B o t a n i c a A c t a Beruhte der Deutschen Botanisehen Gesellschaft • Journal of the German Botanical Society

E d i t o r s E4953F

Ulrich Lüttge, Darmstadt (Editor-in-Chief) Eberhard Schnepf, Heidelberg Andre Läuchli, Davis, California Toshiyuki Nagata, Tokyo i

Volume 105 February 1992 Page 1-80

Contents

1 Editorial A. Läuchli

Biotechnology

3 Osmotic Biosensors. How to Use a Characean Internode for Measuring the Alcohol Content ofBeer M. Rüdinger, P. Hierüng, and E. Steudle

Plant Biochemistry

13 Autumn Leaves of Ginkgo biloba L.: Optical Properties, Pig­ments and Optical Brighteners Ph. Matile, Barbara M.-P. Flach, and B. M. Eller

18 Production of the Phyto-alexin Glyceollin I by Soybean Roots in Response to Symbiotic and Pathogenic Infection Petra E. Schmidt, M. Parniske, and D. Werner

26 Structure of Protein Bodies and Elemental Composition of Phytin from Dry Germ of Maize (Zea mays L.)

M. Mikus, M. Bobäk, and A. Lux

Salinity S t r e s s 34 The Irreversible C 3to CAM Shift in Well-watered and Salt-stressed Plants ofMesembryan-themum crystallinum is under Strict Ontogenetic Control W.Herppich, Margaretha Herp-pich, and D. J. von Willert

41 Changes in the Ultrastruc-ture oiPrasiola crispa ssp. ant-arctica under Salinity Stress A. Jacob, H. Lehmann, G. 0. Kirst, and Chr. Wiencke

Photophysiology

47 Isolation and Characteriza-tion of the Putative Photorecep-tor for Phototaxis in Amoebae of the Cellular Slime Mold, Dicty-ostelium discoideum H.-P. Vornlocher and D.-P. Hader

55 Immunolocalization ofCyto-solic Phytochrome in the Green Mg&Mougeotia Christel Hanstein, F. Grolig, and G. Wagner

Developmental Biology

63 Okadaic Acid as a Probe to Analyse the Cell Cycle Progres­sion in Plant Cells S. Hasezawa and T. Nagata

70 Developmental Changes in the Anatomy of the Sugarcane Stern in Relation to Phloem Unloading and Sucrose Storage Karin Ruth Jacobsen, D. G. Fisher, A. Maretzki, and P. H. Moore

A1 Mitteilungen des Vorstandes der DBG

§Thieme Georg Thieme Verlag Stuttgart • New York

Thieme Medical Publishers, Inc., New York

Bot. Acta ISSN 0932-8629

This Journal is indexed in Current Contents/Life Sciences, Current Contents/Agriculture, Biology & Environmental Sciences, Biosis and CABS

BOACEJ(l), 1-80 (1992)

Instructions for Authors Scope o f t h e J o u r n a l BOTANICA ACTA is an international Journal covering all Gelds of plant science. It aims at bridging the gaps between the different fields of botany.

Submission of papers BOTANICA ACTA accepts

- füll length papers (up to 8 printed pages) - rapid Communications (up to 2 printed pages) - concise review articles (commissioned only) - Botanica Acuta (comments on actual problems)

from all fields of the plant science written in English or Ger­man (review articles in English only). Contributions by non-members are welcome. All manuscripts (in triplicate), except from North America and Japan, to be sent to: Prof. Dr. U. Lüttge Editor-in-Chief of Botanica Acta Institut für Botanik, Technische Hochschule Darmstadt Schnittspahnstrasse 3 D-6100 Darmstadt, FRG or

Prof. Dr. E. Schnepf Zellenlehre, Universität Heidelberg Im Neuenheimer Feld 230 D-6900 Heidelberg 1, FRG

From North America to

Prof. Dr. A. Läuchli Dept. of Land, Air and Water Resources University of California Hoagland Hall Davis CA 95616 USA

From Japan to Prof. Dr. T. Nagata Dept. of Biology Facultyof Science Laboratory of Plant Physiology University of Tokyo. Hongo 113 Tokyo Japan

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The following sections cover the usual contents: Introduc-tion, Materials and Methods, Results, Discussion, Acknowl-

edgements, References (see below), Tables (see below), Fig-ure legends (see below), Illustrations (see below). In Materials and Methods, Results and Discussion subhead-ings are possible. If it is chosen to combine sections Results and Discussion, an additional section Conclusions can be added, but this must be brief.

Rapid Communications Title page and Key words as for füll lengthpapers. For Com­munications written in German give an English abstract only. Follow the flow of reasoning as in Introduction - Ma­terials and Methods - Results - Discussion, leaving out the headings.

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- three or more authors: Use name of first author with: et al.

Two or more references in parentheses should be arranged according to year of publication or in alphabetical order, but consistently throughout your paper. List of references

References should be given at the end of the paper in strict alphabetic order, i.e. first by name of the first author, then by name of the second author if the first author is identical for more references, then by name of the third author if the first two authors are identical, and so on. Order should be by year of publication if all authors and their sequence are identical for more references. If the first author and the year of publication are the same for more references, small letters behind the year must be used both in quotations in the text and in the list of references to allow unambiguous allocation of each reference. Punctuation, number and sequence of items required for the references t a i a n f y » ™ thgfr»iw.n„gexamples;

[ B- - - l e A I Staatsbibliothek 1 l München j

note that in all cases füll titles and first and last page num­ber are required.

A r t i c l e s i n Journals w i t h one, t w o o r m o r e a u t h o r s : Lüttge, U. - Instructions for authors. Botanica Acta 101

(1988), 48-52. Lüttge, U. and Schnepf, E. - Instructions for authors.

Botanica Acta 101 (1988), 48-52. Lüttge, U., Schnepf, E., and Nultsch, W. - Instructions

for authors. Botanica Acta 101 (1988), 48-52. A r t i c l e s i n m u l t i a u t h o r books: Lüttge, U. and Schnepf, E. - Instructions for authors.

In: W. Nultsch, ed., How to write a manuscript, pp. 48-52. Thieme Verlag, Stuttgart • New York,

1988.

Books: Lüttge, U. and Schnepf, E. - How to write a manuscript.

48-52 p. Thieme Verlag, Stuttgart • New York, 1988.

U n i t s BOTANICA ACTA will follow the system of SI units (Systeme International d'Unitees).

Basic units are as follows length: meter m mass: kilogram kg time: second s

Other SI units are energy: Joule J pressure: Pascal Pa radioactivity: Becquerel Bq Use the following prefixes to names of units

gigadO9) G microdO - 6) u. mega(106) M nano(10-9) n kilo(103) k pico(10-12) p millidO-3) m femto(10"15) f

Usage T i m e : Note, that for indicating longer periods of time in-stead of prefixes with the SI unit s the following units should be used: minute min hour h day d year aoryr

C o n c e n t r a t i o n : In SI units concentrations are mmol m"3, mol m - 3 , kmol m - 3 . BOTANICA ACTA also accepts how­ever: |iM, mM, M, etc. Note that (Amol, mmol, m o l . . . are amounts, whereas uM, mM, M . . . are concentrations. P r e s s u r e : bar is accepted in addition to Pa but avoid atm. E x p o n e n t i a l s of l e n g t h : area: cm2 is accepted (but not dm2) volume: cm 3 (cubic centimeter), 1 (liter), ml (milliliter), ul (microliter) are accepted.

L i g h t i n t e n s i t i e s : Use mol photons per unit of area per unit of time for the fluence rate of radiation whenever possible, e.g. Umol photons m 2 s _ 1 or (iE m 2s J . l|xE = 1 microeinst<"n = 1 (imol photons. Watts per area (Wm~2) is also acceptable. But avoiu »ux whenever possible and if unavoidable only use it together with a description including trade name of the lamps. Within a given paper units chosen must be uniform.

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S c i e n t i f i c n a m e s

Scientific names should be cited in their complete form when first mentioned with genus - species - authority -cultivar ( c v . ) or subspecies ( s s p . ) where appropriate. Latin names should be underlined or typed in i t a l i c s . Subse-quently the generic names should be abbreviated, but avoid confusion; e.g. use A . comosus if the only genus with a first letter A in your paper is A n a n a s ; but use unambiguous abbreviations if you have more than one genus with the same first letter, e.g. A n a n a s comosus, Aechmea n u d i c a u -l i s = An. comosus, Ae. n u d i c a u l i s , etc. Common names of organisms, if used, must be accompanied by the correct sci­entific name when first mentioned. For rare or exotic genera it may be useful to give the name of the family and/ or higher taxon in brackets when first mentioned.

Botanica Acta Berichte der Deutschen Botanischen Gesellschaft

Journal of the German Botanical Society

1992

Georg Thieme Verlag Rüdigerstraße 14 D-7000 Stuttgart 30

Postfach 104853 D-7000 Stuttgart 10

Editors Ulrich Lüttge, Darmstadt

(Editor-in-Chief) Eberhard Schnepf, Heidelberg

(Co-Editor) Andre Läuchli, Davis, California

(Editor) Toshiyuki Nagata, Tokyo

(Editor)

Associate Editors E. Beck, Bayreuth H.-D. Behnke, Heidelberg A. J. E. van Bei, Utrecht F. -W. Bentrup, Salzburg T. E. Boller, Basel H. Bothe, Köln D. Cosgrove, University Park/PA C. Dumas, Villeurbanne W. Eschrich, Göttingen G. K. Gottsberger, Gießen H. Griffiths, Newcastle upon Tyne R. Herrmann, München R. L. Jefferies, Toronto J. W. Kadereit, Mainz C. M. Karssen, Wageningen H. Kauss, Kaiserslautern M. Kluge, Darmstadt P. Leins, Heidelberg H. K. Lichtenthaler, Karlsruhe M. Luckner, Halle P. Matile, Zürich E. Medina, Caracas F. Meins, Basel M. Melkonian, Köln I. de Michelis, Messina W. Morawetz, Wien F. Oberwinkler, Tübingen C. B. Osmond, Canberra B. Parthier, Halle D. G. Robinson, Göttingen F. E. Round, Bristol R. Scheibe, Osnabrück H. Schnabl, Bonn H. Senger, Marburg J. A. C. Smith, Oxford L. Taiz, Santa Cruz/CA I. P. Ting, Riverside/CA A. J. Trevawas, Edinburgh J. Wattendorff, Fribourg E. Weiler, Bochum L. Willmitzer, Berlin 0. Wilmanns, Freiburg

661 Figures 103 Tables

Thieme Medical Publishers, Inc. 381 Park Avenue South New York, NY10016

Some of product names, patents and registered designs refered to are in fact registered trademarks or proprie-tary names even though specific reference to this fact is not al-ways made in the text. Therefore, the appearance of a name without designation as proprietary is not to be constructed as a representation by the publisher that it is in the public domain.

All rights, including the rights of publication, distribution and sales, as well the right of translation, are re-served. No part of this work covered by the Copyrights hereon may be reproduced or copied in any form or any means -graphic, electronic or mechanical including photocopying, re-cording, taping, or Information and retrieval Systems-without written permission of the publisher.

© 1992 Georg Thieme Verlag, Rüdigerstraße 14, D-7000 Stutt­gart 30 - Printed in Germany

Contents

No. 1 (February 1992) = Page 1 - 80 No.2 (April 1992) = Page 8 1 - 1 3 2 No. 3 (June 1992) = Page 1 3 3 - 226 No. 4 (August 1992) = Page 2 2 7 - 342 No. 5 (October 1992) = Page 3 4 3 - 394 No. 6 (December 1992) = Page 3 9 5 - 4 6 8

337 Phenotypic Sirnilarity and Genetic Relationship Among Populations of M i c r o s e r i s b i g e l o v i i (Asteraceae: Lactuceae) K. Bachmann

370 Peroxidase Catalyzed Dimerization and Demethyl-ation of Protoberberine Alkaloids W. Bauer, R. Stadler, and M. H. Zenk

260 Selective Reconstitution of the Tonoplast H + -ATPase of the Crassulacean-Acid Metabolism Plant K a l a n c h o e d a i g r e m o n t i a n a Barbara Behre, R. Ratajczak, and U. Lüttge

427 Apoplastic Transport through the Fungal Sheath of P i n u s s y l v e s t r i s / S u i l l u s b o v i n u s Ectomycorrhizae P. Behrmann and W. Heyser

152 Growth Characteristics and Elicitor-induced Re-actions of Photosynthetically Active and Hetero-trophic Cell Suspension Cultures of L y c o p e r s i c o n p e r u v i a n u m (Mill.) Andrea Beimen, L. Witte, and W. Barz

382 Temperature Dependency of Circadian Period in a Single C e M f A c e t a b u l a r i a ) S. Berger, J. Dirk, L. von Lindern, D. Wolff, and D. M ergenhagen

285 Experimental and Modelling Studies of Competition for Light in Roadside Grasses W. Beyschlag, R. J. Ryel, and I. Ulimann

146 Hydroxycinnamic Acid Transferases in the Bio-synthesis of Acylated Betacyanins: Purification and Characterization from Cell Cultures of C h e n o p o d i u m r u b r u m and Occurrence i n Some Other Members of the Caryophyllales Maria Bokern, Susanne Heuer, and D. Strack

273 K + and C l " Conductance of A r a b i d o p s i s t h a l i a n a Plas­ma Membrane at Depolarized Voltages Raffaella Cerana and Roberta Colombo

161 Cyanobacterial Picoplankton from Lake Constance II. Classification of Isolates by Cell Morphology and Pigment Composition Anneliese Ernst, G. Sandmann, Christine Postius, Susanne Brass, U. Kenter, and P. Böger

323 Towards a Revision of the Systematics of the Genus C h l a m y d o m o n a s (Chlorophyta) 1. C h l a m y d o m o n a s a p p l a n a t a Pringsheim H. Ettl and U. G. Schlösser

227 Conservation and Structural Divergence of Organellar D N A and Gene Expression i n Non-Photosynthetic Plastids Düring Ontogenetic Differentiation and Phylogenetic Adaptation J. Feierabend

348 Changes i n the Constitution of Thylakoid Membranes i n Spruce Needles Düring an Open-top Chamber Ex­periment U. Flammersfeld and A. Wild

140 Cryopreservation of Embryogenic Suspension Cul­tures of Barley ( H o r d e u m v u l g a r e L.) A. Fretz, A. Jahne, and H. Lörz

313 Selection of Microspore Derived Embryogenic Struc-tures i n Maize Related to Transformation Potential by Microinjection A. Gaillard, E. Matthys-Rochon, and C. Dumas

292 Nectar Secretion Pattern and Removal Effects in Six Argentinean Pitcairnioideae (Bromeliaceae) L. Galetto and L. Bernardello

213 Homologies i n the Structural Genes Coding for Sul-phate Reducing Enzymes from Higher Plants and Prokaryotes G. Gisselmann, Andrea Niehaus, and J. D. Schwenn

395 Evidence for a G-Protein Regulated Adenylate Cyclase and a Ca 2 + /Calmodulin Controlled Phosphodiesterase i n the Phytoflagellate C h l o r o g o n i u m R. Gromes and K. Zetsche

223 The Effect of Pressurized Gas Transport on Nutrient Uptake Düring Hypoxia of Alder Roots W. Grosse and Doris Meyer

168 Influences of Blue and Red Light on the Photosyn-thetic Apparatus of C h l o r e l l a k e s s l e r i - Alterations i n Pigment-Protein Complexes Regina Grotjohann, Myung-Sook Rho, and W. Kowallik

407 Studies on Sporopollenin Biosynthesis i n T u l i p a Anthers III. Incorporation of Specifically Labeled 1 4C-Phenylalanine in Comparison to Other Precursors S. Gubatz and R. Wiermann

278 Photoinhibition of Photosynthesis and its Recovery in Red Algae D. Hanelt, K. Huppertz, and W. Nultsch

55 Immunolocalization of Cytosolic Phytochrome in the Green Alga M o u g e o t i a Christel Hanstein, F. Grolig, and G. Wagner

63 Okadaic Acid as a Probe to Analyse the Cell Cycle Progression i n Plant Cells S. Hasezawa and T. Nagata

266 Travelling Pattern of Acidity in the Epidermis of Tulip Leaves Z. Hejnowicz

B o t A c t a 105 (1992)

457 A u s t r o p e l t u m g l a r e o s u m gen. et sp. nov., a New Liehen from Moimtain Plateaux in Tasmania and New Zealand A . Henssen, H . Döring, and G. Kantvilas

34 The Irreversible C 3 to C A M Shift in Well-watered and Salt-stressed Plants of M e s e m b r y a n t h e m u m c r y s -t a l l i n u m is under Strict Ontogenetic Control W . Herppich, Margaretha Herppich, and D. J . v o n W ü l e r t

387 Morphology o f H e l i o t r o p i u m (Boraginaceae) Dispersal Units H . H. Hilger

121 The Control of Lateral Root Development in Cultured Pea Seedhngs II. Root Fasciation Induced by Auxin Inhibitors M a u d A . W . Hinchee and T h . L. Rost

127 The Control of Lateral Root Development in Cultured Pea Seedlings III. Spacing Intervals M a u d A . W. Hinchee and T h . L. Rost

441 Altered Pterin Patterns in Photoreceptor Mutants of P h y c o m y c e s b l a k e s l e e a n u s with Defective m a d l Gene N . Hohl , P. Galland, H . Senger, and A . P. Eslava

104 Immunological Evidence of Connexin-like Proteins in the Plasma Membrane of V i c i a f a b a L. Carola Hunte, Heide Schnabl, 0. Traub, K. Willecke, and Margot Schulz

116 External pH Modifies the Intracellular pH and the Mode of Photosynthetic C0 2-Assimilation in Photo-autotrophic Cell Suspension Cultures o f C h e n o p o d i u m r u b r u m L . W. Hüsemann, R. Callies, and D. Leibfritz

41 Changes in the Ultrastructure o i P r a s i o l a c r i s p a ssp. a n t a r e t i c a under Salinity Stress A . Jacob, H . Lehmann, G. 0. Kr ist , and Chr. Wiencke

70 Developmental Changes in the Anatomy of the Sug-arcane Stern in Relation to Phloem Unloading and Suc-rose Storage K a r i n Ruth Jacobsen, D. G. Fisher, A . Maretzki , and P. H . Moore

400 Immunofluorescence Study of Microtubule Organ­ization in Some Polarized Cell Types of Selected Brown Algae Chr. I. Katsaros

355 The Specific Mineral Metabolism of Selected Plant Species and its Ecological Implications H . K i n z e l and Ilse Lechner

174 y-Glutamylcysteinylserine - A New Homologue of Glu-tathione in Plants of the Family Poaceae S. Klapheck, B. Chrost, J . Starke, and H . Z immermann

414 Quantification of Visible Structural Changes of the VoVi-ATPase in the Leaf-tonoplast of M e s e m b r y ­a n t h e m u m c r y s t a l l i n u m by Freeze-fracture Replicas Prepared Düring the C3-Photosynthesis to C A M Transition Rebecca K l i n k and U. Lüttge

343 Geosiphon p y r i f o r m e , an Endosymbiotic Consortium of a Fungus and a Cyanobacterium ( N o s t o c ) , Fixes N i -trogen M . Kluge, D. Mollenhauer, R. Mollenhauer, and R. Kape

246 The Role of the Epidermis in the Control of Elongation Growth in Sterns and Coleoptiles U. Kutschera

435 Investigations of the Blue-green Fluorescence Emis­sion of Plant Leaves M . Lang, P. Siffel , Zuzana Braunovä, and H . K. Lichtenthaler

319 I n V i t r o Ovary Culture of some Apogon Garden Irises { I r i s p s e u d a c o r u s L., /. setosa Pall . , /. v e r s i c o l o r L.) Genevieve Laubl in and M . Cappadocia

1 Editorial A . Läuchli

97 Ferric Ion and Oxygen Reduction at the Surface of Protoplasts and Cells o f A c e r p s e u d o p l a t a n u s F. M a c r i , E. Braidot , E. Petrussa, M . Zancani , and A . Vianello

362 A Comparative Study of Internal Light Environment in Bifacial Leaves of Different Plants A . Martinez v. Remisowsky, J . H . McClendon, and L. Fukshansky

232 Transport Processes i n Vacuoles of Higher Plants E. Mart inoia

13 Autumn Leaves of G i n k g o b i l o b a L.: Optical Prop­erties, Pigments and Optical Brighteners P h . Mati le , B a r b a r a M.-P . F lach , and B. M . El ler

26 Structure of Protein Bodies and Elemental Compo-sition of Phytin from Dry Germ of Maize [ Z e a m a y s L.) M . Miku§, M . Bobäk, and A . L u x

81 Control of Nitrate Reductase and Nitrite Reductase Gene Expression by Light, Nitrate and a Plastidic Factor H . Mohr , A . Neininger, and B. Seith

133 Somatic Hybridization of Dihaploid Potato Protoplasts as a Tool for Potato Breeding C. Möllers and G. Wenzel

421 Bafilomycin Inhibits Vacuolar pH Regulation in a Fresh Water Charophyte, C h a r a c o r a l l i n a Y. Okazaki , M . Tazawa, Y . Moriyama, and N. Iwasaki

B o t . A c t a 105 (1992)

206 Enzymatic Pathways for the Consumption of Carbonyl Sulphide (COS) by Higher Plants G. Protoschill-Krebs and J. Kesselmeier

253 Confocal pH Topography in Plant Cells - Acidic Layers in the Peripheral Cytoplasm and the Apoplast W. Koos

3 Osmotic Biosensors. How to Use a Characean In­ternode for Measuring the Alcohol Content of Beer M. Rüdinger, P. Hierling, and E. Steudle

449 Lichenizing Rhizomorphs and Thallus Development in the Squamulose Liehen A s p i c i l i a c r e s p i a n a Rico i n e d . (Lecanorales, Ascomycetes) W. B. Sanders and V. J. Rico

18 Produktion of the Phytoalexin Glyceollin I by Soybean Roots in Response to Symbiotic and Pathogenic In-fection Petra E. Schmidt, M. Parniske, and D. Werner

367 I n V i t r o and i n V i v o Phosphorylation of Stomatal Phosphoenolpyruvate Carboxylase from V i c i a f a b a L. Heide Schnabl, M. Denecke, and M. Schulz

345 Leaf Surface Microflora May Significantly Affect Studies on Foliar Uptake of Chemicals L. Schreiber and J. Schönherr

111 Immunofluorescent Localization of a Connexin 26-like Protein at the Surface of Mesophyll Protoplasts from V i c i a f a b a L. and H e l i a n t h u s a n n u u s L. Margot Schulz, 0. Traub, Mona Knop, K. Willecke, and Heide Schnabl

375 The Complex-Heterozygotes o f O e n o t h e r a g r a n d i f l o r a L'Her. E. Schumacher, E. Steiner, and W. Stubbe

180 Changes in Sulfur Metabolism Düring Needle Devel­opment of Norway Spruce R. Schupp and H. Rennenberg

197 Enzymatic Characteristics of UDP-sulfoquinovose: Diacylglycerol Sulfoquinovosyltransferase from Chlo-roplast Envelopes U. Seifert and E. Heinz

190 Adenosine 5'-Phosphosulfate Sulfotransferase from Norway Spruce: Biochemical and Physiological Prop­erties Marianne Suter, A. Tschanz, and Chr. Brunold

219 Secondary Carotenoids of E r e m o s p h a e r a v i r i d i s De Bary (Chlorophyceae) Under Nitrogen Deficiency B. Vechtel, U . Kahmann, and H. G. Büppel

90 Immunolocalization and Western Blot Analysis of Nitrogenase in O s c i l l a t o r i a l i m o s a Düring a Light-dark Cycle M. Villbrandt, L. J. Stal, B. Bergman, and W. E. Krumbein

306 Effects of Exogenously Supplied Ammonium on Root Development of Scots Pine ( P i n u s s y l v e s t r i s L.) Seedlings P. Vollbrecht and Helga I. Kasemir

331 Nuclear DNA Polymorphisms Among Strains of M i c r o s e r i s b i g e l o v i i (Asteraceae: Lactuceae) Ampli-fied from Arbitrary Primers A. W. von Heusden and K. Bachmann

47 Isolation and Characterization of the Putative Photo-reeeptor for Phototaxis in Amoebae of the Cellular Slime Mold, D i c t y o s t e l i u m d i s e o i d e u m H.-P. Vornlocher and D.-P. Hader

300 Lipophilic Phenolics from the Leaves of E m p e t r u m n i g r u m - Chemical Structures and Exudate Loca­lization E. Wollenweber, M. Dörr, R. Stelzer, and F. J. Arriaga-Giner

A 7 Mitgliederliste A 1 Mitteilungen des Vorstandes der DBG A 5 Mitteilungen des Vorstandes der DBG A21 Mitteilungen des Vorstandes der DBG A23 Mitteilungen des Vorstandes der DBG A27 Mitteilungen des Vorstandes der DBG

Author's Index

A

Arriaga-Giner, F. J . 300

B Bachmann, K. 331,337 B a r z . W . 152 Bauer, W . 370 Behre, Barbara 260 Behrmann, P. 427 Beiman, Andrea 152 Berger, S. 382 Bergman, B. 90 Bernadello, L. 292 Beyschlag, W . 285 B o b ä k , M . 26 Böger, P. 161 Bokern, Maria 146 Braidot, E. 97 Brass, Susanne 161 Braunovä, Zuzana 435 Brunold, Chr. 190

C Callies.R. 116 Cappadocia, M . 319 Cerana, Raffaella 273 Chrost ,B . 174 Colombo, Roberta 273

D Denecke, M . 367 Dirk, J. 382 Döring, H. 457 Dörr, M . 300 Dumas, C. 313

E Eller, B. M . 13 Ernst, Anneliese 161 Eslava, A . P. 441 Ettl, H. 323

F Feierabend, J. 227 Fisher, D. G. 70 Flach, Barbara M.-P. 13 Flammersfeld, U. 348 F r e t z . A . 140 Fukshansky, L. 362

G Gai l lard ,A. 313 Galetto, L. 292 Galland, P. 441 Gisselmann, G. 213 Grolig, F. 55 Gromes, R. 395 Grosse, W. 223

Grotjohann, Regina 168 Gubatz, S. 407

H Hader, D.-P. 47 Hanelt, D. 278 Hanstein, Christel 55 Hasezawa, S. 63 Heinz, E. 197 Hejnowicz, Z. 266 Henssen, A . 457 Herppich, Margaretha 34 Herppich, W. 34 Heuer, Susanne 146 Heyser,W. 427 Hierling, P. 3 H i l g e r . H . H . 387 Hinchee, Maud A . W.

121,127 H o h l , N . 441 Hunte, Carola 104 Huppertz, K. 278 Hüsemann.W. 116

Iwasaki, N. 421

J Jacob, A . 41 Jacobsen, Karin Ruth 70 Jahne, A . 140

K Kahmann, U. 219 Kantvilas, G. 457 Kape, R. 343 Kasemir, Helga I. 306 Katsaros, Chr. 400 Kenter, U. 161 Kesselmeier, J. 206 Kinzel , H. 355 K i r s t . G . O . 41 Klapheck, S. 174 Klink, Rebecca 414 Kluge, M . 343 Knop, Mona 111 Kowalik, W. 168 Krumbein, W. E. 90 Kutschera, U. 246

L Lang, M . 435 Laublin, Genevieve 319 Läuchli, A . 1 Lechner, Ilse 355 Lehmann, H. 41 Leibfritz, D. 116 Lichtenthaler, H. K. 435

L ö r z . H . 140 Lüttge, U. 260,414 Lux, A . 26

M Macri , F. 97 Maretzki, A . 70 Martinez v. Remisowsky, A .

362 Martinoia, E. 232 Matile, Ph. 13 Matthys-Rochon, E. 313 M c C l e n d o n . J . H . 362 Mergenhagen, D. 382 Meyer, Doris 223 M i k u s . M . 26 M o h r . H . 81 Mollenhauer, D. 343 Mollenhauer, R. 343 Möllers, C. 133 Moore, P. H. 70 Moriyama, Y. 421

N Nagata, T. 63 Neininger, A . 81 Niehaus, Andrea 213 Nultsch, W . 278

0

Okazaki .Y. 421

P Parniske, M . 18 Petrussa, E. 97 Postius, Christine 161 Protoschill-Krebs, G. 206

R Ratajczak, R. 260 Rennenberg, H . 180 Rho, Myung-Sook 168 R i c o , V . J . 449 Roos.W. 253 Rost, T h . L. 121,127 Rüdinger, M . 3 R u p p e l , H . G . 219 R y e l , R . J . 285

S Sanders, W. B. 449 Sandmann, G. 161 Schlösser, G. 323 Schmidt, Petra E. 18 Schnabl, Heide 1 0 4 , 1 1 1 ,

367 Schönherr, J. 345 Schreiber, L. 345

Schulz, Margot 1 0 4 , 1 1 1 , 367

Schumacher, E. 375 Schupp, R. 180 S c h w e n n , J . D . 213 Seifert, U . 197 Seith, B. 81 Senger, H . 441 Sififel,P. 435 Stadler, R. 370 S t a l , L . J . 90 Starke, J . 174 Steiner, E. 375 Stelzer, R. 300 Steudle, E. 3 Strack, D. 146 Stubbe,W. 375 Suter, Marianne 190

T Tazama, M . 421 T r a u b . O . 104,111 T s c h a n z , A . 190

U

Ulimann, I. 285

V van Heusden, A . W . 331 Vechtel, B. 219 Vianello, A . 97 Villbrandt, M . 90 Vollbrecht, P. 306 von Lindern, L. 382 v o n W i l l e r t , D . J . 34 Vornlocher, H.-P. 47

W Wagner, G. 55 Wenzel, G. 133 Werner, D. 18 Wiencke, Chr. 41 Wiermann, R. 407 W i l d , A . 348 Willecke, K. 104,111 Witte, L. 152 Wolff ,D. 382 Wollenweber, E. 300

Z Zancani, M . 97 Z e n k , M . H . 370 Zetsche, K. 395 Zimmermann, H . 174

Index of Names of

A A b r o m e i t i e l l a b r e v i f o l i a 2 9 2 - l o r e n t z i a n a 292 A c a c i a n e o v e r n i c o s a 304 A c e r p s e u d o p l a t a n u s 97 ,119 , 227,

236 A c e t a b u l a r i a a c e t a b u l u m 382,422 - m e d i t e r r a n e a 382 - species 382 A d i a n t u m species 59 A g r o s t i s g i g a n t e a 178 Aizoaceae 149 Algae 4 1 , 5 5 , 1 6 1 , 1 6 8 , 2 1 9 , 2 7 8 ,

323, 395,400 A l n u s g l u t i n o s a 223 A l p i n i a speciosa 304 A m a n i t a m u s c a r i a 146 Amaranthaceae 149,355 A m a r a n t h u s l i v i d u s 355 A n a b a e n a species 95 - v a r i a b i l i s 95 A n a c y s t i s n i d u l a n s 161,214 A n k i s t r o d e s m u s b r a u n i i 221 Annonaceae 304 A p h a n a c a p s a species 214 A r a b i d o p s i s t h a l i a n a 214,273, 395 Ascomycetes 449,457 A s p e r g i l l u s n i g e r 155 - o r y z a e 408 A s p e r u g o species 391 A s p i c i l i a c i n e r e a 450 - c o n t o r t a 449 - c r e s p i a n a 449 A s t a s i a l o n g a 228 - species 227 Asteraceae 304,355 Asteraceae: Lactuceae 331, 337 A t r i p l e x species 238 A u s t r o p e l t u m g l a r e o s u m 457 A v e n a s a t i v a 247 - species 55

B Basidiomycetes 427 B e r b e r i s s t o l o n i f e r a 370 B e t a species 141 - v u l g a r i s 147 Boraginaceae 387 Boraginaceae-Heliotropioideae 387 Boraginoideae 391 Boraginoideae-Cynoglosseae 391 Boraginoideae-Eritricheae 391 B o t r y o c o c c u s b r a u n i i 221 B r a d y r h i z o b i u m j a p o n i c u m 18 Bromeliaceae 292 Bromelioideae 292 B r y o p h y l l u m f e d t s c h e n k o i 367 B u l b o p h y l l u m g y m n o p u s 304 B y r o n i a d i o i c a 355

C C a l l u n a v u l g a r i s 300 C a m p s i s r a d i c a n s 295 C a r p h a c u r v a t a 459

Caryophyllaceae 355,359 Caryophyllales 146 C a t a l p a b i g n o n i o i d e s 362 C a t h a r a n t h u s r o s e u s 237 - species 237

C a y r a t i a j a p o n i c a 295 C e b a l l o s i a f r u t i c o s a 387 - species 387 Celosia a r g e n t e a 147 C e r a t o p t e r i s species 127 C e r i n t h e species 387 C h a r a a u s t r a l i s 235 - c o r a l l i n a 3,421 Characeae 3,421 Chenopodiaceae 149,359 C h e n o p o d i u m r u b r u m 116,146 C h l a m y d o m o n a s acidophüa 324 - a g g r e g a t a 323 - a k i n e t o s 323 - a p p l a n a t a 323 - b i c o n v e x a 329 - b i p a r t i t a 329 - c a l l o s a 324 - c u l l e u s 324 - d e b a r y a n a 324 - dysosmos 324 - g l o e o p h i l a 327 - g r a n d i s t i g m a 327 - h u m i c o l a 323 - i n s a n a 329 - i s o g a m a 327 - k o m a r e k i i 329 - k u t e i n i k o w i i 327 - m a n t o n i i 329 - m i c r o s c o p i c a 327 - m o e w u s i i 324 - m u l t i t a e n i a t a 324 - n o c t i g a m a 324 - o b l o n g a 324 - p e r p u s i l l a 327 - p e t e r f i i 324 - p i t s c h m a n n i i 324 - planoconvexß 329 - r e i n h a r d i y 323 - species 382,395 - s p h a e r o i d e s 324 - zeöra 324 Chlamydophyceae 329 C h l o r e l l a k e s s l e r i 168 - species 4 1 , 1 9 0 Chlorococcales 221 C h l o r o g o n i u m e l o n g a t u m 395 Chlorophyceae 219 Chlorophyta 323 C h o n d r u s c r i s p u s 278, 284 C h o r e l l a f u s c a 397 C i a d i a a g g r e g a t a 463 - f u l i g i n o s a 463 - i n f l a t a 463 - m o n i l i f o r m i s 463 - r e t i p o r a 463 - s u l l v a n i i 463 C l a d i n a c o n f u s a 463 - s o u t h l a n d i c a 463

Cladoniaceae 467 C o c h r a n e a species 387 C o e l o g y n e o v a l i s 304 Combretaceae 304 C o m b r e t u m f a r i n o s u m 295 - species 304 C o n o p h i l u s a m e r i c a n a 228 Crassulaceae 360 C r e p i s capülaris 355 C r o c u s s a t i v u s 320 C u c u m i s s a t i v u s 126 Cucurbitaceae 355 C u r c u r b i t a species 127 C u s c u t a e u r o p a e a 228 - n?/7e*a 228 Cyanobacteria 9 0 , 1 6 1 , 213, 343

D D a c r y d i u m b i f o r m e 459 D a u c u s c a r o t a 146 D e l e s s e r i a s a n g u i n e a 278 D e u t e r o c o h n i a l o n g i p e t a l a 292 D i c t y o s t e l i u m d i s c o i d e u m 47 - species 398 D i c t y o t a d i c h o t o m a 284,404 Dioscoreaceae 304 D i s c o d e r m i a c a l y x 6 7 D o l i c h o s species 125 D o n a t i a n o v a e z e l a n d i a e 463 D r a c o p h y l l u m m i l l i g a n i i 459 D r o s e r a a r c t u r i 463 D r y m o n i a s e r r u l a t a 295 D u n a l i e l l a b a r d a w i l 219 - species 41 D y c k i a f l o r i b u n d a 292 - r a g o n e s e i 292

E E c t o c a r p u s s i l i c u l o s u s 400 E l o d e a c a n a d e n s i s 435 - otewsa 118 E l y m u s r e p e n s 285 Empetraceae 300 E m p e t r u m n i g r u m 300 E n d o c a r p o n p u s i l l u m 449 Enterobacteria 213 E p i f a g u s v i r g i n i a n a 2 2 7 E r e m o s p h a e r a v i r i d i s 219 Eritricheae 391 E r i t r i c h u m c a n u m 387 E s c h e r i c h i a c o l i 213 E s c h s c h o l t z i a c a l i f o r n i c a 374 E u c a l y p t u s p i l u l a r i s 427 E u g l e n a g r a c i l i s 2 2 7 - species 1 9 0 , 2 2 7 , 3 8 2

F Fao/Ms s y l v a t i c a 13 F i c u s p u m i l a 126 F l e m i n g i a c h a p p a 304 F l o u r e n s i a o o l e p i s 304 F r e e s i a species 320 F r i t s c h i e l l a t u b e r o s a 221

VIII B o t A c t a 1 0 5 (1992)

Fungi 3 4 3 , 3 4 5 , 4 2 7 , 4 4 1 F u s a r i u m o x y s p o r u m 152

G G a l i u m a p a r i n e 84 Geosiphon p y r i f o r m e 343 Geum u r b a n u m 355 G i n k g o b i l o b a 13 G l a d i o u l u s species 320 G l o b o d e r a r o s t o c h i e n s i s 133 Gloecapsa species 460 G l o m u s species 343 G l y c i n e m a x 18 G o m p h r e n a g l o b o s a 147 G o n y a u l a x species 382 Gossypium h i r s u t u m 253 - species 238 Graminaceae 360 G y m n o s c h o e n u s species 463 G y p s o p h i l a species 359

H H a e m a t o c o c c u s l a c u s t r i s 221 - species 219 H a k e a species 128 H a l i c h o n d r i a o k a d a i 63 Halobacteria 174 H a l o p t e r i s f i l i c i n a 400 H e l i a n t h u s a n n u u s 111 H e i i c h r y s u m species 463 H e l i o p h y t u m species 387 Heliotropioideae 387 H e l i o t r o p i u m a r b o r e s c e n s 388 - d i g y n u m 388 - e r o s u m 388 - i n d i c u m 388 - m a r i s - m o r t u i 388 - m e s s e r s c h m i d i o i d e s 388 - p e c t i n a t u m 388 - p e r u v a n i u m 388 - species 387 - s u p i n u m 387,388 - z e y l a n i c u m 388 H e v e a b r a s i l i e n s i s 235 H o r d e u m d i s t i c h u m 31 - species 272 - v u l g a r e 3 1 , 1 4 0 , 2 4 7 H y o s c y a m u s n i g e r 318

I I r e s i n e l i n d e n i i 147 I r i s e n s a t a 319 - g e r m a n i c a 319 - h o l l a n d i c a 319 - p s e u d a c o r u s 319 - p u m i l a - setosa 319 - v e r s i c o l o r 319 I s o p h y s i s t a s m a n i c a 459 I x o r h e a t s c h u d i a n a 391

J J a t r o r r h i z a p a l m a t a 370

K K a l a n c h o e d a i g r e m o n t i a n a 236,

260,415

L L a m p r a n t h u s s o c i o r u m 146 L a r r e a n i t i d a 304 L a t h r e a c l a n d e s t i n a 228 Lecanorales 449 Leguminosae 304 L e m n a m i n o r 195 L e p t o s p e r m u m s c o p a r i u m 459 Lichens 449,457 Liliaceae 407 Lithospermeae 387 L o r o g l o s s u m h i r c i n u m 304 L u p i n u s p o l y p h y l l u s 240 L y c h n i s v i s c a r i a 355 L y c o p e r s i c o n e s c u l e n t u m 153 - p e r u v i a n u m 152

M M a r c h a n t i a species 227 M e l a m p y r u m p r a t e n s e 228 M e l i l o t u s a l b a 233 M e m b r a n o p t e r a a l a t a 278 Menispermaceae 370 Mesambryanthemaceae 34 M e s a m b r y a n t h e m u m

c r y s t a l l i n u m 34, 264,414 M e s o t a e n i u m c a l d a r i o r u m 60 - species 57 M i c a r e a a u s t r o t e r n a r i a 463 M i c r o s e r i s b i g e l o v i i 331,337 - e l e g a n s 333,338 M i r a b i l i s j a l a p a 147 M o u g e o t i a s c a l a r i s 56 - species 55 Mühria species 467

N N a r c i s s u s p s e u d o n a r c i s s u s 227 N e o p h y l l i s m e l a c a r p a 463 N e o p o n g i o c o c c u m species 221 N e p h r o p s n o r v e g i c u s 109 N e u r o s p o r a c r a s s a 234 - species 422 N i c o t i a n a species 141 - t a b a c u m 6 4 , 8 1 , 2 1 4 , 4 3 5 N i t e l l a f l e x i l i s 3 - species 248,271 N o s t o c p u n c t i f o r m e 343 Nyctaginaceae 149

0 O e n o t h e r a b i e n n i s 375 - e/ata 377 - g r a n d i f l o r a 375 - n u t a n s 375 - s u a v e o l e n s 377 - v i l l o s a 375 - « ; o p 375 Orchidaceae 304 Orc/ws m i l i t a r i s 304

O r e o b o l u s o l i g o c e p h a l u s 463 O s c i l l a t o r i a l i m o s a 90 - r u b e s c e n s 161 Oxalidaceae 355 Ctea/is s t r i c t a 355

P P e l t i g e r a species 458 P e n i c i l l u m c y c l o p i u m 253 P h a s e o l u s v u l g a r i s 158,195 P h i l o p h o r u s species 467 P h o r m i d i u m p e r s i c i n u m 165 P h y c o m y c e s b l a k e s l e e a n u s 441 P h y c o r y s r u b e n s 278 P h y l l o p h o r a t r u n c a t a 278 P h y t o p h t h o r a m e g a s p e r m a 18 Picea aötes 180,190, 345, 348,435 P i l o p h o r u s species 463 P/hms contorta 306 - r a d i a t a 306 - s y l v e s t r i s 306,427 P i s o l i t h u s t i n c t o r i u s 427 P i s o n i a species 427 Pz'sMm s a t i v u m 1 2 1 , 1 2 7 , 1 9 5 Pitcairnioideae 292 P l a n t a g o m a r i t i m a 239 - raedza 239 - species 238 P l a t y m o n a s s u b c o r d i f o r m i s 44 P l e u r o p h a s c u m g r a n d i g l o b u m 463 Poaceae 174 P o l y c a r p u s d a c r y d i o i d e s 459 Polygonaceae 359 P o l y n e u r a h i l l i a e 278 P o n t e d e r i a species 130 P o r p h y r a p u r p u r e a 44 - u m b i l i c a l i s 41 P o t e n t i l l a r e c t a 355 P r a s i o l a c r i s p a 41 Protaceae 126 P u c c i n e l l i a d i s t a n s 285 Pi/ya s p a t h a c e a 292 P y c n o t h e l i a c a l i g i n o s a 463

R R h i z o b i u m l e g u m i n o s a r u m 18,91 - m e l e l o t i 18, 215 R h o d o s p i r i l l u m r u b r u m 91 ,215 R i c h i a c u r t i s i a e 463 R o c h e l i a species 387 Rosaceae 355

S S a c c h a r o m y c e s c e r e v i s i a e 214 S a c c h a r u m q f f i c i n a r u m 70 - species 177 S a l m o n e l l a t y p h i m u r i u m 214 Scenedesmus species 172 S c l e r o c a r y o p s i s species 391 S e t a r i a f a b e r i 178 S i n a p i s a l b a 81 ,215 Sinorhizobiumfredü 18 S i p h u l a d e c u m b e n s 459 ,463 - f o l i a c e a 463

B o t A c t a 1 0 5 (1992)

- f r a g i l i s 463 - j a m e s i i 463 Solanaceae 152 S o l a n u m n i g r u m 111 - t u b e r o s u m . 1 3 3 , 1 5 8 S o l e n o p s o r a h o l o p h a e a 458 S o r g h u m species 177 - t r i c o l o r 240 S p h a c e l a r i a r i g i d u l a 400 - t r i b u l o i d e s 400 S p i n a c i a o l e r a c e a 81,198 S p i r o g y r a species 61 S t a c h y s s i e b o l d i 233 Stereocaulaceae 457 S t e r e o c a u l o n s o r e d i i f e r u m 467 - species 463 S u i l l u s b o v i n u s 427 Synechococcus e l o n g a t u s 161 - l e o p o l i e n s i s 161 - r u b e s c e n s 161 - species 214 S y n e c h o c y s t i s species 161,214

T T a m u s c o m m u n i s 304 T e t r a c y s t i s species 329 T e t r a s e l m i s species 41 - s u b c o r d i f o r m i s 44 T h a l i c t r u m species 374 T i a r i d i u m species 387 T i l i a a m e r i c a n a 362 Tillandsioideae 292 T r e n t e p o h l i a a u r e a 219 T r i c h o d e r m a r e e s i 155 T r i c h o d e s m i u m t h i e b a u t i i 94 T r i t i c u m a e s t i v u m 3 1 , 8 4 , 1 7 4 , 2 0 6 ,

247 T u l i p a g e s n e r i a n a 266 - species 407 T u s s i l a g o f a r f a r a 355

U Ulotrichales 221 U l v a r o t u n d a t a 2 7 9 U m b i l i c a r i a species 453 U v a r i a a n g o l e n s i s 304

V V a l l i s n e r i a s p i r a l i s 435 V i c i a f a b a 1 0 4 , 1 1 1 , 1 2 1 , 2 7 4 , 3 6 7 - s a t i v a 24 V i m i n a r i a j u n c e a 126 . V i t i s r i p a r i a 362 Volvocales 221,329 V o l v o x species 395

Z Zea d i p l o p e r e n n i s 2 9 - m a y s 2 6 , 5 6 , 2 0 6 , 2 4 7 , 3 1 3 , 3 6 0 ,

422 - species 177 Zingiberaceae 304 Z u c c a g n i a p u n c t a t a 304 Zygnemataceae 57 Zygomycetes 441 Zygophaceae 304

Subject Index

A Absorbance spectra

cyanobacterial picoplankton of Lake Constance 161

Absorption coefficient of plant leaves 362

Acidity pattern in tulip leaves 266

Action spectra gravitropic equilibrium of fungi

441 photoinhibition of photosynthesis

in red algae 278 phototaxis 47

Acylated betacyanins biosynthesis 146 purification from cell cultures

o f C h e n o p o d i u m r u b r u m 146 Acyltransferase

from cell cultures of C h e n o p o d i u m r u b r u m 146

Adenosine 5'-phosphosulfate sulfotransferase in P i c e a a b i e s

190 Adenylate cyclase

activity Stimulation by guanosine-(0-3-thio)triphosphate 395

G-protein regulation in C h l o r o g o n i u m 395

A i r pollutants effect on thylakoid membranes

348 Alcohol content of beer 3 Algae

brown polarized cells 400

green 4 1 , 5 5 , 2 1 9 , 3 2 3 , 3 9 5 cAMP concentration in cells 395

red photoinhibiton of photo­

synthesis 278 recovery of photosynthesis 278

Alkaloid demethylation peroxidase-catalyzed 370

Alkaloid dimerization peroxidase-catalyzed 370

Amaranthin esterification 146

Amino acid transport 234 (R)-(l -amino-2-phenylethyl)

phosphonic acid inhibition of nodulation 18

Ammonium accumulation effects on root development 306

Ammonium toxicity 306 Amoebae

of cellular slime mold 47 Androgenesis 313 Anion transport 235 Antennae chlorophyUs

increased oxidation speed 348 Apoplast 427

acidic layers 253 pH oscillations in tulip 266

tracer experiments 427 Apoplastic transport

oflanthanum nitrate 427 ofsulphorhodamineG 427 through fungal sheath 427

Apothecial development o f A u s t r o p e l t u m g l a r e o s u m 457

Ascocarp ontogeny of A u s t r o p e l t u m g l a r e o s u m 457

Astaxanthin esters 219 Atmospheric trace gases 206 ATPase activity

inhibited by bafilomycin A t 421 Authentic strains

o f C h l a m y d o m o n a s 323 Auxin antagonists

effect on root development 121 p-chlorophenoxyisobutyric acid

121 Auxin(s) 246 Auxin inhibitors

3,3a-dihydro-2-(p-methoxy-phenyl)-8//-pyrazolo[5,1 -a]iso-

indol-8-one 121,127 efffect on root fasciation 121 2,3,5-triiiodobenzoic acid 121

B Bafilomycin A j

inhibitor of vacuolar pH regulation 421

Beer alcohol content 3 osmotic biosensor for alcohol content

3 Berberine alkaloids 372 Berberrubine 372 Biopterin 441 Biosynthesis

acylated betacyanins 146 4,4'-bisjatrorrhizine 370 ofsporopollenin 407

4,4'-Bisjatrorrhizine 370 biosynthesis 370

Blue light effect on photosynthetic apparatus

o f C h l o r e l l a 168 Blue light adaptation

o i C h l o r e l l a 168 Blue-green fluorescence

of plant leaves 435 Boundary tissue

of A u s t r o p e l t u m g l a r e o s u m 457 Buds

of Norway spruce 180,190

C C3-Photosynthesis

transition to CAM 34,414 Ca 2 +/calmodulin

control of Phosphodiesterase activity 395

Caffeoylshikimic acid 152

Calcium plant metabolism 355

Calmodulin Stimulation of Phosphodiesterase

activity 395 Caloxanthin 161 CalyculinA 63 C A M 3 4 , 2 6 0 , 4 1 4

deinduction 34 induction 34 ,414 V 0 V r ATPaseinleaftonoplast 414

cAMP concentration i n algal cells 395

Canadine 372 Canopy photosynthesis

roadside grasses 285 Canthaxanthin 219 Carbohydrate transport 232 Carbonic anhydrase

metabolism of COS 206 Carbonyl sulfide (COS)

metabolism i n plants 206 ß-Carotene 161 Carotenoid(s) 161 Carotenoid esters

in senescent G i n k g o leaves 13 Cation transport 237 Cell(s)

of A c e r p s e u d o p l a t a n u s 9 7 iron reduction 97 oxygen reduction 97

Cell cultures of C h e n o p o d i u m r u b r u m 146 of H o r d e u m v u l g a r e 140 o i L y c o p e r s i c o n p e r u v i a n u m 152 routine maintenance 140

Cell cycle changes in microtubules 63 effect of calyculin A 63 effect of okadaic acid 63 Progression in plant cells 63

Cell morphology cyanobacterial picoplanton of

Lake Constance 161 Cell Suspension cultures

o f L y c o p e r s i c o n p e r u v i a n u m 152 Cell viability

after cryopreservation 140 of H o r d e u m v u l g a r e 140

Cell wall autolysin, of C h l a m y d o m o n a s 323 extensibility 246 formation 313 thickness 247 ultrastructure 247

Cell wall-bound phenolics in cell Suspension cultures of

L y c o p e r s i c o n p e r u v i a n u m 152 Cellufluor 427 Celosianins I and II 146 Chalcone(s)

2,4-dihydroxy- 300 2',4-dihydroxy- 301 2',4'-dihydroxy-4'-methoxy- 302

B o t . A c t a 105 (1992)

2' ,4' -dihydroxy-6' -methoxy-dihydro- 302

o f E m p e t r u m n i g r u m 300 2' -hydroxy-4' ,6' -dimethoxy-

dihydro- 302 2'-hydroxy-4'-methoxy- 301 4'-hydroxy-2'-methoxy- 302

Characean internode alcohol content of beer 3

Cheilanthifoline 372 Chlorogenic acid 152 p-Chlorophenoxyisobutyric acid 121 Chlorophyll breakdown

in senescent G i n k g o leaves 13 Chlorophyll fluorescence

of plant leaves 435 Chloroplast(s) 197, 227,435

contribution to leaf fluorescence 435

Chloroplast movement in M o u g e o t i a 55

Chromoplasts 227 Circadian rhythm

oxygen production o f A c e t a b u l a r i a 382

C l " conduetance o i A r a b i d o p s i s t h a l i a n a plasma

membrane 273 Classification

of cyanobacterial picoplankton from Lake Constance 161

C0 2-assimilation effect of external pH 116 in photoautotrophic cell cultures of

C h e n o p o d i u m r u b r u m 116 Columbamine 372 Comparative physiology

internal hght environment of leaves 362

mineral metabolism 355 Complex-heterozygotes

o f O e n o t h e r a g r a n d i f l o r a 375 Confocal pH topography

of plant cells 253 Conifers 306,345

effect of ammonium on roots 306 effect of surface microflora on

foliar uptake 345 Connexin(s) 104,111 Connexin-like proteins 104,111

immunological detection in V i c i a f a b a 104

in mesophyll chloroplasts of H e l i a t h u s a n n u u s 111

in mesophyll chloroplasts of V i c i a f a b a 111

in plasma membrane of V i c i a f a b a 104

Corygovanine 372 p-Coumaric acid 152 p-Coumaroylamaranthin 146 p-Coujnaroylsliikimic acid 152 p-Coumaroyltryamine 152

Grassulacean acid metabolism; see C A M

Cryopreservation of embryogenic Suspension

cultures 140 o f H o r d e u m v u l g a r e 140

Cryoprotectants, effects of 140 Cyclic adenosine-3' ,5' -

monophosphate, see cAMP cys-gene homologies 213 Cytochromes 348 Cytogenetic analysis

o f O e n o t h e r a g r a n d i f l o r a 375

D Deep-freezing

of embyrogenic Suspension cultures 140

of H o r d e u m v u l g a r e 140 Dehydrocheilanthifoline 372 Dehydrocorygovanine 372 Dehydroscoulerine 372 Developmental control, of CAM 34 Diacylglycerol selectivity

in chloroplasts 197 Diagnosis

of A u s t r o p e l t u m g l a r e o s u m 457 of C h l a m y d o m o n a s a p p l a n a t a

323 Dihydrochalcones, see also chalcones

o f E m p e t r u m n i g r u m 300 Dihydroflavokawin-B 302 3,3a-Dihydro-2-(p-methoxyphenyl)-

8/7-pyrazolo[5,1 -a]isoindol-8-one 121,127

9,10-Dihydrophenanthrenes 4,5-dmydroxy-2,3-dimethoxy- 304 o f E m p e t r u m n i g r u m 300 5-hydroxy-2,3,4-trimethoxy- 302

Dinitrogenase 90 Dinitrogenase reductase 90 Dispersal

o f M i c r o s e r i s b i g e l o v i i 331,337 Dispersal units

o f H e l i o t r o p i u m 387 Diurnal cycle

nitrogenase activity in O s c i l l a t o r i a 90

DNA polymorphisms, nuclear o f M i c r o s e r i s b i g e l o v i i 331,337

E Echinenone 219 Ecophysiology

mineral metaboHsm of plants 355 Ectomycorrhizae

apoplastic transport through fungal sheath 427

o f P i n u s s y l v e s t r i s / S u i l l u s b o v i n u s 427

EGTA inhibition of Phosphodiesterase

activity 395

Elicitation of cell Suspension cultures of

L y c o p e r s i c o n p e r u v i a n u m 152 Elongation growth

roleofepidermis 246 Embryogenic cell suspensions

cryopreservation 140 of H o r d e u m v u l g a r e 140

Endosymbiosis fungus and eyanobacterium 343

Enzymology 146 Epicuticular wax 345 Epidermis 246,266

role in elongation growth 246 of tulip leaves, travelling

acidity pattern 266 Epiphytic fiuigi

effect on foliar uptake of chemicals 345

Ethyleneglycol-bis(2-aminoethyl-ether)-N,N,N' ,N' -tetraacetate,

see EGTA Evolution

o f O e n o t h e r a species 375 External pH

effect on intracellular pH 116 Exudate localization

in E m p e t r u m n i g r u m 300

F Fe-Protein 90 Ferredoxin: sulfite reductase 213 Ferulicacid 152 Feruloylamaranthin 146 Feruloyltyramine 152 Flavine adenine dinucleotide (FAD)

441 Flavins 441 Flavonoids

role in plant development 18 Flowers

of H e l i o t r o p i u m 387 Fluorescence

of senescent G i n k g o leaves 13 Fluorescence spectra 161

cyanobacterial picoplankton of Lake Constance 161

Flushing of Norway spruce 180

Foüar senescence of G i n k g o b i l o b a 13

Foüar uptake of chemicals effect of leaf microflora 345

Freeze fracturing leaftonoplast 414

Fruits o f H e l i o t r o p i u m 387

Fungal sheath of ectomycorhizal roots 427 of P i n u s s y l v e s t r i s / S u i l l u s

b o v i n u s 427

Bot. A c t a 1 0 5 ( 1 9 9 2 )

G G-Protein

regulation of adenylate cyclase in C h l o r o g o n i u m 395

Gel eletrophoresis protein-pigment complex of

D i c t y o s t e l i u m d i s c o i d e u m 47 Gene expression 81, 227

nitrate reductase, control 81 in non-green plants 227

Genotypes of I r i s 319

Ginkgofluor 15 Glandulär cells

o f E m p e t r u m n i g r u m 300 y-Glutamylcysteinylserine

new homologue of glutathione 174

in Poaceae plants 174 in T r i t i c u m a e s t i v u m 174

Glutathione 174 in P i c e a a b i e s 180,190

Glutathione reductase 174 Glyceollin I

content in root exudate 18 content in root hairs 18

Gravitropic equilibrium of fungi action spectra 441

Groenlandicine 372 Growth characteristics

cell Suspension cultures of L y c o p e r s i c o n p e r u v i a n u m 152

Guanosine-(0-3-thio)-triphosphate Stimulation of adenylate cyclase

activity 395 Guard cells

o f V i c i a f a b a 367

H H + -ATPase

selective reconstitution of tono-plast 260

Hartig net distribution of sulphorhodamine G

427 Head and stalk structure

VoV r ATPase 414 Heterologous hybridization 213 Heterotrophic cell Suspension

cultures of L y c o p e r s i c o n p e r u v i a n u m

152 Hill reaction

in C h l o r e l l a 168 Homoglutathione 174 Hummingbird poUination 292 Hybridization

heterologous 213 o f O e n o t h e r a g r a n d i f l o r a 375 ofpotatos 133

p-Hydroxybenzaldehyde 152 Hydroxycinnamic acid(s) 146 Hydroxycinnamic acid transferases

biosynthesis of acylated beta­cyanins 146

in Caryophyllales 146 in cell cultures of C h e n o p o d i u m

r u b r u m 146 1 -O-Hydroxycinnamoyl-ß-glucose:

amaranthin O-hydroxycinnamoyl-transferase 146

Hypothallus 449 Hypoxia

nutrient uptake of roots 223

I Immature inflorescences

o f l r i s 319 Immunoblots

detection of phytochrome i n M o u g e o t i a 55

Immunofluorescence 5 5 , 1 1 1 , 4 0 0 brown algae 400 localization of connexin-like

proteins 111 locahzation o f M o u g e o t i a

p h y t o c h r o m e 55 microtubule Organization in

polarized cells 400 Immunolocalization

nitrogenase activity in O s c i l l a t o r i a 90

Intracellular pH 116, 253 effect of external pH 116

Intramembraneous particles increased size 414

Iodoacetamide 133 Ion Channels 239 Ion currents

o f A r a b i d o p s i s t h a l i a n a plasma membrane 273

Iron reduction in A c e r p s e u d o p l a t a n u s 9 7

Isoamaranthin esterification 146

Isoenzymes 133 analysis in potato hybrids 133

J Jatrorrhizine 370

precursor of 4,4'-bisjatrorrhizine 370

K K + conductance

o f A r a b i d o p s i s t h a l i a n a plasma membrane 273

K m values chloroplast enzyme activities 197

L Lanthanum nitrate

apoplastic distribution 427 Lateral root development

control 121,127 spacing intervals 127

Leafage a n d C 3 t o C A M s h i f t 34

Leaf epidermis blue-green fluorescence 435

Leafexudates o f E m p e t r u m n i g r u m 300

Leaf optics comparative study 362

Leaf surface microflora effect on foliar uptake of

chemicals 345 Leafyellowing

in G i n k g o b i l o b a 13 Leukoplasts 227 Lichen(s) 449,457

occurrence o f A u s t r o p e l t u m g l a r e o s u m 457

Liehen development role of rhizomorphs 449

Lichenization of rhizomorphs 449

Light and growth 246 Light competition

ofroadside grasses 285 Light gradients

of plant leaves 362 Light propagation

in plant tissue 362 Lignification

and sugar transfer 70 of sugarcane stem 70

Lipid bodies content of secondary carotenoids

219 Lipid inhibition

of sulfoo^iinovosyltransferase activity 197

Lipophilic phenolics o f E m p e t r u m n i g r u m leaves 300

Lucifer yellow 313 Lutein 168 Lutein ester 219

M m a d l g e n e mutants

o f P h y c o m y c e s b l a k e s l e e a n u s 441 Magnesium

plant metabolism 355 Magnesium shift

chloroplast enzyme activities 197 Malate formation

effect of external p H 116 in photoautotrophic cell cultures

of C h e n o p o d i u m r u b r u m 116 Mericarpids

o f H e l i o t r o p i u m 387 Mesophyll protoplasts

o f H e l i a n t h u s a n n u u s 111 o f V i c i a f a b a 111 presence of connexin-like proteins

111 Metabolie inhibitors

iodoacetamide 133 rhodamin-6-G 133

B o t . A c t a 1 0 5 ( 1 9 9 2 ) XIII

Methylglucose foliar uptake 345

Metribuzin tolerance of potato hydrids 133

Microinjection of DNA to microspores 313

Microspores microinjection of DNA 313

Microtubule(s) 63,400 changes in cell cycle

Progression 63 cytoskeleton in brown algae 400 Organization 63,400

Mineral metabolism ecological implications 355

MoFe-Protein 90 Molecular phylogeny

o f M i c r o s e r i s b i g e l o v i i 331 Monoclonal antibody Z-3B1 55 Morphogenesis

in lichens 449 Morphology

of H e l i o t r o p i u m dispersal units 387

Multicellular pseudoplasmodia (slugs) 47

Multiflux theory hght gradients in leaves 362

Multispecies canopy model roadside grasses 285

N NADH-dependent peroxidases 97 Nandinine 372 Near-UV/blue-light photoreceptor

441 Nectar

removal 292 secretion pattern 292 sugar content 292

Needle development of P i c e a a b i e s 180,190

Neopterin 441 Neoxanthin 168 Nicotine

inhibition of secondary carotenoid synthesis 219

Nitrate 81,306 effect on nitrate reductase gene

expression 81 plant response 306

Nitrate reductase control of gene expression 81

Nitrite reductase control of gene expression 81

Nitrogen deficiency secondary carotenoids of

Chlorophyceae 219 Nitrogen fixation

by Geosiphon p y r i f o r m e 343 Nitrogenase 90,343

diurnal cycle in O s c i l l a t o r i a 90 of Geosiphon p y r i f o r m e 343

immunolocalization in O s c i l l a t o r i a 90

Nodule initiation inhibited by (R)-(l-amino-2-

phenylethyl)phosphonic acid 18 Norflurazon

inhibition of secondary carotenoid synthesis 219

Norway spruce 180,190 enzyme activity 190 needle development 180

Nostoxanthin 161 Novel forest decline 345, 348 Nutrient uptake 223,355

and mineral metabolism 355 of roots under hypoxia 223

0 Okadaic acid

inhibition of protein phosphatases 63

probe for cell cycle progression 63 Ontogenetic diferentiation 227 Ontogeny

of H e l i o t r o p i u m 387 Open-top Chambers 348 Optical brighteners

in senescent G i n k g o leaves 13 Optical properties

of senescent G i n k g o leaves 13 Organellar DNA 227 Osmotic biosensors 3 Osmotic cells 3 Osmotic stress

effect on P r a s i o l a c r i s p a 41 ultrastructural changes in P r a s i o l a

c r i s p a 41 Ovary(ies), i n v i t r o culture 319 Oxidation speed

increased, of antennae Chlorophylls 348

Oxygen production o f A c e t a b u l a r i a 382

Oxygen reduction in A c e r p s e u d o p l a t a n u s 97

Ozone 348

P P-700 348 Parasitic plants 227 Patch-clamp investigation

o f A r a b i d o p s i s t h a l i a n a plasma membrane 273

Pentachlorophenol foliar uptake 345

Peripheral cytoplasm acidic layers 253

Peroxidase(s) 97,370 from B e r b e r i s s t o l o n i f e r a 370 catalysis of protoberberine alkaloid

demethylation 370 catalysis of protoberberine alkaloid

dimerization 370 NADH-dependent 97

pH effects on root development 306

pH mapping of Gossypium h i r s u t u m cells 253

pH optimum of UDP-sulfoquinovose: diacyl-

glycerol sutfoo^iinovosyltrans-ferase 197

pH regulation 253,421 of C h a r a vacuoles 421

Phenotypic correlations o f M i c r o s e r i s b i g e l o v i i 337

Phenylalanine labeled, incorporation into

sporopollenin 407 specifically labeled 407

Phloem unloading and developmental changes in

anatomy 70 sugarcane stem 70

3' -Phosphoadenylyl sulfate reductase 213

Phosphodiesterase activity inhibition by EGTA 395 activity inhibition by trifluoperazin

395 activity Stimulation by calmodulin

395 control by Ca 2 +/calmodulin 395

Phosphoenolpyruvate carboxylase 206, 367 metabolism of COS 206 phosphorylation 367

Phosphorylation of phosphoenolpyruvate

carboxylase 367 Photoautotrophic cell cultures

116,152 of C h e n o p o d i u m r u b r u m 116 of L y c o p e r s i c o n p e r u v i a n u m 152

Photoautotrophy cell Suspension cultures of

L y c o p e r s i c o n p e r u v i a n u m 152 Photobehavioral mutants

o f P h y c o m y c e s b l a k e s l e e a n u s 441 Photoinhibition 278

of photosynthesis in red algae 278 Photomixotrophic cell Suspension

cultures of L y c o p e r s i c o n p e r u v i a n u m 152

Photoreceptor(s) offungi 441

Photoreceptor pigment o f D i c t y o s t e l i u m d i s c o i d e u m 47

Photosynthesis in red algae 278

Photosynthetic apparatus o f C h l o r e l l a 168

Phototaxis action spectra 47 of multicellular pseudoplasmodia

(slugs) 47 Phototropism 441 Phycobilisomes

XIV Bot. A c t a 105 (1992)

cyanobacterial picoplankton of Lake Constance 161

Phylogenetic adaptation 227 Phytoalexins 18 Phytochrome(s) 55,81

control of nitrate reductase gene expression 81

localization in M o u g e o t i a 55 Phytoflagellate

cellularcAMPlevel 395 Picoplankton

cyanobacterial, from Lake Constance 161

Pigment(s) 1 3 , 1 6 1 , 1 6 8 , 2 1 9 o f E r e m o s p h a e r a v i r i d i s 219 in senescent G i n k g o leaves 13

Pigment composition cyanobacterial picoplankton of

Lake Constance 161 Pigment-protein complexes

o f C h l o r e l l a 168 Plant cells, confocal pH topography

253 Plant cuticle 345 Plant leaves, blue-green fluorescence

emission 435 Plant regeneration

o i l r i s 313,319 of transgenic maize 313

Plant tissue, light propagation 362 Plasma membrane(s) 104, 273

o f A r a b i d o p s i s t h a l i a n a 273 presence of connexin-like

proteins 104 o f V i c i a f a b a 104

PlastidicDNA 227 Plastidic factor

control of nitrate reductase gene expression 81

Plastidic RNA-polymerase 227 Plastids

defective 227 non-green 227 non-photosynthetic 227

3 1 P-NMR spectroscopy i n v i v o 116 of photoautotrophic cell cultures

of C h e n o p o d i u m r u b r u m 116 Polarized cells

immunofluorescence study of microtubules 400

microtubule Organization 400 Pollen 407 Populations

o f M i c r o s e r i s b i g e l o v i i 331, 337

Potassium plant metabolism 355

Potato breeding somatic hydridization 133

Pressurized gas transport effect on nutrient upatke of roots

under hypoxia 223 Principal components

o f M i c r o s e r i s b i g e l o v i i 337

Protein phosphatases 1 and 2A inhibition by calyculin A 63 inhibition by okadaic acid 63

Protein phosphorylation and cell cycle progression 63 inhibited by okadaic acid 63

Prothallus 449 Protoberberine alkaloids 370

peroxidase-catalyzed de-methylation 370

peroxidase-catalyzed dimerization 370

Proton efflux variable in tulip leaves 266

Proton pumps 232 Protoplast(s)

of A c e r p s e u d o p l a t a n u s 9 7 iron reduction 97 oxygen reduction 97

Protoplast fusion of dihaploid potato clones 133

Protoporphyrin and phototaxis o f D i c t y o s t e l i u m

discoideum 47 Pseudopodetium

of A u s t r o p e l t u m g l a r e o s u m 457 Pterins 441

6,7-dimethyl- 441 Pycnidial development

of A u s t r o p e l t u m g l a r e o s u m 457

R Radioimmunoassay

glyceollin I content in soybean roots 18

Reconstitution selective, of tonoplast H +-ATPase

260 Recovery

of photosynthesis in red algae 278 Red light

effect on photosynthetic apparatus o f C h l o r e l l a 168

Red light adaptation o f C h l o r e l l a 168

Reflectance of senescent G i n k g o leaves 13

Rhizomorphs of squamulose lichens 449

Rhodamin-6-G 133 Riboflavin 441 Ribulose-1,5-bisphosphate carboxy­

lase, metabolism of COS 206 Roadside grasses

canopy photosynthesis 285 light competition 285

Root(s) 18 ,121,127, 223, 306 Root development 121,127, 306

effect of auxin inhibitors 121,127 effect of exogenous ammonium

306 lateral 121,127

effect of auxin inhibitors 121,127 o f P i s u m s a t i v u m 121,127

spacing intervals 127 of P i s u m s a t i v u m 121,127

Root exudate content of glyceollin I 18

Root fasciation effect of auxin inhibitors 121 of P i s u m s a t i v u m 121

Root hairs content of glyceollin I 18

Root primordial mass 121

S Salt stress 34,41

and CAM induction 34 ultrastructural changes i n

P r a s i o l a c r i s p a 41 Scattering coefficient

of plant leaves 362 Scoulerine 372 Seasonal changes

sulfur metabolism of P i c e a a b i e s 180,190

Secondary carotenoids of Chlorophyceae under nitrogen

deficiency 219 Secondary metabolite transport 240 Shoot/root ratio

of P i n u s s y l v e s t r i s 306 Slime molds 47 Slugs 47 Solubilization 260 Solute transport 3 Somatic hydridization, for potato

breeding 133 Soybean roots

content of glyceollin I 18 infected 18

Spectral analysis Single cells o f A c e t a b u l a r i a 382

Sporopollenin biosynthesis 407 degradation 407 incorporation of 1 4C-labels

407 Squamules

formation 449 Starch granules

i n P r a s i o l a c r i s p a 41 Stern anatomy

developmental changes on phloem unloading 70

developmental changes on sucrose storage 70

sugarcane 70 Suberization

andsugartransfer 70 of sugarcane stem 70

Substrate induction nitrate reductase gene ex­

pression 81 Sugar storage

and developmental changes i n anatomy 70

in sugarcane stem 70

B o t A c t a 1 0 5 (1992)

Sulfate assimilation i n higher plants 213

i n P i c e a a b i e s 180,190 Sulfate reduction

i n higher plants 213 i n P i c e a a b i e s 180 i n prokaryotes 213

Sulfite reductase 213 Sulfur metabolism of P i c e a a b i e s

seasonal variations 180,190 Sulfur nutrition

i n P i c e a a b i e s 180 Sulphorhodamine G

apoplastic distribution 427 Symbiosis

G l y c i n e m a x / B r a d y r h i z o b i u m j a p o n i c u m 18

Syn-mericarpids o f H e l i o t r o p i u m 387

Systematics revised of C h l a m y d o m o n a s 323

T Temperature compensation

oxygen production o f A c e t a b u l a r i a 382

Temperature dependence 278,382 oxygen production o f A c e t a b u l a r i a

382 of photosynthesis i n red algae 278

Tetrahydrogroenlandicine 372 Tetrahydroprotoberberines 372 Tetrahydrothahfaurine 372 Thalifaurine 372 Thallus morphology

of A u s t r o p e l t u m g l a r e o s u m 457 Thiols

i n P i c e a a b i e s 180 Thylakoid membranes 348,435

contribution to leaf fluorescence 435

effect of air pollutants 348

Tissue tension 246 Tobacco BY-2 cells

cell cycle progression 63 Tonoplast 2 3 2 , 2 6 0 , 4 1 4

freeze fracturing 414 selective reconstitution of

FT-ATPase 260 Tracer experiments

labeled Phenylalanine 407 sporopollenin biosynthesis 407

Transformation by D N A microinjection 313

Transport processes 232 amino acids 234 anions 235 carbohydrates 232

cations 237 secondary metabolites 240 vacuoles of higher plants 232

Travelling wave acidity pattern in tulip leaves 266

Tree roots nutrient uptake under

hypoxia 223 Trifluoperazin

inhibition of Phosphodiesterase activity 395

2,3,5-Triiiodobenzoicacid 121 Tulip leaf

travelling acidity pattern 266 Turgor pressure 246

U UDP-sutfocpiinovose: diacylglycerol

sulfoo^iinovosyltransferase 197 Unicellular cyanobacteria

from Lake Constance 161 Untrastructural changes

of P r a s i o l a c r i s p a under salt stress 41

Uvangoletin 302

V V 0 V r A T P a s e

head and stalk structure 414 structural changes 414

Vacuolar ATP-dependent FT pump inhibited by bafilomycin A i 421

Vacuolar p H regulation inhibited by bafilomycin A t 421

Vacuolar proton pump effect of bafilomycin A t 421

Vacuoles 4 1 , 2 3 2 of higher plants, transport pro­

cesses 232 of P r a s i o l a c r i s p a under salt

stress 41 Vanill in 152 Vegetation

global sink for carbonyl sulfide 206

Violaxanthin 168

W Water relations 3 Western blot analysis 9 0 , 1 0 4

localization of connexin-like proteins i n V i c i a f a b a 104

localization of nitrogenase i n O s c i l l a t o r i a 90

Wetlandtrees 223 X

Xanthophylls 161 Xanthopterin 441

Z Zeaxanthin 161

XVI Bot. A c t a 1 0 5 ( 1 9 9 2 )

Production of the Phytoalexin Glyceollin I by Soybean Roots in Response to Symbiotic and Pathogenic Infection

P e t r a E. S c h m i d t , M . P a r n i s k e , a n d D . W e r n e r Fachbereich Biologie der Philipps-Universität Marburg, Marburg, FRG

Received: July 18,1991; Accepted: November 25,1991

Abstract

The amount of the phytoalexin glyceollin I in root exudate and root hairs of individual seedlings of G l y c i n e m a x (L. Merr. cv. Preston) was analysed using a radioimmunoassay. B r a d y r h i z o b i u m j a p o n i c u m 110spc4, which is able to form nitrogen fixing nodules with this plant, caused an increase of up to 50-fold in glyceollin I levels in root exudate relative to uninfected control seedlings. Maximum glyceollin I levels were reached within 10 h of incubation. Elevated glyceollin I levels were also observed after incubation of soybean roots in sterile bacterial supernatant, a Suspension of autoclaved bacteria or the supernatant from broken cells of B r a d y ­r h i z o b i u m j a p o n i c u m . Increased glyceollin I production is not due to the process of active root hair penetration by the microsymbiont since living bacterial cells are not necessary for the induction. The observed glyceollin I production in response to B r a d y r h i z o b i u m j a p o n i c u m is several times lower than that after pathogenic infection. Infection with zoospores of the phytopathogenic oomycete, P h y t o p h t h o r a m e g a s p e r m a f. sp. g l y c i n e a race 1, leads within 20 h to an accumulation of 7nmol glyceollin I/seedling in the root exudate of the compatible cultivar Kenwood and 48nmol glyceollin I/seedlings in that of the incompatible cultivar Maple Arrow. These re­sults support the idea that phytoalexins are implicated in determination of compatibility in pathogenic interactions. Crude cell extracts of different symbiotic bacteria { B r a d y r h i z o b i u m j a p o n i c u m 110s/?c4, R h i z o b i u m m e l i l o t i 2011, R h i z o b i u m l e g u m i n o s a r u m PRE 8, S i n o r h i z o b i u m

f r e d i i H H 103) were found to induce different amounts of glyceollin I in the root exudate. The observed glyceollin I levels could not be correlated with the ability of these rhizobial strains to nodulate G l y c i n e m a x . Inhibition of flavonoid and phytoalexin synthesis by (R)-(l-amino-2-phenylethyl)phosphonic acid (APEP), a specific inhibitor of the phenylalanine-ammonia-lyase (PAL), during the first 20 h of the symbiotic interaction dramatically de-creased the number of nodules formed in root regions that had been in contact with the inhibitor. This effect was observed at concentrations that inhibited neither bacterial nor plant growth. The implications of these findings for the process of nodule initation are discussed.

Key words

Flavonoids, nodule initiation, G l y c i n e m a x , B r a d y r h i z o b i u m j a p o n i c u m , P h y t o p h t h o r a m e g a s p e r m a f. sp. g l y c i n e a , root exudate, root hairs.

Abbreviations and Symbols

APEP: (R)-(l-amino-2-phenylethyl)phosphonic acid

cfu: colony forming units CHS: chalcone synthase MES: 5 m M morpholinethanesulfonic acid,

1 m M CaCl 2 , pH 6.2 PAL: phenylalanine-ammonia-lyase rtm: root tip mark X M M : xylose minimal medium

Bot. Acta 105 (1992) 18-25 © Georg Thieme Verlag Stuttgart • New York

G l y c e o l l i n I A c c u m u l a t i o n i n Soybean R h i z o s p h e r e Bot. A c t a 105 (1992)

Introduction

The symbiosis between the soybean G l y c i n e m a x and the Gram-negative soil bacterium B r a d y ­r h i z o b i u m j a p o n i c u m (110spc4) results in the formation of root nodules in which the bacteria actively fix atmospheric nitrogen. It is well established that the early stages of the rhizobia-legume interaction can be subdivided into two fundamentally different physiological processes (Long, 1989): on the one hand, rhizobia infect root hairs and thereby resemble plant pathogens invading host tissues (Djordjevic et al . , 1987). On the other hand, rhizobia in-duce changes in the developmental program of root cells so that they become meristematic (Rolfe and Gresshoff, 1988).

One group of secondary plant products, the flavonoids, has been implicated both in plant defense re-actions as well as in plant developmental processes. The isoflavonoid glyceollin I is an antibiotic phytoalexin found in G l y c i n e m a x . Phytoalexins are considered to play an im­portant role in pathogenic interactions as an early plant de­fense response (Hahn et al . , 1985; Ebel and Grisebach, 1988). A well investigated plant-pathogen system is root and stem rot of soybean caused by P h y t o p h t h o r a m e g a ­s p e r m a f. sp. g l y c i n e a . Two lines of evidence indicate that glyceollin I is an important early defensive barrier erected by the plant against the invading pathogen and involved in determination of cultivar/race specific compatibility. Firstly, a detailed analysis of the distribution of fungal hy-phae in plant tissue and the spatial and temporal pattern of glyceollin I accumulation was performed by Hahn et al. (1985). They found that limitation of fungal growth in the incompatible interaction is correlated with a rapid accumulation of glyceollin I at the infection site. Secondly, treatments that reduce the amount of phytoalexin present in resistant plants cause a corresponding increase i n sensitivity to pathogens. For example, Waldmüller and Grisebach (1987) found that inhibition of glyceollin produc­tion with the PAL inhibitor APEP enables a normally in­compatible race of P h y t o p h t h o r a m e g a s p e r m a f.sp. g l y c i n e a to infect soybean roots. By using indirect im-munofluorescence of hyphae in cryotome cross-sections of roots, they could show that the growth pattern of the in­compatible race 1 of P h y t o p h t h o r a m e g a s p e r m a f .sp. g l y c i n e a changed to that of the compatible race 3 under conditions where APEP caused loss of resistance against P h y t o p h t h o r a m e g a s p e r m a f. sp. g l y c i n e a race 1.

In contrast to their well documented function in plant defense, less attention has been paid to another aspect of flavonoid physiology, namely their possible role in plant development. Flavonoids can function as modulators of polar auxin transport (Jacobs and Rubery, 1988). The isoflavonoid glyceollin acts at low concentrations synergistically with auxin to stimulate adventitious root formation on mung bean (Yoshikawa et al . , 1986). Root primordia formation is also stimulated. Therefore, glyceollin appears to enhance early processes of root initiation rather than simply promoting development of already existing primordia. It is tempting to speculate that the induction of root formation by glyceollin is due to the ability of flavonoids to influence lateral auxin transport. Recently, Hirsch et al . (1990) found that certain flavonoids elicit the formation of nodule-like structures on alfalfa

roots, an effect that was also found with synthetic auxin transport inhibitors (Hirsch et al . , 1989).

Taken together, these findings suggest that flavonoids might be involved in meristem induction in both developmental processes, nodule and lateral root mor-phogenesis. If this is true, then one should expect an alteration of local flavonoid concentration preceeding meristem formation. It has already been shown that the flavonoid composition of root hairs undergoes changes during the symbiotic infection process (Parniske et al . , 1988). In this paper we restricted our measurement to glyceollin I concentrations in root hairs and root exudate and report a significant glyceollin I induction by B r a d y ­r h i z o b i u m j a p o n i c u m 1 1 0 s p c 4 . A possible physiological role for the symbiotic glyceollin I production is discussed.

Materials and Methods

C h e m i c a l s

The PAL inhibitor (RMl-amino-2-phenylethyl)-phosphonic acid a generous gift of Dr. E. K. Baylis, Ciba-Geigy, Manchester, USA. The antiserum against glyceollin I was a gift from Prof. Dr. H. Grisebach, Freiburg, Germany. Anti-rabbit-solid-phase was obtained from Fa. Laboserv, Gießen, Germany. Perlite was obtained from the Deutsche Perlite GmbH, Dortmund, Germany and Vermiculite from the Deutsche Vermiculite Dämmstoff GmbH, Sprockhövel (Haßlinghausen), Germany.

G r o w t h o f s e e d l i n g s

Soybean seeds {Glycine max cv. Preston, Pioneer Hi-Bred Intern. Inc., Iowa, USA, Glycine max cv. Maple Arrow, Agriculture Canada Forage section, Ottawa, Ontario, Canada, Glycine max cv. Kenwood, Iowa State University, Arnes, USA) were surface sterilized for 10 min in 30% H 2 0 2 , washed 7-10times with sterile H 2 0 and soaked for 6-8hours in sterile H 2 0. Seedlings were grown in Vermiculite/Perlite (1:1) for 2 days at 25 °C, 13Wm" 2, 75% humidity and a day night regime of 14:10h.

B a c t e r i a a n d c u l t u r e c o n d i t i o n

Bradyrhizobium j a p o n i c u m 110spc4 (Regens­burger and Hennecke, 1983), Rhizobium meliloti 2011 (Casse et a l , 1979), R h i z o b i u m l e g u m i n o s a r u m PRE 8 (Dr. Lie, Agricultural University of Wageningen, Netherlands) and Sinorhizobium fredii HH 103 (Dowdle and Bohlool, 1985) were grown at 28 °C on a rotary shaker in 20E-Medium (Werner et al., 1975) or xylose minimal medium (XMM), a Variation of the medium described by Tully (1985), containing 10 mM xylose as sole C-source and the following Vitamins: 2 x 10"6 M 4-aminobenzoic acid, 5 x 10"6 M pyridoxine HCl, 1 x 10"4 M mesoinositol, 2 x 10' 5 thiamine-di-HCl,2x 10"6MCa-D-pantothenate,3x 10"6MD(+)-biotin, 1 x 10" 5M nicotinic acid.

B a c t e r i a l i n o c u l u m

Bacteria, grown in appropriate medium to mid log phase (max. 109 cfu ml"1), were centrifüged and resuspended in 5 mM MES (pH 6.2) plus 1 mM CaCl2 and adjusted to the desired cell densities. Bacteria were killed by autoclaving a buffer washed Suspension (8 x 10 1 0 cfu ml"1) for 15 min at 120 °C. This treatment resulted in 100% killing as confirmed by plating aliquots on 20E agar plates. A cell free bacterial exudate was obtained from a cell Suspension of Bradyrhizobium japonicum in buffer (8 x 10 1 0 cfu ml"1), stirred at room temperature for l h . Cells were removed by centrifugation and the supernatant was filtered through a 0.2 um cellulose-nitrate-filter. Crude cell extracts of rhizobia grown in

Bot. Acta 105 (1992) Petra E. Schmidt, M. Parniske, andD. Werner

XMM were prepared using a French press. Cell debris were re-moved by centrifiigation. The extracts were adjusted to an equiv-alent concentration of 2 x 10 1 0 cfu ml" 1 with MES buffer.

Culture andzoospore induction of Phytophthora megasperma / sp. glycinea

Phytophthora megasperma f. sp. glycinea race 1 was obtained from T. Waldmüller and J. Ebel (Freiburg) and cultured on Lima-Bean-Agar (Difco) in petri dishes at 25 °C in the dark. Zoospores were obtained from 6 day old cultures according to Eye et al. (1978) with the exception that agar plates with Phytophthora megasperma f. sp. glycinea mycelia were washed with mineral Solution (0.58g l " 1 Ca(N03)2, 0.15g l " 1 MgS04 x 7 H 20,1.15 g l " 1 KH 2P0 4 ,0.06 g l " 1 KCl) instead of distilled water.

Inoculation procedure

Roots of 2 day old soybean seedlings were trans­ferred to 2.3 ml test tubes containing either buffer, the respective bacteria-derived inducing agents or a Suspension of Phytophthora megasperma f. sp. glycinea zoospores (2.5 x 104/plant). After an in­cubation time of 20 h (except where otherwise indicated) under the conditions described above, the volume of the liquid in the test tubes was made up to 2.3 ml with buffer (compensation for differ­ent transpiratory losses) and aliquots were removed for quantification of glyceollin I. The root hairs were harvested by scraping them with a sharpened spatula into an Eppendorf-cup.

Quantification of Glyceollin I

Freeze-dried root hairs were extracted with 1 ml methanol, 0.5 ml of the extract was evaporated to dryness and re-dissolved in 10% methanol. An aliquot of the root exudate was adjusted to 10 % methanol. Glyceollin I concentrations in root hair extracts and root exudate were determined by a modification of the radioimmunoassay of Hahn et al. (1985), in which anti-rabbit-solid-phase was used to precipitate Anti-Glyceollin I. Appropriate dilutions were assayed in duplicate.

Application of PAL inhibitor APEP during seedling infection

The effect of the PAL inhibitor APEP on nodule formation was assayed on plants growing in growth pouches (Northrup, King Seed Co., Minneapolis, MN, USA). Examples and detailed descriptions of applications of the growth pouch technique for the analysis of early events in the soybean-Bradyrhizobium japonicum symbiosis are given by Bhuwanesvari et al. (1981) and Caetano-Anolles et al. (1991). Two-day-old seedlings of Glycine max cv. Preston were incubated for 20 h in 2.3 ml test tubes containing a Bradyrhizobium japonicum WOspcA Suspension (5.2 x 103 cfu/plant) in MES buffer and the inhibitor at different concentrations. Subsequently, the seedlings were trans­ferred into sterile growth pouches and the position of the root tip was marked with a felt-tip pen on the transparent pouch. This root tip mark represents the time point at which the roots were re­moved from the inhibitor Solution. When necessary, growth pouches were wetted with sterile half concentrated nutrient Solution (Werner et al., 1975). After 11 days of growth under con-trolled environmental conditions (Werner et al., 1975), position and number of the nodules were determined.

Results

Bradyrhizobium japonicum 110spc4 induced production of glyceollin I

Effect of inoculum cell density on glyceollin I production

The initial studies focussed on the effect of Bradyrhizobium japonicum on phytoalexin concentration i n roots and root exudate of Glycine max. When buffer-washed cells o f B r a d y r h i z o b i u m japonicum were applied to soybean roots, we observed that glyceollin I accumulated during the symbiotic interaction. Phytoalexin levels in root exudate increased with increasing infection titers (Fig. 1). Beginning with inoculation titers of 1 0 1 0 cfii/ml, Brady­rhizobium japonicum induced significant amounts of glyceollin I in the root exudate which were up to 50 fold higher than those of control seedlings (Fig. 1), whereas no glyceollin I accumulated in root hairs.

To exclude the possibility that the induction was caused by traces of medium-derived yeast extract which might contain fungal elicitors of phytoalexin accumulation, we incubated the seedlings with bacteria grown in yeast-free xylose minimal medium. These bacteria also induced elevated glyceollin I production. The effects were independent of the bacterial growth phase (data not shown).

Kinetics of glyceollin I accumulation during symbiotic interaction

The kinetics of glyceollin I accumulation in response to inoculation with Bradyrhizobium japonicum is shown in Fig. 2. At every time point analysed, the major Proportion of the glyceollin I produced appeared in the root exudate (Fig. 2). Maximum glyceollin I levels were reached after 10 h of incubation and this level was maintained for up to 43 h.

c

1 — 9 9 10 i 2 X 1 0 5 X 1 0 10 3X10

inoculum density (c fu /mi )

Fig. 1 Glyceollin I (pmol/seedling) in root hairs and root exudate of Glycine maxcv. Preston after 20 h of incubation in cell suspensions of Bradyrhizobium japonicum 1 \0spcA grown in 20E medium, washed and resuspended in MES buffer. Each point represents the mean of data from 7-10 seedlings. The MES control seedlings contained on average 20 pmol glyceollin I/seedling in root hairs and 22 pmol glyceollin I/seedling in the root exudate. Bars indicate SEM values.

G l y c e o l l i n I A c c u m u l a t i o n i n Soybean R h i z o s p h e r e B o t . A c t a 105 (1992)

C7> c

250 • in root hairs O in root exudate

0 10 20 30 40

incubation time (h)

Fig. 2 Glyceollin I (pmol/seedling) in root hairs and root exudate of Glycine max cv. Preston at different times after exposure to a Sus­pension of 3.9 x 1010 cfu/ml Bradyrhizobium japonicum UOspcA cells, grown in 20E medium, washed and resuspended in MES buffer. Each point represents the mean of data from 8 seedlings. The MES control seedlings contained on average 5 pmol glyceollin I/seedling in root hairs and 20 pmol glyceollin I/seedling in the root exudate. Bars indicate SEM values.

800

700

600

500

400

300

200

100

0

M in root hairs i i in root exudate I

1 i

Fig. 3 Glyceollin I (pmol/seedling) in root hairs and root exudate of Glycine max cv. Preston after 20 h of incubation in different preparations of a Bradyrhizobium japonicum UOspcA cell Suspension. 1 = Bacteria grown in 20E medium, washed and adjusted to 8 x 1010 cfu/ml with MES buffer. 2 = same as 1, but cells were autoclaved for 15 min prior to application to the seedlings. 3 = same as 1, but cells were removed by centrifugation and the supernatant, filtered through a 0.2 jum pore size filter, was used for the plant assay. Each column represents the mean of data from 8-10 seedlings. The MES control seedlings contained on average 3 pmol glyceollin I/seedling in root hairs and 10 pmol glyceollin 1/ seedling in the root exudate. Bars indicate SEM values.

Glyceollin I induction by living cells, dead cells and cell-free exudate of B r a d y r h i z o b i u m j a p o n i c u m 1 1 0 s p c 4

Glyceollin I production by soybean roots after incubation with B r a d y r h i z o b i u m j a p o n i c u m might be due to the infection of epidermal root cells by rhizobia. Alternatively, it may be a response to bacterial elicitors, in-dependent of root hair penetration. In order to assess the probability of these alternatives, we tested dead bacteria, which are unable to infect the root, for their inducing capability.

G l y c e o l l i n I p r o d u c t i o n by s o y b e a n r o o t s d u r i n g p h y t o p a t h o g e n i c i n t e r a c t i o n s

Symbiotic and pathogenic interactions were compared with respect to glyceollin I accumulation which they induced in the rhizosphere of soybean. Since there are no well characterised soybean root pathogenic bacterial strains available, zoospores of P h y t o p h t h o r a m e g a s p e r m a f .sp. g l y c i n e a , a soybean root pathogenic oomycete, were tested for their effect on glyceollin I pro­duction by soybean roots. This oomycete has commonly

As shown in Fig. 3, autoclaved rhizobia in­duced a glyceollin I response that was fully comparable with that obtained by living bacteria. Both the magnitude of the response as well as the distribution pattern of phytoalexin between root hairs and exudate were similar. Obviously, the plant reaction seems to be independent of the presence of living bacteria. Based on this Observation we concluded that a penetration of the root surface is not necessary for the induction of glyceollin I accumulation.

During the incubation time of the plant assay, the dead bacteria sedimented to the bottom of the test tubes, leaving the roots submerged in a clear super­natant. It was therefore suspected that a soluble factor might be sufficient for the induction of phytoalexin accumulation. In order to test this hypothesis, we repeated the experiment with cell-free bacterial exudate. As shown in Fig. 3, the bacterial exudate induced a similar reaction as did living or dead cells. This result shows that the elicitor of the plant reaction is a soluble factor < 0.2 p m .

cr> c

T3 <D CD

O CD O

60

50

40

30

20

_ 10

• • in root hairs I I in root exudate

Maple Arrow Kenwood

Fig. 4 Glyceollin I (nmol/seedling) in root hairs and root exudate of an incompatible (Maple Arrow) and a compatible (Kenwood) Glycine max cultivar after 20 h of incubation with 2.5 x 104 zoospores of Phytophthora megasperma f. sp. glycinea race 1 /seedling. Each column represents the mean of 10 seedlings. Bars indicate SEM values.

Bot. A c t a 105 (1992) P e t r a E. S c h m i d t , M . P a r n i s k e , a n d D . Werner

been used to investigate phytoalexin responses of soybean roots and it is well established that P h y t o p h t h o r a m e g a s p e r m a f. sp. g l y c i n e a infection induces high concen­trations of glyceollin I in root tissue (Hahn et a l . , 1985). In addition, our data show that glyceollin I accumulates in the root exudate (rhizosphere) of such seedlings. As illustrated in Fig. 4, a seedling of the cultivar Kenwood (compatible with P h y t o p h t h o r a m e g a s p e r m a f .sp. g l y c i n e a ) exuded about 7 nmol glyceollin I during the first 20 h of a successful infection with P h y t o p h t h o r a m e g a s p e r m a f .sp. g l y c i n e a race 1. Roots of these seedlings became brown, lost turgidity and developed no secondary roots. At later stages, hyphae could often be observed growing out of the cotyledons. In contrast, the incompatible interaction with seedlings of cultivar Maple Arrow was characterized by a darkening of the root tip and the development of numerous secondary roots. The shoots could not be distinguished from those of buffer-treated plants. Those seedlings, which were able to resist pathogenic attack, produced about 48 nmol glyceollin I/seedling during the first 20 h after in­fection with zoospores of P h y t o p h t h o r a m e g a s p e r m a f.sp. g l y c i n e a race 1. These glyceollin I concentrations in the root exudate are considerably higher than those induced by all other treatments applied in this study.

TD CD CD

O 1 E

WM in root hoirs l l in root exudate

B. jap S. fredii R. mel R. leg

Fig. 5 Glyceollin I (nmol/seedling) in root hairs and root exudate of Glycine max cv. Kenwood after 20 h of incubation with cell extracts of different rhizobia. Cells were grown in XMM and broken using a French Press. Cell debris were removed by centrifugation and the resulting ex­tracts were adjusted to an equivalent concentration of 2 x 1010 cfu/ml. Each column represents the mean of data from 7-9 seedlings. The MES control seedlings contained on average 8 pmol glyceollin l/seedling in root hairs and 24 pmol glyceollin l/seedling in the root exudate. Bars in­dicate SEM values.

G l y c e o l l i n I i n d u c t i o n by r h i z o b i d l s t r a i n s

B r a d y r h i z o b i u m j a p o n i c u m induces the production of low but significant amounts of glyceollin I by G l y c i n e m a x . These glyceollin I levels remain low even at high inoculum densities, indicating that the microsymbiont is capable of suppressing or preventing this plant defense response. Such a reduction in the plant defense response could be a prerequisite for a successful infection. We were interested whether rhizobia that are unable to nodulate soybean differ from B r a d y r h i z o b i u m j a p o n i c u m with re-spect to the amounts of glyceollin I/seedling they induce. When soybean roots were infected with suspensions of R h i z o b i u m m e l i l o t i (unable to nodulate G l y c i n e m a x ) , highly variable responses were observed. These ranged from no response up to a production of 3 nmol/seedling in the root hairs. It was determined that the Variation was not due to the length of the incubation time (data not shown). However, the glyceollin I concentration seemed to be de-pendent on the batch of bacterial cells used. In order to analyse the factors responsible for the Variation, we tested crude cell extracts for their inducing capacity. Inter-estingly, incubation in such a preparation of R h i z o b i u m m e l i l o t i cells always led to very high glyceollin I concentra­tions in the nmol-range.

Since it was found that the use of cell ex­tracts led to more reproducible reactions by plant roots, these conditions were chosen to test two additional rhizobial strains (Fig. 5). S i n o r h i z o b i u m f r e d i i , another G l y c i n e m a x nodulating bacterium, and R h i z o b i u m l e g u m i n o s a r u m , a bacterium which is unable to nodulate G l y c i n e m a x , were used. The two strains tested that are unable to nodulate soybean, induced different glyceollin I levels. The glyceollin I levels increased significantly after incubation with the supernatant from a crude R h i z o b i u m m e l i l o t i cell extract, whereas no enhancement could be observed after incubation with a comparable preparation from R h i z o b i u m l e g u m i n o s a r u m cells. In addition, there

were strong differences between the two soybean nodulating rhizobia analysed in this study. Disrupted B r a d y r h i z o b i u m j a p o n i c u m cells failed to induce any more glyceollin I accumulation than whole cells (compare data from Fig. 5 with Figs. 1,2, 3). As seen in Fig. 5, a crude ex­tract of S i n o r h i z o b i u m f r e d i i cells induced glyceollin I con­centrations in soybean root exudate which are in the same order of magnitude as those found after compatible in­fection with zoospores of the root pathogen P h y t o p h t o r a m e g a s p e r m a f. sp. g l y c i n e a race 1.

No correlation could be found between the ability of the rhizobial strains to infect soybeans and the glyceollin I levels induced by cell preparations from these strains.

Absolute glyceollin I concentrations showed considerable variations between different tests i.e. differ­ent batches of seedlings. This Variation in phytoalexin re­sponse between different batches of seedlings was also observed by other workers (e.g. Bonhoff and Grisebach, 1988; Hahn et al . , 1985). Consequently each figure con-tains data derived from the same test.

I n h i b i t i o n o f n o d u l a t i o n b y t h e P A L - i n h i b i t o r A P E P

Having observed low but significant in­duction of the phytoalexin glyceollin I after infection with B r a d y r h i z o b i u m j a p o n i c u m , we were interested to assess the physiological significance of this in the early stages of the symbiotic interaction. Our approach was to analyse the effect of an inhibitor of flavonoid synthesis on nodule formation.

G l y c e o l l i n I A c c u m u l a t i o n i n Soybean R h i z o s p h e r e B o t A c t a 1 0 5 (1992)

0 0.01 0.1 1

A P E P ( m M )

Fig. 6 Effect of the PAL inhibitor APEP on the number of nodules formed above the root tip mark after infection of Glycine max cv. Preston with Bradyrhizobium japonicum 1 lOspcA (5.2 x 103 cfu/plant). The pri-mary roots were incubated for 20 h in 2.3 ml test tubes containing in­hibitor and bacteria, then transferred to growth pouches. The position of the root tip was marked. After 11 days, position and number of the nodules were determined. Each point represents the mean of data from 12-16 plants. Bars indicate SEM values.

Table 1 Effect of preculture of Bradyrhizobium japonicum 110spc4 in isoflavonoid-containing medium on the inhibition of nodulation by APEP.

Nodule number2 above rtm Preculture1 after incubation of roots in of bacteria | MES APEP

control 8.5a 1.5b

isoflavonoid 6.5a 1.0b

1 Bradyrhizobium japonicum was precultured in 20E medium containing either 10 uM each of daidzein and coumestrol (isoflavonoid) or 0.02 % (v/v) dimethylform-amide (control). Plants were infected with 8 x 103 cfu/plant. All other conditions were the same as described in Fig. 6. 2 Values with different indices differ with a significance of < 5 % (t-test).

A key enzyme in flavonoid synthesis is the phenylalanine-ammonia-lyase (PAL). A specific inhibitor of this enzyme is (R)-(l-amino-2-phenylethyl)phosphonic acid (APEP) (Laber et al . , 1986). The effect of the PAL in­hibitor on nodulation was tested with seedlings growing in growth pouches. In this experiment, the position of the root tip mark (rtm) corresponded to the time of removal of the root from the inhibition Solution. Starting with APEP con­centrations of 330 JUM, the number of nodules over the rtm was found to be drastically reduced (Fig. 6). On APEP treated plants, a burst of nodulation was typically observed directly below the rtm (data not shown) indicating that nodulation was possible as soon as the inhibitor was re-moved. The highest APEP concentration applied in this ex­periment (ImM) inhibits neither bacterial nor plant growth (data not shown). These observations show that the reduced nodule initiation in the presence of APEP is not due to a non-specific toxic effect. The nodulation of APEP-treated seedlings could not be increased by using rhizobia

that had been previously grown in a medium containing coumestrol and daidzein, the major isoflavonoid con-stituents in root exudate of soybean seedlings (D'Arcy-Lameta, 1986) (Table 1). These results suggest that the re­duced nodule number in root regions that have been in contact with the inhibitor results from a specific inhibition of a metabolic function which is indispensable in the early symbiotic interaction.

Discussion

In this study, glyceollin I production by Single seedlings of G l y c i n e m a x in symbiotic and pathogenic interactions was analysed. Axenically grown seedlings produce very low amounts of glyceollin I in root hairs as well as in root exudate. In contrast, when seedlings were incubated with living cells of B r a d y ­r h i z o b i u m j a p o n i c u m 110spc4, a bacterium that is able to form effective nodules on G l y c i n e m a x roots, a significant production of glyceollin I was observed. Typically, most of the glyceollin I produced during the symbiotic interaction accumulated in the root exudate, whereas a smaller portion was found in root hairs. The elicitor of this reaction appears to be a soluble, heat stable factor of a size < 0.2/im since autoclaved cells as well as filtered cell-free bacterial exudate or a crude cell extract had the same in-ducing capacity. The maximum glyceollin I accumulation was reached after 10 h. The concentration of glyceollin I in root exudate did not increase during longer incubation.

For the pathogenic interaction between G l y c i n e m a x and P h y t o p h t h o r a m e g a s p e r m a f .sp. g l y c i n e a , evidence has accumulated that the production of glyceollin plays an important role in resistance of the host plant towards incompatible races of P h y t o p h t h o r a m e g a s p e r m a f .sp. g l y c i n e a (Waldmüller and Grisebach, 1987; Hahn et al . , 1985). Our data confirm this idea since during the first 20 h of the incompatible interaction { G l y c i n e m a x cv. Maple Arrow - P h y t o p h t h o r a m e g a s p e r m a f. sp. g l y c i n e a race 1), much higher amounts of phytoalexins accumulated than during the compatible interaction { G l y c i n e m a x cv. K e n w o o d - P h y t o p h t h o r a m e g a s p e r m a f. sp. g l y c i n e a race 1).

In contrast to pathogenic Systems, where incompatibility is correlated with glyceollin I levels, no connection could be found between the ability of a rhizobial strain to form nodules on G l y c i n e m a x and the amount of glyceollin I produced in root hairs or exudate. Unexpectedly, the highest glyceollin I levels, induced by crude bacterial cell extract, were obtained with S i n o ­r h i z o b i u m f r e d i i (able to nodulate G l y c i n e m a x ) , whereas the lowest levels were obtained with R h i z o b i u m l e g u ­m i n o s a r u m (unable to nodulate G l y c i n e m a x ) (Fig. 5). However, there was a strong Variation observed amongst the strains that were unable to nodulate G l y c i n e m a x , since R h i z o b i u m m e l i l o t i induced higher levels of glyceollin I than R h i z o b i u m l e g u m i n o s a r u m . The comparison of the four rhizobial strains, with respect to their glyceollin I in-ducing capacity, showed no correlation between their abil­ity to infect G l y c i n e m a x and the glyceollin I levels induced by crude cell extracts from these strains. Certainly, these conclusions have to be prefaced with the remark that these results were obtained by the use of crude cell extracts since

B o t A c t a 105 (1992) P e t r a E. S c h m i d t , M . P a r n i s k e , a n d D . Werner

it was impossible to obtain reproducible plant reactions with living R h i z o b i u m m e l i l o t i cells. It is possible that these conditions do not reflect the i n v i v o Situation.

In this study we observed that B r a d y ­r h i z o b i u m j a p o n i c u m \ \ 0 s p c 4 induces a small but signifi­cant glyceollin I exudation by G l y c i n e m a x roots. Estabrook and Sengupta-Gopalan (1991) analysed mRNA levels for PAL and CHS in roots of G l y c i n e m a x after symbiotic in­fection with B r a d y r h i z o b i u m j a p o n i c u m USDA 110. They used these key enzymes of the phenylpropanoid and flavonoid biosynthetic pathway to investigate if an in­duction of the pathway, leading to symbiotic signal molecules and phytoalexins like glyceollin I, had taken place and were able to show significant increases in the quantity of specific mRNAs even on the first day after in­oculation. This finding is consistent with the glyceollin I exudation observed in our study.

Flavonoid accumulation in the root exudate of leguminous plants in response to symbiotic infection was also described by Recourt et al. (1991). They reported that inoculation with the homologous symbiont R h i z o b i u m l e g u m i n o s a r u m bv. v i c i a e results in an increase of n o d gene inducing flavonoids in root exudate of V i c i a s a t i v a subsp. n i g r a .

The increased glyceollin I levels, induced by the symbiotic microorganism, are in line with the pre-viously observed increase in flavonoid synthesis (Parniske et al . , 1988). Nevertheless, the physiological significance of glyceollin I exudation by soybean roots is still unclear. An increase in phenylpropanoid derivative concentration seems to be indispensible for a successful symbiotic inter­action since its inhibition leads to a significant reduction in nodule formation. That PAL inhibition in alfalfa roots leads to a reduced synthesis and release of flavonoids was re­ported by Maxwell and Phillips (1990). We observed that in­hibition of flavonoid and glyceollin I synthesis with the PAL inhibitor APEP, drastically reduced induction of nodule formation. The negative effect of PAL inhibition on nodule formation could not be explained by an absence of n o d gene inducers since nodule formation was still inhibited when the bacteria were grown in an isoflavonoid-(= n o d gene inducers) containing medium (Tab. 1). The effective inhibitor concentrations were the same as described by Waldmüller and Grisebach (1987). They used a similar assay design and observed a 46 % inhibition of glyceollin I production with 330 p M APEP and a 67% reduction with 1 mM APEP. Based on their data, we conclude that PAL and glyceollin I synthesis were also inhibited in our ex-periment. The reduction in nodule formation caused by APEP suggests that flavonoid Compounds, induced after symbiotic infection, are important mediators of early symbiotic events.

Some recent results from several inde-pendent laboratories indicate a possible role of flavonoids in meristem induction. For the induction of root formation, local concentrations of plant hormones, like auxin and cytokinin, have been suggested to be critical parameters (Libbenga et al . , 1973). It was shown by Jacobs and Rubery (1988) that certain flavonoids can specifically compete with a synthetic auxin transport inhibitor for binding to its re-

ceptor. These flavonoids disturb polar auxin transport in a manner similar to that of synthetic auxin transport in­hibitors and therefore may have an influence on auxin dis­tribution and local concentrations. In that connection it is an interesting finding that enhanced expression of a bean CHS8-GUS gene fusion was found in root areas of trans-genic tobacco where the formation of lateral roots had be­en initiated (Schmid et al . , 1990). CHS is a key enzyme of flavonoid synthesis.

Since both the initiation of lateral roots and that of symbiotic root nodules (Rolfe and Gresshoff, 1988) require the induction of meristematic activity, the oc-currence of similar physiological processes during the ontogenesis of the two root-derived plant organs seems possible. The close ontogenic relationship between nodules and lateral roots is also evidenced by the Observation that some R h i z o b i u m m e l i l o t i strains stimulate both normal nodules and "hybrid" structures intermediate between a nodule and a lateral root (Dudley et al . , 1987). The finding that auxin transport inhibitors, as well as certain flavonoids, elicit nodule-like structures on alfalfa, which closely resemble R h i z o b i u m - i n d u c e d legume nodules (Hirsch et a l . , 1989,1990), is evidence for the critical role of auxin during nodule development.

The glyceollin I levels induced in soybean roots infected with the compatible microsymbiont B r a d y ­r h i z o b i u m j a p o n i c u m 1 1 0 s p c 4 were low when compared to those characteristic of a pathogenic interaction. In both types of pathogenic interactions, compatible and in­compatible, the levels of glyceollin I production were several times higher than those observed as a result of the symbiotic interaction. It is likely that at these low concen­trations glyceollin I has plant hormone characteristics and plays a role in nodule induction rather than in plant de­fense. Evidence for glyceollin I as a plant hormone comes from the experiments of Yoshikawa et al . (1986) who observed an increased meristem induction by mung bean roots in response to glyceollin I/auxin treatments. Our re­sults indicate that the plant is in need of PAL products during the early stages of nodule formation. Experiments are in progress to analyse whether this is due to the in-volvement of flavonoids in meristem induction.

Acknowledgement

We thank the laboratory of the late Prof. Dr. H. Grisebach for the supply of glyceollin antibody. We thank Dr. E. K. Baylis for the gift of APEP. We thank the Deutsche Forschungs­gemeinschaft (Bonn) for continuing support in the SFB 305 "Ökophysiologie, Verarbeitung von Umweltsignalen".

G l y c e o l l i n I A c c u m u l a t i o n i n Soybean R h i z o s p h e r e B o t . A c t a 1 0 5 (1992)

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P e t r a E. S c h m i d t

Philipps-Universität Marburg Fachbereich Biologie Karl-v. Frisch-Straße D(W)-3550 Marburg Federal Republic of Germany