A Journal of Zeitschrift für Naturforschung C

Biosciences Founded 1946 in the Institutes Volume 48 1993 of the Max-Planck-Gesellschaft

Verlag der Zeitschrift für Naturforschung Tübingen

ZEITSCHRIFT FÜR NATURFORSCHUNG

Section C

A Journal of Biosciences

ISSN 0939-5075

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V E R L A G D E R Z E I T S C H R I F T F Ü R N A T U R F O R S C H U N G , T Ü B I N G E N P. O. Box 2645, Uhlandstraße 11, D-W-7400 Tübingen Fax: 07071-360571 • Telefon: 07071-3 1555

Managing Editor:

H . Hausen, Tübingen

Council:

A . Butenandt, München M . Eigen, Göttingen H . Haken, Stuttgart

Editors:

A . Hager, Tübingen K . Hahlbrock, Köln W. Hasselbach, Heidelberg P. Karlson, Marburg J. Kle in . Tübingen J. St. Schell, Köln

Associate Editors:

N . Amrhein, Zürich B. A . Askonas, London W. Barz, Münster P. Böger, Konstanz G . Bornkamm, Freiburg D . Bückmann, U l m H . Budzikiewicz, Köln K . G . Götz , Tübingen G . Gottschalk, Gött ingen P. Gruss, Heidelberg G . Isenberg, Köln G . M a a ß , Hannover V . ter Meulen, Würzburg G . F . Meyer, Tübingen M . Rajewsky, Essen D . Schulte-Frohlinde,

Mülheim/R. G . Schulz, Freiburg F. F . Seelig, Tübingen J. Seelig, Basel H . Simon, München W. Steglich, München H . Stieve, Aachen J. Suko, Wien A . Trebst, Bochum G . Weissenböck, Köln G . Wiek, Innsbruck F . Wieland, Heidelberg V . Zimmermann, Würzburg

Section a Physics, Physical Chemistry, Cosmic Physics

Section b Inorganic and Organic Chemistry

Anschrift des Verlages: Postfach 26 45, D-72016 Tübingen Satz und Druck: Allgäuer Zeitungsverlag GmbH, Kempten

ISSN 0939-5075

Nachdruck - auch auszugsweise - nur mit schriftlicher Genehmigung des Verlages

B a y e r i s c h e

Band 48 c Zeitschrift für Naturforschung 1993

Contents

C o n t e n t s o f N u m b e r 1/2

O r i g i n a l C o m m u n i c a t i o n s

5-Hydroxyaloin A in the Genus A l o e . Thin Layer Chromatographie Screening and High Perform­ance Liquid Chromatographie Determination H . W . R A U W A L D and A . B E I L 1

Epicuticular Leaf Wax of E u p h o r b i a h e l i o s c o p i a L . (Euphorbiaceae) M . N A Z I R , W . A H M A D , N . A . R A B I , and S. A . K H A N 5

Studies on Sporopollenin Biosynthesis in C u c u r ­b i t a m a x i m a I: The Substantial Labeling of Sporopollenin from C u c u r b i t a m a x i m a after Application of [1 4C]Phenylalanine S. G U B A T Z and R. W I E R M A N N 10

7-O-Methyl-luteone Metabolism in B o t r y t i s c i n e ­r e a : Identification of the Epoxy-Intermediate and Absolute Configuration of the Pyrano-iso-flavone Metabolite S. T A H A R A , F . S A I T O H , and J . M I Z U T A N I 16

Different Forms of Fructose 1,6-Bisphosphatase in C h l o r e l l a N . G R O T J O H A N N , G . S C H N E I D E R , and W. K o W A L L I K 22

Presence of Both Hemidiscoidal and Hemiellip-soidal Phycobilisomes in a P h o r m i d i u m Species (Cyanobacteria) M . W E S T E R M A N N , W. R E U T E R , C H . S C H I M E K , and W . W E H R M E Y E R 28

A n Auxin Binding Protein is Localized in the Sym-biosome Membrane of Soybean Nodules A . J A C O B I , R . Z E T T L , K . P A L M E , and D . W E R N E R

35

Inactivation and Reactivation of the Hydroge-nases of the Green Algae Scenedesmus o b l i q u u s and Chlamydomonas r e i n h a r d t i i T H . U R B I G , R. S C H U L Z , and H . S E N G E R 41

Changes of Excitation Spectra of i n v i v o Chloro­phyll Fluorescence during Induction of Photo-synthesis W. I. G R U S Z E C K I and Z . K R U P A 46

Microbial Reduction of Aromatic Carboxylic Acids H . - A . A R F M A N N and W . - R . A B R A H A M 52

Biochemical Model Reactions on the Prooxidative Activity of Homocysteine G . PREIBISCH, C H . K Ü F F N E R , and E . F . E L S T N E R

58

The Effect of Neighboring Bases on Miscoding Properties of N 2,3-Ethenoguanine M . M . M R O C Z K O W S K A and J. T. K U S M I E R E K 6 3

Flavonoids from A p i s m e l l i f e r a Beeswax F. A . T O M Ä S - B A R B E R Ä N , F . F E R R E R E S , F . T O M Ä S - L O R E N T E , and A . O R T I Z 68

Artifacts and Pheromone Blends from N e z a r a spp. and Other Stink Bugs (Heteroptera: Pentato-midae) J. R. A L D R I C H , H . N U M A T A , M . B O R G E S , F . B I N , G . K . W A I T E , and W . R. L U S B Y 73

Cast Skin Lipids of the Indian Python { P y t h o n m o l u r u s b i v i t t a t u s , Kühl , 1820) J . J A C O B , B. Z I E M S E N , and U . H O P P E 80

Glycogen Synthesis in Rat Liver from a Pool of Free Glucose H . SCHIMASSEK 85

Two Different Bump Types of the Ventral Photo-reeeptor of L i m u l u s H . R E U S S and H . STIEVE 92

Spectral Categories in the Learning Behaviour of Blowflies N . T R O J E 96

IV Contents

Notes

New Investigations on Flavonoids from V i s c u m a l b u m L . ssp. a b i e t i s , a l b u m and a u s t r i a c u m (In German) E . L O R C H 105

Aromatic Amino Acids in the Venom of the Bra-conid Parasitoid Apanteles k a r i y a i T . S H I M I Z U , Y . T A T S U K I , and N . T A K E D A 108

Pheromones, 88 . Sex Pheromone Components of Female E u z o p h e r a p u n i c a e l l a M . (Lepidoptera, Pyralidae) H . J. B E S T M A N N , F . K E R N , G . G . M E L I K Y A N , D . SCHÄFER, O . V O S T R O W S K Y , E . V . B A B A Y A N , and S H . O . B A D A N Y A N 110

Deoxyribonucleotide Synthesis in Phycovirus-In-fected Green Algae. A New Virus-Induced Ribonucleotide Reductase C. B O R N E M A N N and H . F O L L M A N N 113

C o n t e n t s o f N u m b e r 3 / 4

C h l o r o p l a s t M e t a b o l i s m a n d I t s I n h i b i t i o n by H e r -b i c i d e s

Preface S. Y O S H I D A , W . OETTMEIER, and P. B Ö G E R 119

Review

Research on Biochemistry of Herbicides: A n His-torical Overview D. E . M O R E L A N D 121

/. I n h i b i t o r s of Photosystem I I

Hydrogen Bonding of Cyanoacrylates with the D 1 Peptide J . N . P H I L L I P S and W . K . B A N H A M 132

N-Methylanilinocyanoacrylate Photosystem II In­hibitors. Structure-Activity Relationships W . K . B A N H A M , J . L . H U P P A T Z , and J . N . PHILLIPS 136

Understanding the Topography of the Photosys­tem II Herbicide Binding Niche: Does Q S A R Help? J. L . H U P P A T Z A N D H . G . M C F A D D E N 140

On the Orientation of Photosystem II Inhibitors in the Q B -Binding Niche: Acridones, Xanthones and Quinones W . O E T T M E I E R , K . M A S S O N , R . K L O O S , and E. R E I L 146

Halogenation Enhances the Photosystem II Inhib-itory Activity of 4-Hydroxypyridines: Struc­ture-Activity Relationships and Their Mode of Action T. A s A M I , M . B A B A , H . K O I K E , Y . I N O U E , and S. Y O S H I D A 152

Photosystem II Inhibition by Phloroglucinol De­rivatives Having Both Phenol and Urea Func-tionalities I. H O N D A , M . S I B A G A K I , K . Y O N E Y A M A , Y . N A K A J I M A , M . K O N N A I , N . T A K A H A S H I , and S. Y O S H I D A 159

Photosystem II Inhibition by Pyran-enamine Derivatives K . Y O N E Y A M A , Y . N A K A J I M A , M . O G A S A W A R A , H . K U R A M O C H I , M . K O N N A I , H . I W A M U R A , F . S A T O , K . ICHINOSE, T. A S A M I , N . T A K A H A S H I , and S. Y O S H I D A 163

Interactions of Halogenated Benzoquinones with the Non-Heme Iron (Q 4 0 0 ) in Photosystem II H . K O I K E , Y . K A S H I N O , and K . S A T O H 168

Electron Transport from Q A to Thymoquinone in a Synechococcus Oxygen-Evolving Photo­system II Preparation: Role of Q B and Binding Affinity of Thymoquinone to the Q B Site K . S A T O H , Y . K A S H I N O , and H . K O I K E 174

Comparison of Experimental and Calculated Hy­drogen Bonding Properties of Some Urea and Triazine Inhibitors of Photosystem II S. C R E U Z E T , T. M I R A N D A , and J . - M . D U C R U E T

179

//. The I n h i b i t o r B i n d i n g Site of Photosystem I I

Protein Modifications in the D 2 Protein of Photo­system II Affect Properties of the Q B/Herbicide-Binding Environment

Contents V

H . K L E S S , M . O R E N - S H A M I R , I. O H A D , M . E D E L -M A N , and W . V E R M A A S 185

Molecular Modelling of the Interaction between D C M U and the Q B -Binding Site of Photo­system II S. P. M A C K A Y and P. J . O ' M A L L E Y 191

Structural Analysis of the Q B Pocket of the D 1 Subunit of Photosystem II in Synechocystis P C C 6714 and 6803 C H . ASTIER, I. P E R E W O S K A , M . P I C A U D , D . K I R I L O V S K Y , and C. V E R N O T T E 199

Binding of Triazines and Triazinones in the Q B -Binding Niche of Photosystem II K . G . T I E T J E N , W . D R A B E R , J . GOOSSENS, J . R . J A N S E N , J . F . K L U T H , M . S C H I N D L E R , H . - J . W R O B L O W S K Y , U . H I L P , and A . T R E B S T 205

Inhibition of Photosynthesis by 4-Nitro-6-alkylphenols: Structure-Activity Studies in Wi ld Type and Five Mutants of C h l a m y d o m o n -as r e i n h a r d t i i Thylakoids W. D R A B E R , U . H I L P , H . L I K U S A , M . S C H I N D ­L E R , and A . T R E B S T 213

Kinetics of Electron Transfer between Q A and Q B

in Wi ld Type and Herbicide-Resistant Mutants of C h l a m y d o m o n a s r e i n h a r d t i i A . R. C R O F T S , I. B A R O L I , D . K R A M E R , and S. T A O K A 259

Mutational Analysis of the PsbL Protein of Photo­system II in the Cyanobacterium Synechocystis sp. P C C 6803 P. R. A N B U D U R A I and H . B. P A K R A S I 267

Tolerance of Cultured A m a r a n t h u s r e t r o f l e x u s Cells to Atrazine Y . S H I G E M A T S U , S. C H A I C H A R O E N , F . S A T O , and Y . Y A M A D A 275

Comparison of Photosynthetic Activities in Tria-zine-Resistant and Susceptible Biotypes of C h e n o p o d i u m a l b u m V . B. C U R W I E L , G . S C H A N S K E R , O. J . D E V O S , and J . J . S. V A N R E N S E N 278

Pleiotropy in Triazine-Resistant B r a s s i c a napus: Leaf and Environmental Influences on Photo­synthetic Regulation J . D E K K E R 283

F u n c t i o n a l Aspects of Photosystem I I

Characterization of the Light-Induced Oxygen Gas Exchange from the IC 2 Deletion Mutant of Synechocystis P C C 6803 Lacking the Photo­system II 33 k D a Extrinsic Protein V . A . B O I C H E N K O , V . V . K L I M O V , S. R. M A Y E S , and J . B A R B E R 224

Photosystem II: Thermodynamics and Kinetics of Electron Transport from Q A ~ to Q B ( Q B ~ ) A N C I

Deleterious Effects of Copper(II) G . R E N G E R , H . M . G L E I T E R , E . H A A G , and F . R E I F A R T H 234

Significance of Photosystem II Core Phosphoryla-tion Heterogeneity for the Herbicide-Binding Domain M . T. G I A R D I 241

The Role of D l * in Light-Induced D l Protein Turnover in Leaves A . J . S Y M E , H . R. B O L H Ä R - N O R D E N K A M P F , and C H . C R I T C H L E Y 246

Bicarbonate-Reversible Inhibition of Plastoqui-none Reductase in Photosystem II G O V I N D J E E 251

I V . C a r o t e n o i d a n d L i p i d B i o s y n t h e s i s

Effects and Absorption of Sethoxydim on Excised Root Tips of C o m ( Z e a mays) and Pea ( P i s u m s a t i v u m ) M . K . T A K A G I and H . H O S A K A 288

Studies on the Inhibition of Biotin-Containing Carboxylases by Acetyl-CoA Carboxylase In­hibitors A . M O T E L , S. G Ü N T H E R , M . C L A U S S , K . K O B E K , M . F O C K E , and H . K . L I C H T E N T H A L E R 294

Synthesis and Bleaching Activity of 1-Ethyl- and 1 -Propyl-5-Substituted Imidazoles N . Y A M A D A , E . K U W A N O , and M . E T O 301

Differential Inhibition of Phytoene Desaturases from Diverse Origins and Analysis of Resistant Cyanobacterial Mutants G . S A N D M A N N and P. D . F R Ä S E R 307

Structure-Activity Correlations of Substituted 3(2//)Furanones Chemically Related to the Bleaching Herbicide Flurtamone G . S A N D M A N N and P. B Ö G E R 312

Contents VI

V. P e r o x i d i z i n g H e r b i c i d e s

H P L C and i n v i v o Spectrophotometric Detection of Porphyrins in Plant Tissues Treated with Porphyrinogenic Herbicides ST. O. D U K E , M . V . D U K E , and H . J. L E E 317

Molecular Aspects of Herbicide Action on Proto-porphyrinogen Oxidase B . N I C O L A U S , G . S A N D M A N N , and P. B Ö G E R 326

New Peroxidizing Herbicides: Activity Compared with X-Ray Structure H . K O H N O , K . H I R A I , M . H O R I , Y . S A T O , P. B Ö G E R , and K . W A K A B A Y A S H I 334

Isolation and Characterization of a C h l a m y d o -monas r e i n h a r d t i i Mutant Resistant to Photo-bleaching Herbicides H . OSHIO, H . S H I B A T A , N . M I T O , M . Y A M A M O TO, E. H . H A R R I S , N . W . G I L L H A M , J. E . B O Y N -T O N , and R. S A T O 339

A Quantum Chemical Study of "Light-Dependent Herbicides" T. A K A G I and N . S A K A S H I T A 345

Localization of Target-Site of the Protoporphyri-nogen Oxidase-Inhibiting Herbicide, S-23142, in S p i n a c i a o l e r a c e a L . F.-S. C H E , Y . T A K E M U R A , N . S U Z U K I , K . ICHI -NOSE, J . - M . W A N G , and S. Y O S H I D A 350

VI. A m i n o A c i d a n d N - M e t a b o l i s m ; M i s c e l l a n e o u s T o p i c s

Vinylogous Sulfonylureas: A New Class of Aceto-hydroxyacid Synthase Inhibitors Incorporating a Large Bridging Moiety H . G . M C F A D D E N , J. L . H U P P A T Z , and C. H . L . K E N N A R D 356

A Herbicidal Inhibitor of Isopropylmalate Iso-merase T. R. H A W K E S , J. M . Cox, T. E. M . F R Ä S E R , and T. L E W I S 364

Inhibitory Action of Glufosinate on Photosyn-thesis A . W I L D and C H . W E N D L E R 369

Paraquat (Methylviologen): Its Interference with Primary Photochemical Reactions T. H I Y A M A , A . O H I N A T A , and S. K O B A Y A S H I 374

Differential Response of Two Soybean Cultivars to Paraquat S. K I M and K . K . H A T Z I O S 379

Comparative Effects of Paraquat on Antioxidant Components and Scavenging Enzymes in Kwangkyo and Hood Soybean S. K I M and K . K . H A T Z I O S 385

The Effect of Free Radical Enhancers and Scaven-gers on Accumulation of Early Light-Inducible Protein during Light Stress I. A D A M S K A , K . K L O P P S T E C H , and I. O H A D 391

Chlorophyll Fluorescence Applied in the Analysis on Vertical Movement of Herbicides in Soil D . Y A N A S E , A . A N D O H , M . C H I B A , and S. Y O S H I D A 397

Energy Transfer from N A D P H to Protochloro-phyllide in Isolated Protochlorophyllide Holo-chrome as Determined by UV-Fluorescence Excitation Spectroscopy at 77 K N . N . L E B E D E V and E. D U J A R D I N 402

C o n t e n t s o f N u n i b e r 5/6

O r i g i n a l C o m m u n i c a t i o n s

Molecular Mobil i ty of Sucrose in Aqueous Solu­tion Studied by 1 3 C N M R Relaxation D . G I R L I C H and H . - D . L Ü D E M A N N 407

Alkaloid Composition of L u p i n u s albescens (Faba-ceae) from South America A . M . P L A N C H U E L O - R A V E L O and M . W I N K 414

On the Occurrence of Caffeoyltartronic Ac id and Other Phenolics in C h o n d r i l l a j u n c e a M . C. T E R E N C I O , R. M . G I N E R , M . J . S A N Z , S. M Ä N E Z , and J . L . Rios 417

Rare Flavonoid Aglycones from A n a p h a l i s m a r -g a r i t a c e a and Two G n a p h a l i u m Species E. W O L L E N W E B E R , H . F R I T Z , B. H E N R I C H , J . J A K U P O V I C , G . S C H I L L I N G , and J . N . R O I T M A N

420

Contents

Pseudoverdin, a Compound Related to the Pyo-verdin Chromophore from a Pseudomonas a e r u ­g i n o s a Strain Incapable to Produce Pyoverdins I. L O N G E R I C H , K . T A R A Z , H . B U D Z I K I E W I C Z , L . T S A I , and J . - M . M E Y E R 425

Acylated Anthocyanins from Flowers of Cine-raria, Senecio c r u e n t u s , Red Cultivar N . T E R A H A R A , K . T O K I , and T. H O N D A 430

Uptake and Transport of Quinolizidine Alkaloids in C u s c u t a r e f l e x a Parasitizing on L u p i n u s a n g u -s t i f o l i u s P. B Ä U M E L , W . D . J E S C H K E , L . W I T T E , F . - C . C Z Y G A N , and P. P R O K S C H 436

Partial Purification and Characterization of Mem­brane-Associated 3-Hydroxy-3-methylglutaryl-Coenzyme A Lyase from Radish Seedlings T H . W E B E R and T H . J . B A C H 444

Asymmetrie Reduction of Synthetic Ketones by Marine Microorganisms Y . Y A M A Z A K I , A . M A R U Y A M A , K . H O S O N O , T . H I G A S H I H A R A , and H . K O B A Y A S H I 451

Hysteresis and Reversible Cold-Inactivation of ADP-Glucose Pyrophosphorylase from Barley Seeds L . A . K L E C Z K O W S K I , P. V I L L A N D , and O . -A . O L S E N 457

Reconstitution of Light-Harvesting Complexes from C h l o r e l l a f u s c a (Chlorophyceae) and M a n -t o n i e l l a s q u a m a t a (Prasinophyceae) M . M E Y E R and C H . W I L H E L M 461

Molecular Modelling of the Interactions between Optically Active Triazine Herbicides and Pho­tosystem II S. P. M A C K A Y and P. J . O ' M A L L E Y 474

Photoproduction of Hydrogen by Purple Bacteria: A Critical Evaluation of the Rate Limiting Enzymatic Steps J . - H . K L E M M E 482

Differential Effect of Hg(II) on [d(A) n • d(T)n] and [d(A-T) n • d(A-T) n] Sequences: Circular Dichro-ism (CD) Measurements and Endonuclease Digestion Studies Using Poly[d(A) • d(T)] and Poly[d(A-T) • d(A-T)] as Substrates S. R . O K and D . W . G R U E N W E D E L 488

VII

Azadirachtin Inhibits Proliferation of Sf9 Cells in Monolayer Culture H . R E M B O L D and R. S. A N N A D U R A I 495

Spin Label Study of Apomembranes and Purple Membranes T. R . L A Z A R O V A and M . Y . V E L I T C H K O V A 500

Inter- and Intraspecific Variation of the Nucleo-tide Sequence of the Cytochrome b Gene in Cory's ( C a l o n e c t r i s d i o m e d e a ) , Manx Shear-water (Puffinus puffinus) and the Fulmar ( F u l -m a r u s g l a c i a l i s ) M . W I N K , P. H E I D R I C H , U . K A H L , I. S W A -T S C H E K , H . - H . W I T T , and D . R I S T O W 504

Pheromones, 92. Odorous Substances from the Abdominal Hair Brushes of the Male Sphingid M o t h A c h e r o n t i a a t r o p o s L . (Lepidoptera: Sphingidae) (In German) H . J . B E S T M A N N , J . E R L E R , O. V O S T R O W S K Y , and L . T H . W A S S E R T H A L 510

Pheromones, 93. The Sex Pheromone of the Cosmopterigid M o t h L i m n a e c i a p h r a g m i t e l l a (Lepidoptera: Cosmopterigidae) (In German) H . J . B E S T M A N N , F . K E R N , E . JANSSEN, and I. H A S E N F U S S 515

Monte Carlo Simulation of Shot Noise Analysis for Reconstructing Elementary Events: Quan­tum Bumps in Photoreceptor Cells ST. J O E K E N and J . S C H N A K E N B E R G 519

Notes

The Biflavonoid Pattern of A n a c o l i a w e b b i i T. SEEGER, H . G E I G E R , R. M U E S , and H . D . ZINSMEISTER 529

The Biflavonoid Pattern from the Moss B a r t r a m i a h a l l e r i a n a (In German) R. S A L M , T. SEEGER, and H . D . ZINSMEISTER 531

Exudate Flavonoids from G r i n d e l i a t a r a p a c a n a of Chile E. W O L L E N WEBER, M . D Ö R R , B. N . T I M M E R ­M A N N , J . S T R A N D , and E. R. F U E N T E S 533

Contents VIII

C o n t e n t s o f N u m b e r 7/8

I n v i t e d Trends A r t i c l e

Interaction and Compartmentalization of the Components of Bacterial Enzyme Systems In-volved in Cell Energetics H . G E R B E R D I N G and F . M A Y E R 535

O r i g i n a l C o m m u n i c a t i o n s

Antioxidative Properties of Phenazone Deriva­tives: Differentiation between Phenylbutazon and Mofebutazon I. S C H N E I D E R and E . F . E L S T N E R 542

Phellodonic Acid , a New Biologically Active Hir-sutane Derivative from P h e l l o d o n melaleucus (Thelephoraceae, Basidiomycetes) M . S T A D L E R , T. A N K E , J . D A S E N B R O C K , and W . S T E G L I C H 545

Phytoalexin Accumulation in O r n i t h o p u s sativus as a Response to Elicitor Treatment K . SEIFERT, S. H Ä R T L I N G , A . P O R Z E L , S. J O H N E , and G . K R A U S S 550

Two Forms of Phosphoenolpyruvate Carboxylase in C h l o r e l l a kessleri N . G R O T J O H A N N and C. H I P P E 556

UDP-D-Glucose: Flavonol 3-0- and 7-O-Glucosyl Transferases from Young Leaves of P a e d e r i a scandens var. m a i r e i N . I S H I K U R A , Z . Y A N G , and S. T E R A M O T O 563

Photoregulation of Carotenoid Biosynthesis in Mutants of N e u r o s p o r a crassa: Activities of En-zymes Involved in the Synthesis and Conversion of Phytoene G . S A N D M A N N 570

Elicitor-Active Oligosaccharides from Algal Lami-naran Stimulate the Production of Antifungal Compounds in Alfalfa A . K O B A Y A S H I , A . T A I , H . K A N Z A K I , and K . K A W A Z U 575

A Protein Cross-Reacting with Anti-Spectrin A n ­tibodies is Present in Higher Plant Cells A . F . S IKORSKI , W . S W A T , M . B R Z E Z I N S K A , Z . W R Ö B L E W S K I , and B. BISIKIRSKA 580

Synthesis of Protoheme via Both, the C 5 - and the Shemin-Pathway, in the Pigment Mutant C-2 A ' of Scenedesmus o b l i q u u s B. D R E C H S L E R - T H I E L M A N N , D . D Ö R N E M A N N , and H . S E N G E R 584

Study on the Influence of Temperature on the Mortality of T r i b o l i u m confusum J . du Va l . Ex­posed to Carbon Dioxide or Nitrogen L . A . B U S C A R L E T 590

Survey of the Taxonomic and Tissue Distribution of Microsomal Binding Sites for the Non-Host Selective Fungal Phytotoxin, Fusicoccin C H . M E Y E R , K . W A L D K Ö T T E R , A . S P R E N G E R , U . G . SCHLÖSSER, M . L U T H E R , and E. W. W E I L E R

595

Preferential Deactivation of the S 3 State of the Water-Oxidizing Complex, Favoured by Plasto-quinone Reduction in Barley Chloroplasts F . F R A N C K and G . H . S C H M I D 603

Immunological Identification of Organ Specific Proteins and Transcripts in Developing Barley Grains H . K L U N G L A N D and M . BOSNES 609

Factors Controlling Medium-Chain Fatty Acid Synthesis in Plastids from Maturing Cuphea Embryos J . F U H R M A N N and K . - P . HEISE 616

Determination of Glycolipids, Sulfolipid and Phospholipids in the Thylakoid Membrane R. H A A S E , M . U N T H A N , P. C O U T U R I E R , A . R A D U N Z , and G . H . S C H M I D 623

X - R a y Studies on Phospholipid Bilayers. XII I . In-teractions with Gentamicin M . S U W A L S K Y and J . F R J A S 632

Brominated Secondary Compounds from the Marine Sponge V e r o n g i a a e r o p h o b a and the Sponge Feeding Gastropod T y l o d i n a p e r v e r s a R. T E E Y A P A N T , P. K R E I S , V . W R A Y , L . W I T T E , and P. P R O K S C H 640

Identification and Cytogenetic Analysis of an A b ­normal Pig Chromosome for Flow Cytometry and Sorting M . H A U S M A N N , C. P. P O P E S C U , J . BOSCHER, D . KERBceuF, J . D Ö L L E , and C H . C R E M E R 645

Contents I X

Ion Transport through Gramicidin A . Water Structure and Functionality M . P O X L E I T N E R , J . S E I T Z - B E Y W L , and K . H E I N ­Z I N G E R 654

Quinolizidine Alkaloid Profiles of South Ameri­can Lupins: L u p i n u s l i n e a r i s and the L u p i n u s g i b e r t i a n u s Complex A . P L A N C H U E L O - R A V E L O , L . W I T T E , and M . W I N K 702

Notes

Characterization of N O 2 Bound to the Plant Cuti-cle by F T - I R Spectroscopy P. L U Q U E , A . H E R E D I A , F . J . R A M I R E Z , and M . J . B U K O V A C 666

Electron Microscopical Morphology of Cyto-plasmic Granules from Horse Eosinophil Leu-cocytes J . C. S T O C K E R T , C. I . TRIGOSO, A . T A T O , and J . M . F E R R E R 669

C o n t e n t s o f N u m b e r 9/10

I n v i t e d T r e n d s Ärticle

Genetic Engineering of Disease and Pest Resist­ance in Plauts: Present State of the Ar t G . S T R I T T M A T T E R and D . W E G E N E R 673

O r i g i n a l C o m m u n i c a t i o n s

Epicuticular Leaf Waxes of the Hop ( H u m u l u s l u -p u l u s ) . Chemical Composition and Surface Structures P . - G . G Ü L Z , E . M Ü L L E R , T. H E R R M A N N , and P. L Ö S E L 689

Comparative F T I R and , 3 C C P / M A S N M R Spec-troscopic Investigations on Sporopollenin of Different Systematic Origins S. W I L M E S M E I E R , S T . S T E U E R N A G E L , and R. W I E R M A N N 697

Fructose 1,6-Bisphosphatase in C h l o r e l l a k e s s l e r i Grown in Red or Blue Light N . G R O T J O H A N N 707

3a-Hydroxysteroid-5ß-oxidoreductase in Tissue Cultures of D i g i t a l i s l a n a t a U . S T U H L E M M E R , W . H A U S S M A N N , F . M I L E K , W . K R E I S , and E . R E I N H A R D 713

Effect of Different Condensed Tannins on T r i c h o -p l u s i a n i Performance A . G O N Z A L E Z - C O L O M A , C H . S. W I S D O M , and P H . W . R U N D E L 722

6 ß-Hydroxylation of 17a-Acetoxycortexone by F l a v o b a c t e r i u m G . SPASSOV and W . - R . A B R A H A M 727

Study of Canadian Propolis by G C - M S and H P L C C. G A R C I A - V I G U E R A , F . F E R R E R E S , and F . A . T O M Ä S - B A R B E R Ä N 731

Glandulär Trichomes and the Volatiles Obtained by Steam Distillation of Q u e r c u s r o b u r Leaves R. E N G E L , P . -G. G Ü L Z , T H . H E R R M A N N , and A . N A H R S T E D T 736

The Unexpected Reduction of the Vinyl Group of Chlorophyll b by Sodium Borohydride in Meth-anolic Extracts of Maize Leaves and Its Inhibi­tion by 8-Hydroxyquinoline R. J . P O R R A , W . S C H Ä F E R , E . C M I E L , I. K A T H E ­D E R , and H . S C H E E R 745

Metabolie Reduction of Phenylpropanoid Com­pounds in Primary Leaves of Rye {Seeale ce-r e a l e L.) Leads to Increased U V - B Sensitivity of Photosynthesis S. R E U B E R , J . L E I T S C H , G . H . K R A U S E , and G . W E I S S E N B Ö C K 749

Naturally Occurring Antidotes against Benzimid-azole Fungicides S. T A H A R A , Y . M A T S U K U R A , H . K A T S U T A , and J . M I Z U T A N I 757

Contents X

Oxidative Stress of Crops Monitored by E P R H . B. S T E G M A N N , P. S C H U L E R , ST . W E S T P H A L , and E. W A G N E R 766

Molecular Modelling of the Interaction of Cyano-acrylate Inhibitors with Photosystem II. Part 1. The Effect of Hydrophobicity of Inhibitor Bind­ing S. P. M A C K A Y and P. J . O ' M A L L E Y 773

Molecular Modelling of the Interaction of Cyano-acrylate Inhibitors with Photosystem II. Part 2. The Effect of Stereochemistry of Inhibitor Bind­ing S. P. M A C K A Y and P. J . O ' M A L L E Y 782

Photoregulation of Nitrogen Metabolism and Pro­tein Accumulation in the Red Alga C o r a l l i n a e l o n g a t a Elbs et Soland F. L . F I G U E R O A 788

Translocation of Adenine Nucleotides in the Mi to -chondria of Male Sterile and Male Fertile Sorghum J . A R O R A , P. N A T H , and P. V . S A N E 795

Complete Sequence of One Copy of the psbA Gene from the Thermophilic Cyanobacterium Syne-chococcus e l o n g a t u s R. K L O O S , E. STEVENS, and W . OETTMEIER 799

Linear Free Energy Relationships for N(7)-Substi-tuted Guanosines as Substrates of Calf Spleen Purine Nucleoside Phosphorylase. Possible Role of N(7)-Protonation as an Intermediary in Phosphorolysis A . B Z O W S K A , E. K U L I K O W S K A , and D . S H U G A R

803

Notes

Isolation and Properties of Trimethylamine N-Oxide/Dimethylsulfoxide Reductase from the Purple Bacterium R h o d o s p i r i l l u m r u b r u m P. S A J I T Z , J . - H . K L E M M E , H . - G . K O C H , and M . M O L I T O R 812

Inhibition of Dihydrofolate Reductase by Mofe-butazon E . S T R E H L , I. S C H N E I D E R , and E . F . E L S T N E R 815

Antitumoral Effect of Bleomycin + Dolomite Combination Treatment, in Mice Bearing Ehr­lich Ascites Carcinoma S. S C H E L L E R , W . K R O L , K . S K I R M U N T T , G . Z Y D -O W I C Z , and J . S H A N I 818

C o n t e n t s o f N u m b e r 11/12

O r i g i n a l C o m m u n i c a t i o n s

l H N M R Assignments in Biflavonoid Spectra by Proton-Detected C - H Correlation H . G E I G E R , T. SEEGER, H . H A H N , H . D . Z I N S -MEISTER, K . R. M A R K H A M , and H . W O N G 821

10-Hydroxymajoroside, an Iridoid Glucoside from P l a n t a g o c o r n u t i Gouan L . N . H A N D J I E V A , R. T A S K O V A , and S. P O P O V 827

Segregation of Activity Profile in Benzimidazoles: Effect of Spacers at 5(6)-Position of Methyl Benzimidazole-2-carbamates S. K . A G A R W A L , f S. S H A R M A , A . P. B H A D U R I , J . C . K A T I Y A R , and R. K . C H A T T E R J E E 829

Bisbibenzyl Formation in Aseptic Cultures of M a r c h a n t i a p o l y m o r p h a L . K . - P . A D A M and H . B E C K E R 839

New Biologically Active Compounds from the Nematode-Trapping Fungus A r t h r o b o t r y s o l i -g o s p o r a Fresen. M . S T A D L E R , O. S T E R N E R , and H . A N K E 843

Biochemical Activities of Propolis Extracts. I. Standardization and Antioxidative Properties of Ethanolic and Aqueous Derivatives R. V O L P E R T and E . F . E L S T N E R 851

Biochemical Activities of Propolis Extracts. II. Photodynamic Activities R. V O L P E R T and E . F . E L S T N E R 858

Diurnal Fluctuations of Cocaine and Potential Precursors in Leaves of E r y t h r o x y l u m c o c a E. L . J O H N S O N 863

Contents

Transport of Oxidized Glutathione into Barley Vacuoles: Evidence for the Involvement of the Glutathione-S-Conjugate ATPase R. T O M M A S I N I , E . M A R T I N O I A , E . G R I L L , K . - J . D I E T Z , and N . A M R H E I N 867

Cu(II)-Induced Oxidation of Catechols, Ascorbate and ö-Phenylenediamine is Promoted by D N A W . A . P R Ü T Z 872

Interaction of Microsomal Cytochrome P-450s and N-Phenylcarbamates that Induce Flower-ing in A s p a r a g u s Seedlings F . T A N I G A K I , A . I S H I H A R A , K . Y O S H I D A , T. H A R A , M . S H I N O Z A K I , and H . I W A M U R A 879

Isoprenoid Biosynthesis and Stability in Develop-ing Green and Achlorophyllous Leaves of Rye {Seeale c e r e a l e L.) P. G Ö L Z and J. F E I E R A B E N D 886

Modification of Histidine Residues of Photosys­tem II by Diethyl Pyrocarbonate Inhibits the Electron Transfer between the Primary (Q A ) and Secondary (Q B) Quinone Acceptors U . H E G D E , S. P A D H Y E , L . K O V Ä C S , A . V O Z Ä R , and S. D E M E T E R 896

Seasonal Changes in the Activities of Apoplastic, Cytoplasmic, Ionically and Covalently Bound Isoperoxidases from Norway Spruce { P i c e a abies (L.) Karst.) Needles: A Comparison be­tween Three Collection Sites with Different A m -bient Ozone Concentrations D . I K E M E Y E R , P. B Ü T T N E R , and W . B A R Z 903

Studies of Components of the Thylakoid Mem­brane of Undamaged and Damaged Spruce Trees at Different Mountain Sites A . W I L D , P. S T R O B E L , a n d U . F L A M M E R S F E L D 911

XI

X - R a y Studies on Phospholipid Bilayers. X I V . In-teractions with the Antiarrhytmic Asocainol M . S U W A L S K Y , I. S Ä N C H E Z , and F . N E I R A 930

Antibiotic and Cytotoxic Activity of Brominated Compounds from the Marine Sponge V e r o n g i a a e r o p h o b a R. T E E Y A P A N T , H . J. W O E R D E N B A G , P. K R E I S , J. H A C K E R , V . W R A Y , L . W I T T E , and P. P R O K S C H

939

Differences between Thymic and Splenic Cells of the Rat: Biochemical and Physico-Chemical In-vestigations i n v i t r o on D N A Topoisomerase II - Inhibitors and Thiyl Radicals K . T E M P E L 946

Notes

The Biflavones of D i c r a n u m s c o p a r i u m (Dicrana-ceae) H . G E I G E R , A . V O I G T , H . - D . ZINSMEISTER, JOSE-A N T O N I O L Ö P E Z - S Ä E Z , M . - J O S E P E R E Z - A L O N S O , and A R T U R O V E L A S C O - N E G U E R U E L A 952

Further Analysis of Lipids from the Scent Gland Secretions of Dumeril's Ground Boa { A c r a n t o -p h i s d u m e r i l i Jan) J. T. S IMPSON, T H . R . S H A R P , W . F . W O O D , and P . J. W E L D O N 953

Flavonoid Aglycones as Glandulär Products in Rosa c e n t i f o l i a cv. muscosa and in Rubusphoeni-c o l a s i u s (In German) E. W O L L E N W E B E R , M . D Ö R R , and S. A R M ­BRUSTER 956

Occurrence of Vesicular-Arbuscular Mycorrhizal Fungi in Alberta, Canada S. M . B O Y E T C H K O and J. P. T E W A R I 923

Subject Index

Authors Index

959

979

Contents Z. Naturforsch. Vol. 48c, No. 9/10, September/October 1993

Invited Trends Article ^

Genetic Engineering of Disease and Pest Resist­ance in Plants: Present State of the Art G . S T R I T T M A T T E R and D . W E G E N E R 673

Original Communications

Epicuticular Leaf Waxes of the Hop ( H u m u l u s l u -p u l u s ) . Chemical Composition and Surface Structures P . - G . G Ü L Z , E. M Ü L L E R , T. H E R R M A N N , and P. L Ö S E L 689

Comparative F T I R and , 3 C C P / M A S N M R Spec-troscopic Investigations on Sporopollenin of Different Systematic Origins S. WILMESMEIER, ST . S T E U E R N A G E L , and R. W I E R M A N N 697

Quinolizidine Alkaloid Profiles of South Ameri­can Lupins: L u p i n u s l i n e a r i s and the L u p i n u s g i b e r t i a n u s Complex A . P L A N C H U E L O - R A V E L O , L . W I T T E , and M . W I N K 702

Fructose 1,6-Bisphosphatase in C h l o r e l l a k e s s l e r i Grown in Red or Blue Light N . G R O T J O H A N N 707

3a-Hydroxysteroid-5 ß-oxidoreductase in Tissue Cultures of D i g i t a l i s l a n a t a U . S T U H L E M M E R , W. H A U S S M A N N , F. M I L E K , W. K R E I S , and E. R E I N H A R D 713

Effect of Different Condensed Tannins on T r i c h o -p l u s i a n i Performance A . G O N Z Ä L E Z - C O L O M A , C H . S. W I S D O M , and P H . W. R U N D E L 722

6ß-Hydroxylation of 17a-Acetoxycortexone by F l a v o b a c t e r i u m G . SPASSOV and W.-R. A B R A H A M 727

Study of Canadian Propolis by G C - M S and H P L C C. G A R C I A - V I G U E R A , F . FERRERES, and F. A . T O M A S - B A R B E R Ä N 731

Glandulär Trichomes and the Volatiles Obtained by Steam Distillation of Q u e r c u s r o b u r Leaves R. E N G E L , P . -G. G Ü L Z , T H . H E R R M A N N , and A . N A H R S T E D T 736

The Unexpected Reduction of the Vinyl Group of Chlorophyll b by Sodium Borohydride in Meth-anolic Extracts of Maize Leaves and Its Inhibi­tion by 8-Hydroxyquinoline R. J. P O R R A , W . SCHÄFER, E. C M I E L , I. K A T H E ­DER, and H . SCHEER 745

Metabolie Reduction of Phenylpropanoid Com­pounds in Primary Leaves of Rye {Seeale ce-r e a l e L.) Leads to Increased U V - B Sensitivity of Photosynthesis S. R E U B E R , J. L E I T S C H , G . H . K R A U S E , and G . WEISSENBÖCK 749

Naturally Occurring Antidotes against Benzimid-azole Fungicides S. T A H A R A , Y . M A T S U K U R A , H . K A T S U T A , and J. M I Z U T A N I 757

Oxidative Stress of Crops Monitored by E P R H . B. S T E G M A N N , P. S C H U L E R , S T . W E S T P H A L , and E. W A G N E R 766

Molecular Modelling of the Interaction of Cyano-acrylate Inhibitors with Photosystem II. Part 1. The Effect of Hydrophobicity of Inhibitor Bind­ing S. P. M A C K A Y and P. J. O ' M A L L E Y 773

Molecular Modelling of the Interaction of Cyano-acrylate Inhibitors with Photosystem II. Part 2. The Effect of Stereochemistry of Inhibitor Bind­ing S. P. M A C K A Y and P. J. O ' M A L L E Y 782

Photoregulation of Nitrogen Metabolism and Pro­tein Accumulation in the Red Alga C o r a l l i n a e l o n g a t a Ellis et Soland F. L . F I G U E R O A 788

Translocation of Adenine Nucleotides in the Mi to -chondria of Male Sterile and Male Fertile Sorghum J. A R O R A , P. N A T H , and P. V . S A N E 795

Continued overleaf

ISSN 0939-5075

Contents Z. Naturforsch. Vol. 48 c, No. 9/10, September/October 1993

Complete Sequence of One Copy of the psbA Gene from the Thermophilic Cyanobacterium Syne-chococcus e l o n g a t u s R. K L O O S , E . STEVENS, and W . OETTMEIER 799

Linear Free Energy Relationships for N(7)-Substi-tuted Guanosines as Substrates of Calf Spleen Purine Nucleoside Phosphorylase. Possible Role of N(7)-Protonation as an Intermediary in Phosphorolysis A . B Z O W S K A , E . K U L I K O W S K A , and D . S H U G A R

803

Notes

Isolation and Properties of Trimethylamine N-Oxide/Dimethylsulfoxide Reductase from the Purple Bacterium R h o d o s p i r i l l u m r u b r u m P. S A J I T Z , J . - H . K L E M M E , H . - G . K O C H , and M . M O L I T O R 812

Inhibition of Dihydrofolate Reductase by Mofe-butazon E. S T R E H L , I. S C H N E I D E R , and E . F . E L S T N E R 815

Antitumoral Effect of Bleomycin + Dolomite Combination Treatment, in Mice Bearing Ehr­lich Ascites Carcinoma S. S C H E L L E R , W . K R O L , K . S K I R M U N T T , G . Z Y -D O W I C Z , and J . S H A N I 818

The Unexpected Reduction of the Vinyl Group of Chlorophyll b by Sodium Borohydride in Methanolic Extracts of Maize Leaves and Its Inhibition by 8-Hydroxyquinoline R. J. Porra*-*, W. Schäfer b , E. Cmiel c , Ingrid Katheder 3, and H . Scheera

a Botanisches Institut der Universität, D-80638 München b Max-Planck-Institut für Biochemie, D-82152 Martinsried c Institut für Physikalische Chemie, Technische Universität München, D-85748 Garching

Z. Naturforsch. 48c, 745-748 (1993); received May 14/July 9, 1993

Photosynthesis, Chlorophyll b, Reductive Extraction, Borohydride, 8-Hydroxyquinoline

Dür ing rapid extraction of Chlorophylls from maize leaves under reducing conditions with methanol containing N a B H 4 , Chlorophyll a remained unchanged but Chlorophyll b yielded [7-hydroxymethyl]-chlorophyll b. The 3-vinyl group of Chlorophyll b was also reduced forming significant amounts, up to 60%, of [3-ethyl]-[7-hydroxymethyl]-chlorophyll b. This was unex­pected since this reduction of the 3-vinyl group does not occur when isolated Chlorophyll b is treated in an identical manner with methanolic borohydride. The vinyl-group of Chlorophyll a is not reduced during the same extraction conditions suggesting that the presence of a formyl or hydroxyethyl group at C-7 is necessary. The presence of 8-hydroxyquinoline and N a B H 4 in equimolar (16.5 mM) concentrations strongly inhibits the reduction of the 3-vinyl group of Chlorophyll b in leaf extracts.

Introduction

The reduction of pure Chi a (Fig. 1; structure I) and Chi b (II), and of bacteriochlorophylls by N a B H 4 has been extensively studied [1-6]. In the plant Chlorophylls, the 7-formyl group of Ch i b is rapidly reduced within seconds to form [7-hy-droxymethyl]-Chl b (III): the reduction of the 131-oxo group of both Chls a and b is much slower tak-ing several hours at room temperature for comple-tion [6]. N o concomitant reduction of conjugated double bonds, a well known side-reaction of bo-rohydrides [7], has been reported so far. We have now found that as much as 60% of the Chi b was reduced to [3-ethyl]-[7-hydroxymethyl)-Chl b (IV) during extraction of the newly-formed Chloro­phylls in greening maize seedlings with methanol containing 16.5 mM N a B H 4 : this reductant was employed to convert the formyl group to a hy-droxymethyl group in biosynthetic l 8 0-incorpora-

A b b r e v i a t i o n s : Chi , Chlorophyll; DEAE-cellulose, di-ethylaminoethyl-cellulose; E D T A , ethylenediamine-tetra-acetic acid; N M R , nuclear magnetic resonance.

* P e r m a n e n t address: CSIRO-Divis ion of Plant Indus-try, G P O Box 1600, Canberra A C T 2601, Australia.

Reprint requests to Hugo Scheer, Botanisches Institut, Universität München, Menzinger Straße 67, D-80638 München, Germany.

Verlag der Zeitschrift für Naturforschung, D-72072 Tübingen 0939-5075/93/0900-0745 $ 01.30/0

tion studies which showed that the precursor of the formyl-group oxygen of Chi b is molecular oxygen [8].

R} R 2

C O O C 2 0 H 3 g COOCH3

R, R

=CH, I Chlöf - C H II Chi/? - C H III [7-hydroxymethyl]-

C h \ b IV [3-ethyl]-[7-hy-

droxymethyl]-Chlb - C H 2 - C H 3

V [7-hydroxymethyl]-[13'-hydroxy]-ChlÄ - C H = C H 2

C H 2

C H = C H 2

2

- C H 3

-CHÖ

- C H 2 O H

C H . O H

R 3 =0 =0

=0

=0

- O H

Fig. 1. The structures of Chlorophyll derivatives referred to in this paper. The I U B - I U P A C approved numbering System for tetrapyrroles [14] has been used with bracket [ ] nomenclature for substitutions. [7-hydroxymethyl]-Chl b (III) is identical to [7-hydroxymethyl]-Chl a but will be referred to here as a Chi b derivative to indicate its origin.

746 R. J. Porra et a l . • Vinyl Group Reduction of Chlorophyll b

Experimental

C h e m i c a l s

N a B H 4 and 8-hydroxyquinoline (technical grade) were obtained from Merck-Schuchardt, Darmstadt, Germany. N a B 2 H 4 was obtained from Cambridge Isotope Laboratories, Cambridge, M A . , U . S . A . Solvents and other chemicals were analytical reagent grade or purified by Standard techniques. DEAE-cellulose (DE 52), supplied by Whatman Laboratory Division, Maidstone, Eng­land, was prepared as a methanolic Suspension [9] which was then equilibrated with C H C 1 3 . Pure Chls a and b were prepared from green maize leaves as previously described [10].

O r g a n i s m s , g r o w t h a n d g r e e n i n g c o n d i t i o n s

Etiolated maize seedlings { Z e a mays hybrid var. Dekalb XL689) were grown in the dark at 18 °C for 18 days [11]. Etiolated leaves, excised from these seedlings, were then placed in H 2 0 and greened by illumination (50-60 j iE*m~ 2 -s _ 1 ) with white light (Philips T L D 18 W/84 tubes) for up to 26 hat 27 °C[12].

E x t r a c t i o n of C h l o r o p h y l l s f r o m g r e e n e d maize leaves

Leaves were finely chopped with scissors into a mortar and extracted by grinding with a pestle to a translucent pulp in a freshly prepared Solution (14 ml/g fresh wt. of leaves) of N a B H 4 (16.5 IHM) in methanol. Where specified in the text, the ex­traction was carried out with methanol containing either 16.5 mM N a B 2 H 4 or equimolar (16.5 mM) concentrations of N a B H 4 and 8-hydroxyquino­line. Grinding was completed in three minutes when an excess of glucose (150 mg) was added to remove unspent N a B H 4 . The clear methanolic su-pernatant contained Chi a and reduced derivatives o fCh lA.

C o l u m n c h r o m a t o g r a p h y of e x t r a c t e d C h l o r o p h y l l d e r i v a t i v e s

The Chlorophylls in the methanolic supernatant were transferred to diethylether by adding large volumes of saturated brine. The ether Solution was then washed with a further large volume of satu­rated brine, dried over solid N a C l and evaporated to dryness at approximately 40 °C under vacuum

in a rotary evaporator. The dry Chlorophylls were redissolved in a minimum quantity of C H C 1 3 and applied to a 4 x 65 mm high column of D E A E - c e l ­lulose equilibrated with C H C 1 3 (see above). The carotenoids and Chlorophylls were eluted with C H C 1 3 or CHCI3 containing either 2 or 10% meth­anol as described in the Resuits section. The elu-tion of the Chlorophylls was monitored by adsorp-tion spectroscopy of each ml of chlorophyll-con-taining eluant between 680-630 nm and 480-400 nm.

Spectroscopy

A l l absorption spectroscopy was performed in quartz cuvettes (1 ml capacity and 1 cm light path) using a Shimadzu U V 1202 Spectrophotometer. Mass spectra were obtained by fast atom bom-bardment ionization (m-nitro-benzylic alcohol matrix) with a M A T 900 Mass Spectrometer (Fin-nigan M A T , Bremen, Germany). ' H N M R spectra were recorded in 2 H 5 -pyridine with a model A M 360 M H z instrument (Bruker, Karlsruhe, Ger­many).

Resuits and Discussion

The C h l o r o p h y l l s present i n m e t h a n o l i c - N a B H 4

e x t r a c t s of g r e e n e d d a r k - g r o w n maize leaves as d e t e r m i n e d by c h r o m a t o g r a p h y a n d a b s o r p t i o n spectroscopy

When the pigments extracted in methanol con­taining 16.5 mM N a B H 4 were applied to a D E A E -cellulose column and developed with C H C 1 3 , a yel-low carotenoid fraction (A,m a x = 481, 454 and 426 nm) was followed by three blue Chlorophyll bands. The first contained Chi a (I) (A,m a x = 666 nm). The second, split band contained two pigments with similar absorption spectra (Fig. 2). The first one (A,m a x = 659 and 428 nm, spectrum 2) contained an unknown pigment, the second half (̂ max = 659 and 434 nm, spectrum 3), eluting only slowly with C H C 1 3 , and better with 2% methanol added, contained the expected C-7 1 reduction product of Chi by v i z . [7-hydroxymethyl]-Chl b (III) [cf 6]. The slowest-moving third blue band ( \ m a K = 653 and 415 nm), which eluted with C H C 1 3

containing 10% methanol, contained [7-hydroxy-methylHB'-hydroxyJ-Chl b (V), which is addi-tionally reduced at the 13 ]-oxo group [cf 6].

R. J. Porra et a l . • Vinyl Group Reduction of Chlorophyll b 141

A " [a.u]

/ / M i ' 659

i '

! 647̂ 1

f 1

/ V ' / / /

/ / 19 \ \ \ \ \ 1 \ \ \ \ \ \

400 500 X[nm] 600 700

Fig. 2. U V - V i s absorption spectra o f Chi b and its reduc­tion products in C h l o r o f o r m . Chi b (II) ( , spectrum 1); [3-cthyl]-[7-hydroxymethyl]-Chl b (IV) ( , spec­trum 2); and [7-hydroxymethyl]-Chl b (III) ( , spectrum 3). Peak positions of the main bands (nra) are indicated.

Consistent with previous studies [2-4, 6], we showed that pure Chi b (II), when treated rapidly with N a B H 4 in methanol, was all reduced to [7-hy-droxymethyl]-Chl b (III). We speculated that the additional band in extracts of leaves ( X m a x = 659 and 428 nm) was [3-ethyl]-[7-hydroxymethyl]-Chl b (IV) in which both the 3-vhiyl and 7-formyl groups were reduced: both the more rapid elution of the 428 nm-absorbing material from the column and the shift of the Soret peak by 6 nm to shorter wavelengths relative to that of [7-hydroxymethyl]-Chl b (III) are consistent with the reduction of a vinyl group. Assuming equal absorption coeffi-cients of III and IV at 659 nm, the latter comprised up to 60% of reduction products.

P r o d u c t i d e n t i f i c a t i o n

The ' H N M R spectrum of [7-hydroxymethyl]-Chl b (III) agreed with the structure. The 3-vinyl Signals occur at 8.06 (H x ) , 6.38 (H A ) and 6.06 ppm (H B ) . The O H signal, which is somewhat variable and known to be very solvent dependent, occurs at approximately 7.4 ppm and was identified by ] H / 2 H exchange with 2 H 2 0 . The mass spectrum showed both M + (base at 906 m/z) and ( M + H ) +

ions (base at 907 m/z) and the corresponding iso-tope peaks in an approximately 4:1 ratio. Upon reductive extraction with N a B 2 H 4 in methanol,

there was an increase in mass by 1 m/z, and a de-crease in the intensity of the 7 - C H 2 signal at 6.08 ppm by 50% in the ] H N M R spectrum.

In the ' H N M R spectrum of [3-ethyl]-[7-hy-droxymethyl]-Chl b (IV), the ring current was slightly reduced, and the vinyl Signals were no longer present. The mass spectrum showed again M + (base at 908 m/z) and ( M + H ) + ions (base at 909 m/z) in an approximately 4:1 ratio. Reductive extraction with N a B 2 H 4 in methanol increased the mass by 2 m/z indicating the incorporation of one 2 H atom into the hydroxymethyl group and also the 3-ethyl group.

I n v e s t i g a t i o n of t h e r e d u c t i o n of t h e v i n y l g r o u p of C h i b i n m e t h a n o l i c - b o r o h y d r i d e e x t r a c t s of maize leaves

Consideration of which components of the leaf extract initiated reduction of the vinyl group of Chi b led to investigation of the effect of 10, 20 and 40% water on the reaction of pure Chi b with methanolic N a B H 4 . It was always completely re­duced to the [7-hydroxymethyl]-Chl b (III) with Soret absorption at 434 nm: no [3-ethyl]-[7-hy-droxymethyl]-Chl b (IV), the "3,8-diethyl" deriva­tive with Soret absorption at 428 nm was detected.

To investigate the possibility that metal ions in the cell sap of the maize leaves are involved in the vinyl group reduction, the effect of three metal chelating agents on ethyl group formation during Chlorophyll extraction with methanolic N a B H 4

were investigated: 8-hydroxyquinoline, 8-hydroxy-quinoline-5-sulphonic acid (sodium salt) or E D T A (tetra-sodium salt) were added in equimolar con-centrations with the N a B H 4 . The two 8-hydroxy­quinoline derivatives, but not the E D T A , strongly inhibited the formation of the 428 nm-absorbing "3,8-diethyl" derivative (IV). However, when the concentration of the N a B H 4 exceeded that of the chelating agent, the "3,8-diethyl" Compound was again formed. Further, metal ions such as F e 2 + , M g 2 + , M n 2 + , C o 2 + , C u 2 + and C a 2 + did not induce the reduction of the vinyl group of pure Chi b. We concluded, therefore, that the inhibition is not due to chelation of an activating metal ion; rather, the 8-hydroxyquinoline derivatives, in equimolar pro-portions with N a B H 4 , were complexing the reduc-tant and modifying its reducing properties. Indeed the chelator prevented also reduction of the

748 R. J. Porra et a l . * Vinyl Group Reduction of Chlorophyll b

B' -oxo group. In the presence of equimolar chela-tor, the N a B H 4 formed a third slow-moving Chlo­rophyll band but it no longer had principal peaks of the doubly-reduced Chi b (V) (see above) but absorbed at 652.5, 612.5 and 431.5 nm with a Shoulder at 411 nm and moved considerably faster during chromatography.

C o n c l u d i n g r e m a r k s

The presence of a formyl- or hydroxymethyl-group at C-7 appears to be needed for the reduc­tion of the nearby 3-vinyl group as no evidence could be found for the reduction of the 3-vinyl group of Chi a during extraction. Since the 7-formyl and the 3-vinyl substituents of Chi b are close to each other and conjugated, a concerted mechanism is feasible. However, the identity of the agents in these extracts which catalyze the reduc­tion are unknown.

Because the above ' H N M R investigations have shown that 2 H from N a B 2 H 4 was incorporated not

only into the 7-hydroxymethyl group but also into the 3-ethyl group, it is clear that the majority (if not all) of the "3,8-diethyr derivative arose by borohydride reduction during extraction and was not originally present in the leaves. This fact, cou-pled with the failure to find any "3,8-diethyr de­rivative of Ch i a in extracts, Supports the view of Rebeiz et a l . [13] that the natural heterogeneity of Chls a and b in nature, which includes the so-called "3-mono-vinyl-" and "3,8-di-vinyl-" derivatives, does not extend to "3,8-diethyr forms.

A c k n o w l e d g e m e n ts

One of us (RJP) thanks the Deutscher Akade­mischer Austauschdienst ( D A A D ) for financial support during his visit to the Botanisches Institut der Universität München to complete this work. This work was also in part supported by the Deutsche Forschungsgemeinschaft (SFB 143, Pro­jekt A 9 ) and the CSIRO-Divis ion of Plant Indus-try, Australia.

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