a journal of zeitschrift für naturforschung c biosciences
TRANSCRIPT
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
Managing Editor:
H . Hausen, Tübingen
Council:
A . Butenandt, München M . Eigen, Gött ingen H . H a k e n , Stuttgart
Editors:
A . Hager, Tübingen K . Hahlbrock, Köln W . Hasselbach, Heidelberg P. Karlson, Marburg J . Kle in , Tübingen J . St. Schell, Köln
Associate Editors:
Information for Contributors
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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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