ontogenesis F. Ferron and A. Sauvesty

Development of the photosynthetic apparatus i n e u c a r y o t i c algae H. Senqer

Biochemical aspects of regreening of streptomycin-bleached Chlamydomonas reinhardii sr 3 A. B o s c h e t t i

I s o l a t i o n of c h l o r o p l a s t s from Chlamydomonas r e i n h a r d i i CW 15 mutant L. Mendiola-Morgenthaler and A. B o s c h e t t i

Development of t h y l a k o i d s and photosynthetic a c t i v i t y i n thermo-s e n s i t i v e and light-dependent mutants of Chlorella p y r e n o i d o s a G. G a l l i n g

C h l o r o p h y l l a f l u o r e s c e n c e and O2 e v o l u t i o n of synchronized Chlorella fusca D. Mende, A. Heinze and W. Wiessner

The e f f e c t of t r a n s l a t i o n and t r a n s c r i p t i o n I n h i b i t o r s on the development of the photosynthetic apparatus i n c e l l c y c l e s o f Scenedesmus quadricauda I . S g t l i k , E. S e t l i k o v a , J . Masojidek, V. Zachleder, T. K a i i n a , and P. Mader

Cytochromes i n orange, c h l o r o p h y l l - d e f i c i e n t c e l l s and during the r e g r e e n i n g of the green alga Chlorella fusoa L.H. Grimme and E. Lorenz

PSI and PSII u n i t growth i n stroma and grana t h y l a k o i d s during c h l o r o p l a s t development i n Fhaseoulus v u l g a r i s : a c r i t i c a l reassessmeht o f the PSII u n i t s i z e i n stacked and unstacked reg i o n s A. C a s t o r i n i s and J.H. Ärgyroudi-Akoyounoglou

Formation and growth of photosystem I and I I u n i t s i n developing t h y l a k o i d s of Fhaseoulus vulgaris S. T s a k i r i s and G. Akoyunoglou

xi

491

Independent growth of the photosystem I and I I u n i t s . The r o l e of the l i g h t - h a r v e t i n g pigment-protein complexes G. Akoyunoglou, S. T s a k i r i s and J.H. Argyroudi-Akoyunoglou 5 2 3

A c r i t i c a l reassessment of the photosystem I I content i n bündle sheath c h l o r o p l a s t s of young leaves of Zea mays J.H. Golbeck, I.F. M a r t i n , B.R. Velthuys and R. Radmer

L i n c o c i n e f f e c t on the development of photosystem I I E. S a r v a r i , P. Simon and F. Lang

S t r u c t u r a l O r g a n i z a t i o n and development of the l i g h t - h a r v e s t i n g pigment p r o t e i n s f o r photosystem I and I I J.E. M u l l e t , K. Leto and C.J. Arntzen

Synthesis of c h l o r o p h y l l - p r o t e i n complexes i n i s o l a t e d c h l o r o ­p l a s t s of Hkglena W. O r t i z and E. S t u t z

533

547

557

The formation of the pigment-protein complexes i n t h y l a k o i d s of Ihaseolus vulgaris during c h l o r o p l a s t development K. Kaiosakas, J.H. Argyroudi-Akoyunoglou and G. Akoyunoglou 569

The appearance of a new s o l u b l e Polypeptide and the prevention of t h y l a k o i d assembly during i n h i b i t i o n of C h l o r o p h y l l synthesis i n maize leaves* Y. Konis, E. Harel and S. K l e i n 581

591

Calcium i n h i b i t i o n of amino a c i d i n c o r p o r a t i o n by pea c h l o r o p l a s t s and the question of l o s s of a c t i v i t y w i t h age P-Y. Bouthyette and A.T. Jagendorf 599

Monoclonal a n t i b o d i e s : a novel approach to enzyme and p l a s t i d ontogenesis and phylogenesis. R e s u l t s and prospects W. Liedgens, Hj.A.W. Schneider and R. Grützmann 611

New concepts f o r the formation of photosynthetic membranes i n e t i o p l a s t s C. Lütz 619

xi i Changes i n the photosynthetic apparatus i n ageing Scenedesmus c e l l s H. Daniel1 and G. Kulandaivelu 631

F u n c t i o n a l and s t r u c t u r a l changes of i s o l a t e d c h l o r o p l a s t s during'ageing N. Laasen and W. Urbach

DEVELOPMENT AND DIFFERENTIATION OF THE PHOTOSYNTHETIC PROCARYOTES 653

Or g a n i z a t i o n , l o c a l i z a t i o n and assembly of the pigment-protein complexes i n membranes of the phototrophic bacterium Rhodopseu-domonas capsulata

G. Drews, A.F. G a r c i a , R. D i e r s t e i n and J . Shiozawa 655

Development of the b a c t e r i a l photosynthetic apparatus R.A. Niederman, C.N. Hunter, G.S. Inamine and D.E. Mallon 663 The d i f f e r e n t i a t i o n of the photosynthetic apparatus i n pro-caryotes J . P e l z e , K. Arnheim, I . K a i s e r and E. Post 675

Genetic engineering i n a photosynthetic bacterium B. Marrs, S. Cohen and D. Taylor 687

C o n t r o l of R h o d o p s e u d o m o n a s sphaeroides Y 5-aminolaevulinic acid-synthetase by reduced t h i o r e d o x i n J.D. Clement-Metrai 695

An increased carotenoid s h i f t on transference of R h o d o p s e u d o m o n a s c a p s u l a t a from darkness to l i g h t e i t h e r a e r o b i c a l l y or anaerobic-a l l y E.H. Evans, J . Manwaring and G. B r i t t o n 7 0 1

Photosynthesis and r e s p i r a t i o n i n blue-green algae: e l e c t r o n -t r a n s p o r t i n g funetions of t h y l a k o i d s and plasmamembrane, and occurrence of cytochrome a.a3, i n A n a c y s t i s nidulans G.A. Peschek, G. Schmetterer, W. Lockau and U.B. S l e y t r 707

Re v e r s i b l e degradation of photosynthetic apparatus and t h y l a k o i d s i n the blue-green a l g a Anacystis nidulans G. Schmetterer' and G.A. Peschek 721

The photosynthetic apparatus of v e g e t a t i v e and heteroeystous c e l l s o f the filamentous eyanobacterium N o s t o c muscorium G.C. Papageorgiou and J . Isaakidou 727

Composition and f u n e t i o n of the photosynthetic apparatus of heteroeysts i s o l a t e d from the blue-green a l g a N o s t o c m u s c o r u m H. Almon and H»-Böhme 737

GENETIC CONTROL OF CHLOROPLAST DEVELOPMENT

C h l o r o p l a s t development: a p e r s p e c t i v e J.W. Bradbeer

R e g u l a t i o n o f r e p l i c a t i o n and t r a n s c r i p t i o n i n the c h l o r o p l a s t R. Hagemann

M e t a b o l i t e r e g u l a t i o n of c h l o r o p l a s t genome expression and of the a c t i v i t y of the photosynthetic apparatus V.E. Semenenko

Comparative s t u d i e s on c h l o r o p l a s t t r a n s f e r RNAs: tRNA sequences and tRNA gene l o c a l i z a t i o n i n the rDNA u n i t s J.H. W e i l , P. Guillemaut, G. Burkard, J . Canaday, M. Mubumbila, M.L. Osorio, M. K e l l e r , R. G l o e c k l e r , A. Steinmetz, G. K e i t h , D. H e i s e r and E.J. Crouse

The e f f e c t o f a high non-permissive temperature on c h l o r o p l a s t development i n barley D. A l l s o p , Y.E. Atkinson and J.W. Bradbeer

C h l o r o p l a s t development i n normal and mutant l i n e s of b a r l e y Y.E. A t k i n s o n , D. A l l s o p and J.W. Bradbeer

P h o t o r e g u l a t i o n of the synthesis of r i b u l o s e bisphosphate carbo-x y l a s e and l o c a t i o n of the gene f o r i t s l a r g e subunit on a spe­c i f i c Eiglena c h l o r o p l a s t DNA r e s t r i c t i o n fragment G. F r e y s s i n e t , T. Gallagher, R. E i c h h o l z , E.A. Wurtz, M. F r e y s s i n e t and D.E. Buetow

Nuclear gene mutation e f f e c t s on the p l a s t i d s t r u c t u r e and RUBP-Case p r o p e r t i e s i n N i c o t i a n a tabacum (CV XANTHI) A. Nato, J . Brangeon and H. D u l i e u

Evidence f o r p o s t - t r a n s l a t i o n a l processing i n the c h l o r o p l a s t by p r o t e i n s synthesized i n the cytoplasm E. M. Reardon and C A . P r i c e

In v i t r o t r a n s l a t i o n o f exogenous messengers w i t h i s o l a t e d c h l o r o ­p l a s t s of spinach A. Gnanam, T. Mariappan and J.D. Manjula Devi

Biochemical and u l t r a s t r u c t u r a l a n a l y s i s of the e f f e c t of nuclear genome d u p l i c a t i o n on c h l o r o p l a s t development and b i o s y n t h e t i c a c t i v i t y i n e uploid Ricinus c e l l s .. M.P. Timko, R.E. Triemer and A.C. Vasconcelos

Evidence f o r a t r a n s l a t i o n a l c o n t r o l mechanism i n the s y n t h e s i s of the major t h y l a k o i d Polypeptides i n Chlamydomonas r e i n h a r d t i i J.K. Hoober

Xiv CONTROL OF CHLOROPLAST DEVELOPMENT (PHOTO, ENVIRONMENTAL, ETC.) 8 6 7

C o n t r o l of c h l o r o p l a s t development by l i g h t - Some recent aspects H. Mohr 869

The e f f e c t of blue and red l i g h t on the development of the p h o t o s y n t h e t i c u n i t s during greening of e t i o l a t e d bean leaves H. Anni and G. Akoyunoglou 889

Development of the s t r u c t u r e of dimorphic c h l o r o p l a s t s of Zea mays i n the presence of blue and red l i g h t D. M i l i v o j e v i c 395

E f f e c t s of blue l i g h t on r e s p i r a t i o n and enzyme a c t i v i t y i n a y e l l o w C h l o r e l l a mutant G. Ruyters 905

The e f f e c t of continuous i r r a d i a t i o n on C h l o r o p h y l l accumulation i n s e e d l i n g s of Pinus sylvestris H. . Kasemir and H. Mohr 915

Two steps i n i n i t i a l phytochrome a c t i o n on C h l o r o p h y l l s y n t h e s i s H. Oelze-Karow and H. Mohr 923

Synergism between red l i g h t and yellow-green l i g h t w i t h respect to C h l o r o p h y l l synthesis i n Rtglena L.S. Kaufman and H. Lyman 933

D i f f e r e n c e s i n u l t r a s t r u c t u r e and composition of c h l o r o p l a s t s from r a d i s h seedlings grown i n strong l i g h t , weak l i g h t and under the i n f l u e n c e of bentazon D. Meier and H.K. L i c h t e n t h a l e r 939

L i g h t r e g u l a t i o n of the s y n t h e s i s of two major nuclear-coded c h l o r o p l a s t Polypeptides i n Lenma gibba E. M. Tobin , . ' 949

I n t e r a c t i o n between p l a s t i d s , mitochondria and cytoplasm d u r i n g c h l o r o p l a s t development A.R. Wellburn and R. Hampp 961

Adenylate pools, energy C h a r g e , and phosphorylation p o t e n t i a l of p l a s t i d s , mitochondria and cytoplasm during development of photo­s y n t h e t i c c a p a c i t y R. Hampp and M. R i e h l 9 6 9

C o n t r i b u t i o n of photosynthesis to the growth and d i f f e r e n t i a t i o n o f c u l t u r e d tobacco c e l l s . The r e g u l a t o r y r o l e of i n o r g a n i c phosphate M. Miginiac-Maslow, Y. Mathieu, A. Nato and A. Hoarau 977

Comparison of 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 s y n t h e t i c Performance i n A c e t a b u l a r i a t r e a t e d w i t h auxin and w i t h a n t i - a u x i n T. Vanden Driessche, M. Geuskens, M. Glory and E. Cerf 985

The content of p h y t o l i n the green alga Chlorella f u s c a and the e f f e c t of the pyridazinone h e r b i c i d e SAN H 6706 on p h y t o l b i o s y n t h e s i s M.M. Tantawy and L.H. Grimme

C h l o r o p h y l l b accumulation and grana formation i n normal and SAN-9789 grown wheat see d l i n g s i n low i n t e n s i t i e s of red l i g h t H. V i r g i n , L. Axelsson, H. Ryberg and K-0. W i d e l l

E f f e c t of cadmium on l i g h t r e a c t i o n s of i s o l a t e d p l a s t i d s from greening b a r l e y seedlings F. Roynet, R. Lannoye and J . Barber

INDICES TO VOLUMES I - VI

Author index I - 1 P l a n t index 1-11 Subject index 1-15

Photosynthesis V. Chloroplast Development Edited by George Akoyunoglou © ]981 Balaban International Science Services, Philadelphia, Pa.

THE PLASTIDIC SHI Kl MATE PATHWAY AND ITS ROLE IN THE SYNTHESIS OF PLASTO-

QUINONE-9, « - T 0 C 0 P H E R 0 L AND PHYLLOQUINONE IN SPINACH CHLOROPLASTS

Schul t z , G., B i c k e l , H. , Buchholz, B. and S o l l , J .

In s t i t u t f ür T i e r e r n ä h r u n g , Arbeitsgruppe f ür Phytochemie und Futtermit­

telkunde, T i e r ä r z t l i c h e Hochschule, D 3000 Hannover 1 (FRG)

ABSTRACT

The p l a s t i d i c SkA pathway is operative in the synthesis of aromatic amino

acids and of the prenylquinones PQ -9, c*T and phyl loquinone. Neither exo-genous Substrates nor coenzymes are needed under photosynthetic c o n d i t i o n s .

However, ad d i t i o n of PEP - and for Trp formation Gin and Ser - enhances

the rates of synthe s i s . The pathway e x h i b i t s a s p e c i f i c feedback: Trp in-

h i b i t s the pathway at the Steps between SkA and chorismate and not at the

KDAHP-step as in some microorganisms, whereas Phe and Tyr only i n h i b i t

t h e i r cwn syn t h e s i s .

The introductory step in PQ-9 and c*T-synthes i s is the oxidation of p-hydro-

xypheny1pyruvate to homogentisate / ! / . It is prenylated to the methyl - 6 -prenylquinol by the corresponding prenyl-PP under simultaneous e l i m i n a t i o n

of the ca rboxy lgroup of homogent i sate. The only s i t e of c*T synthesis is the envelope membrane of the c h l o r o p l a s t , whereas that of PQ-9 synthesis is the envelope and the thylakoid membrane too. The sequence in ocT syn­thesis in spinach is ( F i g . 3 . ) :

Homogentisate Phytyl-PP^ Me-6-PQH2 2,3-Me

2-PQH

2

C

yc l i z a t i o n

.

_ SAM _ _A , . .

u Solanesyl-PP

yj a

T ; for the PQ-9 biosynthesis i t i s : Homogent i sate 1

+~

Me-6-SQH2 PQH

2.

Abbreviations: E^P - erythrose-^-P; GGPP - gerany1gerany1-PP; HPP - p-hydro-xyphenylpyruvate; KDAHP - 2-keto -3"deoxyarabinoheptonic acid - 7-P; Me-6-GGQH - 2-methyl - 6-geranylgeranylquinol; Me-6-PQH and isomers - 2-methyl -6-phy-t y l q u i n o l and isomers; 2,3-Me

2-PQH

2 - 2 , 3-dimethyl - 5-phytylquinol; M e ^ - P Q H «

- t r i m e t h y l p h y t y l q u i n o l ; Me-6-SQH2 - 2-methyl - 6-solanesyIquinol; PGA ^ 3"D-

phosphoglycerate; PQ-9*" plastoquinone - 9 ; PQH« - p l a s t o q u i n o l - 9 ; PEP -

phosphoenolpyruvate; SkA - shikimate; PRPP - 5-P-ribosyl - 1-PP; SAM - S-ade-

nosylmethionine; ocT, ß T , j/T, <5T - e r , ß - , p-, 5-tocopherol.

311

312 Schultz et al.

From i s o t o p i c studies using CO^ /2/,SkA /3/ and o-benzoy1succinate /k/ i t

has been proved that higher plants are able to synthesize phylloquinone

(2-methyl-3~phytylnaphthoquinone vitamin K1) . The envelope membrane is the

s i t e o f prenylation of 1 .k-d\hydroxy -2-naphthoateto form 2-methyl-naphtho-quinol which is methylated by SAM to y i e l d p h y l l o q u i n o l . Consequent1y, the

sequence in i t s biosynthesis seems to correspond with that in microorgan-

isms / 5 , 6, I I : S k A ( C h o r i smate Succinylsemialdehyde-TPP^ } q

_S u c

.

cinylbenzoate Pr e n

Y^ 2-Preny1naphthoquinol —SAM^ 2-Methy1~3~preny1-naphthoquinol. In plants phytyl-PP is preferred as prenylCompound.

The SkA pathway operates in the synthesis of aromatic amino acids in plants.

It is a l s o involved in the synthesis of prenylquinones by synthesizing the

aromatic moiety. The prenyl sidechain o r i g i n a t e s from the p l a s t i d i c meva-

lonate pathway /8/. These prenylquinones operate in d i f f e r e n t ways in the

photosynthetic t i s s u e : PQ-9 acts in the photosynthetic e l e c t r o n transport

/9/> c*T i n a c t i v a t e s e n e r g e t i s i z e d oxygen species formed by l i g h t (by sca-

venging r a d i c a l s and a l s o by quenching 0^ / 1 0 , 11/). a J is a l s o an im-

portant membrane co n s t i t u e n t . The funetion of phylloquinone in plants is

not yet c l e a r . The f i r s t problem was to study the compartmentation of the

SkA pathway involved in these syntheses.

I d e n t i f i c a t i o n of a p l a s t i d i c SkA pathway

Biosynthesis of aromatic amino acids in spinach c h l o r o p l a s t s under pho­

tosynthetic conditions: The findings that isoenzymes of SkA dehydrogenase

/12, 13/ and enzymes of Tryp synthesis / 1 V are enriched in c h l o r o p l a s t

f r a c t i o n s , strongly indicate the occurence of enzymes of SkA pathway in

t h i s o r g a n e i l e . To prove the funetion of t h i s pathway, p u r i f i e d i n t a c t chlo-

14

roplasts were illuminated under photosynthetic conditions with CO^ and

the label in the expected aromatic amino acids and prenylquinones was de-

termined / 15 , 16, 17/ (see F i g . 1). In a l l experiments c h l o r o p l a s t s suspen-

sions with at least 1 mg chlorophy11/ml were used; t = 30 min; control

expt. disproved microbial contamination / 1 6 / . The p u r i t y of the chloro­

plasts i s o l a t e d acc. to /18/ was tested for marker enzymes / 1 9 , 20/.

PEP PEP GluNH2 PRPP Ser

co2 •

Choris- Y Anthra- Y Indol- V T

nil - ' ' ~ 1

Prephenate

? \ e Ty

PEP v • . ^ c u - f c - J W w l u ' l i _ ^ . n i i m i ' a - m i i i u u i - ^ -r

Photosynthetic^ E 4 p

mate nilate glycerpl-P p

C-f1xation i I i \^ Prephenate

CHLOROPLASTS

E4P SkA Chorismate Anthranilate

F i g . 1. SkA pathway in spinach c h l o r o p l a s t s and Substrates tested 1 2 h l

Schultz et al. 313

i J, T a b l e 1. C - I n c o r p o r a t i o n from CO^ i n t o amino a c i d s o f i l l u m i n a t e d

i n t a c t s p i n a c h c h l o r o p l a s t s o f d i f f e r e n t p u r i t y /21/.

P u r i f i e d Non p u r i f i e d

c h l o r o p l a s t s c h l o r o p l a s t s

% ~ o f p h o t o s y n t h e t i c a l l y f i x e d CO,,

Sum o f amino a c i d s o . l k O.kk

% o f l a b e l i n amino a c i d s

Asn+ A s p + G i n + G l u 16.6 1.8

Ser + G l y 38.9 79-9

A l a "40.3 12.9

Phe + T y r + T r p 10.2 5-^

Increasing the p u r i t y of the c h l o r o p l a s t s , the label of Gly and Ser decrea-

sed, whereas that of the aromatic amino acids (and of Ala) increased /21/.

Decrease of Gly and Ser synthesis is due to the e l i m i n a t i o n of the peroxy-

somes and mitochondria /21/ (Table 1).

As pointed out by /22/ at the onset of i l l u m i n a t i o n the exchange of d i -

hydroxyacetonephosphate from the ch l o r o p l a s t s versus P. of the Suspension

medium by the phosphate translocator /23/ of the envelope membranes resul t s

in a l a g p h a s e o f photosynthetic carbon f i x a t i o n . This is caused by a lack

of PGA which is needed by the Ca 1 v i n - c y c l e . The same should be v a l i d to

metabolic pathways adjacent to the C a l v i n - c y c l e , e.g. the SkA pathway in-

vestigated here. Experiments with and without P. in the medium proved t h i s

assumption / 1 7 / : Omission of P. of the medium e f f e c t s in a manifold i n -14

1

corporation from CO^ into aromatic acids (Table 2 ) .

Requirement of exogenous Substrates: As shown above, the SkA pathway

operates under conditions of photosynthetic carbon f i x a t i o n by intact

c h l o r o p l a s t s at considerable rates. In additional studies the ~inf1uence

of exogenously added Substrates on the p l a s t i d i c SkA pathway was determi-

ned 1 2 h / . To narrow the study, the metabolic flow of the SkA pathway was

di r e c t e d only to the Trp branch by i n h i b i t i n g the other two branches by

\ k \k

T a b l e 2. C - I n c o r p o r a t i o n from CO- i n t o a r o m a t i c amino a c i d s and p r e n y l ­

q u i n o n e s o f i l l u m i n a t e d i n t a c t Spinaen c h l o r o p l a s t s a d d i n g and o m i t t i n g P.

t o the S u s p e n s i o n medium .

P. added P. o m i t t e d ' « I i .

TO * % o f p h o t o s y n t h e t i c a l l y f i x e d C02

Phe 35 66

T y r 0.*»5

T r p 2.3 h PQ-9 0.^2 1.3

« T 0.73 1.3

314 Schultz et al.

T a b l e 3. C - l n c o r p o r a t i o n from XO* o r / I , 6 - C/-SkA i n t o a r o m a t i c

amino a c i d s o f i l l u m i n a t e d i n t a c t s p i n a c h c h l o r o p l a s t s i n the p r e s e n c e o f

Phe, T y r and T r p , r e s p e c t i v e l y /25/.

+ Phe + T y r + T r p

- each 5 mM - f

% o f c o n t r o

T y r

T r p

82 386 5 5

163 25

212

18 37 12

+ / i t 6 - 7-SkA

5 1*7 120

152 8A

208

7 10 38

Phe

T y r

T r p

w i t h o u t a d d i t i o n o f a r o m a t i c amino a c i d s

adding Phe and Tyr (see below). An addition of PEP, Gin and Ser increased

14 the incorporation from CO^ into Trp. The optimal concentrations were

(without added Substrate = 1.0): ca 3-5 f o l d increase at 5 x 10 ^ M PEP;

-6 14 ca 2.5 f o l d increase at 10 M Gin and Ser, resp. . Furthermore, /1- C/-

PEP is incorporated into aromatic amino acids of c h l o r o p l a s t s in consider-

able y i e l d s /24/. When E4P, SkA, chorismate and anthrani1ate, r e s p e c t i v e l y ,

14

were applied in C0^ experiments in increasing concentrations, the label

in the aromatic amino acids more or less decreased /24/. This might be cau-

sed by i s o t o p i c d i l u t i o n of endogenous intermediates and/or regulatory phe-

nomena.

14

Feedback control by endproducts: From C ü ^ - e x p e r iments in the presen­

ce of Phe, Tyr or Trp (each 5 mM) i t could be revealed that the SkA path­

way is subject to feedback control by endproducts /25/ (Table 3). Phe and

Tyr exert feedback control over t h e i r own rates of s y n t h e s i s , whereas Trp

co n t r o l s the rate of synthesis of a l l three aromatic amino a c i d s . To deter-

14

mine a point of attack more e x a c t l y , /1.6- C/-SkA was fed as a more d i r e c t

precursor (Table 3) • These r e s u l t s indicate thatTrp attacs a Step between

the synthesis of SkA and chorismate ( F i g . 2) and not the KDAHP Step as in

some microorganisms (for survey see /26/).

Co ^ KDAHP SkA +*• Chorismate 2

V ^Tr p

Prephenate

F i g . 2. Feedback control of SkA pathway by Phe and Tyr and Trp in spinach

c h l o r o p l a s t s /25/.

Schultz et al. 315

Transfer of aroroatic amino acids across the envelope membranes: Feeding

/ 1 . 6 - C/-SkA to spinach c h l o r o p l a s t s , the main portion of label in the

aromatic amino acids was found in the Suspension medium a f t e r a period of

30 min / 1 6 / . This f i n d i n g as well as the considerable incorporation of

14

/ß- C/-Tyr applied to endosperm, into PQ and flavonoids of leaves of bar­

ley seedlings 1 2 1 / indicates a r e l a t i v e l y rapid transfer of aromatic amino

acids across the envelope membranes in v i t r o and in v i v o .

Oxidation of HPP to homogentisate: This step which is a p r e r e q u i s i t e

for the synthesis of PQ and a l was shown in the thylakoid f r a c t i o n of Lem-14

na gibba / 2 8 / . Furthermore, from the incorporation of C l a b e l l e d CO^,

SkA and Tyr into PQ and oflT, i t could be conclouded that the HPP oxidation

takes place in the c h l o r o p l a s t s / 16 , 17/.

Biosynthesis of <a-tocophero1 and plastoqu 1 none -9

The aromatic moiety of both derives from homogentisate /!/ which is f o r ­

me d by an oxydase System from HPP /28/.

ot-Tocopherol biosynthesis: The only s i t e of c*T biosynthesis in spinach

c h l o r o p l a s t s is the envelope membrane / 2 9 , 3 0 / . Homogentisate is s o l e l y

prenylated with phytyl-PP to form Me-6-PQH2 / 3 0 / . There i.s no Stimulation

by other c h l o r o p l a s t f r a c t i o n s l i k e thylakoid membranes or stroma / 3 0 / . The

prenyltransferase in spinach shows a strong s p e c i f i c i t y for phytyl-PP

(26 pmol/h mg envelope p r o t e i n ) ; GGPP is i n a c t i v e in t h i s System / 3 0 / . From

the possible p o s i t i o n s isomers only Me-6-PQH^ is formed; Neither Me-5" nor

Me-3"PQH2 could be found / 3 0 / .Consequently,the pathway is strongly d i r e c t e d

at this step. A kinase which forms phytyl-PP from phytol plus ATP is loca-

l i z e d in the stroma / 3 0 / . Phytol and i t s pyrophosphate a r i s e s by reduction

from GGPP / 3 1 / which is synthesized by a recombinated System of epvelope

or thylakoid membranes plus stroma protein / 3 2 / .

The following methylation Steps with SAM as methyl-group donor to form

cxT from Me-6-PQH2 are a l s o performed by enzyme Systems l o c a l i z e d in the

envelope membranes / 2 9 / (see F i g . 3 ) . The quinol is the Substrate of the

methylation and not the quinone. Comparison of the methylation rates to

y i e l d the corresponding dimethyl-Compound shows that Me-6-PQH2 is not only

s t r o n g l y preferred to i t s isomers Me -5- and Me-3-PQH^ but a l s o t o i t s chromanol

stage S T ( r a t i o s are 100 : 10 : 5 : 5) / 3 3 / - Thus, the raain product is

2,3-Me2~PQH

2 which undergoes ringclosure to y l and further methylation by

SAM to « T . The chromanol stage is the p r e r e q u i s i t e for the second methyl­

a t i o n , no Me^-PQH2 occurs / 3 3 , 3 4 / . y l is preferred to ßT to y i e l d ocT

(100 : 35) / 3 3 / . In marked contrast to the prenylation enzyme, the t r a n s f e r -

316 Schultz et al.

ase for the f i r s t methylation step e x h i b i t s a preference for Me-6-GGQH2

(2 nmol/h mg envelope protein) in comparison to Me-6-PQH^ (0.7 nmol mg

envelope protein) / 2 9 , 3 4 / .

The ring closure of the dimethy1prenylquinol to the corresponding chro­

manol (in t h i s case 2.3 M e2~ P Q H

2y T ) takes place only in int a c t chloro­

p l a s t s / 3 3 , 34/ but not in i s o l a t e d envelope membranes. The concentrations

of PQ-9 and o<T in the envelope are: PQ 0.53 ug/mg envelope protein (7.1 x

10_ i* M) , c*T 1.03 ug/mg envelope p r ö t e i n (2.4 x 10~^ M) ( S o l l , Douce unpbl.).

Plastoquinone - 9 biosynthesis: PQ-9 b i o s y n t h e s i s , both prenylation and

methylation, is not only performed by the envelope membranes (1.2 pmol/h mg

protein and 10 pmol/h mg p r o t e i n , respectively) but a l s o at low rates by

the thylakoid membranes (0.013 pmol/h mg protein and 0.35 pmol/h mg protein,

respectively) / 3 0 7 . However, i f one takes into account the rate of t h y l a ­

koid protein to that of envelope protein per mg C h l o r o p h y l l , the y i e l d s in

total are not as d i f f e r e n t as they are ca l c u l a t e d on the basis of prot e i n

i t s e l f . The sequence of reactions involved in PQ-9 biosynthesis is s i m i l a r

to ocT. Solanesyl-PP (Cj^) serves as prenyl Compound in the prenylation re-

action to form Me-6-SQH2 with homogentisate. In the following steps Me -6-

SQH2 is methylated with SAM to y i e l d PQ-9-

Shikimate pathway Prenyl-PP-synthesis

env + str + thyl

Tyr -*^p-riydroxyphenyl pyruvate y

' (HPP)

'ipL

Geranylgeranyl-PP (GGPP)

str

oh Homogentisate

ch 2-cooh Phytyl -PP I Phytol Sol anesyl -PP

thyl

Me-6-Phytylquinol (Me-6-PQH

2)

| SAM env

2,3-Me9-Phytylquinol ^(2,3-Me

2PQH

2)

| Cyclization

y-Tocopherol {yl)

SAM env

a-Tocopherol {al)

~~| env t

Me-6-Solanesylquinol (Me-6-SQH

2)

SAM env thyl

Plastoquinol-9 (PQH

2)

str Stroma

env Envelope

thyl Thylakoids

F i g . 3. Biosynthesis of &1 and PQ in spinach c h l o r o p l a s t s / 2 9 , 30, 33, 34/

Schultz et al. 317

M III IV

Fig . k. Proposed scheme for the biosynthesis of phylloquinone in spinach

c h l o r o p l a s t s . I - o-succinylbencoic acid; II - 1 ,4-dihydroxy-2-naphthoic

aci d ; III - 2-phytyl-l,4-naphthoquinol; IV - 2-methyl-3-phyty1-1,4-naph-

thoqu i nol

Phylloquinone biosynthesis

As mentioned above, biosynthesis of phylloquinone in plants could be detec-

ted by feeding some S u b s t r a t e s including SkA by the group of T h r e l f a l l /3,

k l . In studies on spinach c h l o r o p l a s t (Schultz and E l l e r b r o c k , unpublished

data), 1 ,4-dihydroxy-2-naphthoateis prenylated by phytol plus ATP to form

2- phytylnaphthoquinol (60 pmol/h mg C h l o r o p h y l l ) . (For the biosynthesis

of phytyl-PP by c h l o r o p l a s t s see /307). The quinol is methylated by SAM to

y i e l d phylloquinol (6 pmol/h mg C h l o r o p h y l l ) . There is a strong s p e c i f i c i t y

of the sequence of both r e a c t i o n s . 2-Phyty1naphthoquinol is preferred to

i t s GG-and f a r n e s y l - homologue. The s i t e of prenylation is the envelope

membrane; thylakoid membranes as well as stroma protein seems to be inac-

t i v e . Both reactions need Mg (2.5 mM); l i g h t i s not required.

CONCLUSIONS

The p l a s t i d i c SkA pathway is involved in the biosynthesis of aromatic amino

acids as well as of prenylquinones of algae and higher plants (for d i s t r i -

bution /35/) but how i t is combined with the generally occuring secon-

dary metabolism of aromatics in plants is not c l e a r /36/.

ACKNOWLEDGEMENTS

Financial support by the Deutsche Forschungsgemeinschaft and the Centre

Nationale de la Recherche S c i e n t i f i q u e (ERA 847 to Prof. Dr. R. Douce, Bio­

lo g i e Vegetale, CENG and USM-G, Grenoble, France) are g r e a t f u l l y acknow-

ledged.

318 Schultz et al.

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