Botanica Acta

Beruhte der Deutschen Botanisehen Gesellschaft • Journal of the German Botanical Society

Editors E4953F

Ulrich Lüttge, Darmstadt (Editor-in-Chief) Eberhard Schnepf, Heidelberg Volume 105 Andre Läuchli, Davis, California February 1992 Toshiyuki Nagata, Tokyo i Page 1-80

Contents

1 Editorial 26 Structure of Protein Bodies Photophysiology Developmental Biology A. Läuchli and Elemental Composition of Phytin from Dry Germ of Maize 47 Isolation and Characteriza- 63 Okadaic Acid as a Probe to Biotechnology (Zea mays L.) tion of the Putative Photorecep- Analyse the Cell Cycle Progres• tor for Phototaxis in Amoebae of sion in Plant Cells 3 Osmotic Biosensors. How to M. Mikus, M. Bobäk, and A. Lux the Cellular Slime Mold, Dicty- S. Hasezawa and T. Nagata Use a Characean Internode for Salinity Stress ostelium discoideum Measuring the Alcohol Content H.-P. Vornlocher and 70 Developmental Changes in

ofBeer 34 The Irreversible C3to CAM D.-P. Hader the Anatomy of the Sugarcane M. Rüdinger, P. Hierüng, and Shift in Well-watered and Salt- Stern in Relation to Phloem E. Steudle stressed Plants ofMesembryan- 55 Immunolocalization ofCyto- Unloading and Sucrose Storage themum crystallinum is under solic Phytochrome in the Green Karin Ruth Jacobsen, Plant Biochemistry Strict Ontogenetic Control Mg&Mougeotia D. G. Fisher, A. Maretzki, and W.Herppich, Margaretha Herp- Christel Hanstein, F. Grolig, P. H. Moore 13 Autumn Leaves of Ginkgo pich, and D. J. von Willert and G. Wagner biloba L.: Optical Properties, Pig• A1 Mitteilungen des ments and Optical Brighteners 41 Changes in the Ultrastruc- Vorstandes der DBG Ph. Matile, Barbara M.-P. Flach, ture oiPrasiola crispa ssp. ant- and B. M. Eller arctica under Salinity Stress A. Jacob, H. Lehmann, 18 Production of the Phyto- G. 0. Kirst, and Chr. Wiencke alexin Glyceollin I by Soybean Roots in Response to Symbiotic and Pathogenic Infection Petra E. Schmidt, M. Parniske, and D. Werner

This Journal is indexed in Current Contents/Life Sciences, Current Contents/Agriculture, Biology & Environmental Sciences, §Thieme Biosis and CABS Georg Thieme Verlag Stuttgart • New York Thieme Medical Publishers, Inc., New York BOACEJ(l), 1-80 (1992) Bot. Acta ISSN 0932-8629 Instructions for Authors

Scope of the Journal edgements, References (see below), Tables (see below), Fig- note that in all cases füll titles and first and last page num• BOTANICA ACTA is an international Journal covering all ure legends (see below), Illustrations (see below). ber are required. Gelds of plant science. It aims at bridging the gaps between In Materials and Methods, Results and Discussion subhead- Articles in Journals with one, two or more authors: the different fields of botany. ings are possible. If it is chosen to combine sections Results Lüttge, U. - Instructions for authors. Botanica Acta 101 and Discussion, an additional section Conclusions can be (1988), 48-52. Submission of papers added, but this must be brief. Lüttge, U. and Schnepf, E. - Instructions for authors. BOTANICA ACTA accepts Rapid Communications Botanica Acta 101 (1988), 48-52. - füll length papers (up to 8 printed pages) Title page and Key words as for füll lengthpapers. 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Berichte der Deutschen Botanischen Gesellschaft Editors Ulrich Lüttge, Darmstadt Journal of the German Botanical Society (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

1992

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Thieme Medical Publishers, Inc. 381 Park Avenue South New York, NY10016 Some of product names, patents and registered © 1992 Georg Thieme Verlag, Rüdigerstraße 14, D-7000 Stutt• designs refered to are in fact registered trademarks or proprie- gart 30 - Printed in Germany 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. Contents

No. 1 (February 1992) = Page 1- 80 227 Conservation and Structural Divergence of Organellar No.2 (April 1992) = Page 81-132 DNA and Gene Expression in Non-Photosynthetic No. 3 (June 1992) = Page 133- 226 Plastids Düring Ontogenetic Differentiation and No. 4 (August 1992) = Page 227- 342 Phylogenetic Adaptation No. 5 (October 1992) = Page 343- 394 J. Feierabend No. 6 (December 1992) = Page 395-468 348 Changes in the Constitution of Thylakoid Membranes in Spruce Needles Düring an Open-top Chamber Ex• 337 Phenotypic Sirnilarity and Genetic Relationship Among periment Populations of Microseris bigelovii (Asteraceae: U. Flammersfeld and A. Wild Lactuceae) K. Bachmann 140 Cryopreservation of Embryogenic Suspension Cul• tures of Barley (Hordeum vulgare L.) 370 Peroxidase Catalyzed Dimerization and Demethyl- A. Fretz, A. Jahne, and H. Lörz ation of Protoberberine Alkaloids W. Bauer, R. Stadler, and M. H. Zenk 313 Selection of Microspore Derived Embryogenic Struc- tures in Maize Related to Transformation Potential by 260 Selective Reconstitution of the Tonoplast H+-ATPase Microinjection of the Crassulacean-Acid Metabolism Plant Kalanchoe A. Gaillard, E. Matthys-Rochon, and C. Dumas daigremontiana Barbara Behre, R. Ratajczak, and U. Lüttge 292 Nectar Secretion Pattern and Removal Effects in Six Argentinean Pitcairnioideae (Bromeliaceae) 427 Apoplastic Transport through the Fungal Sheath of L. Galetto and L. Bernardello Pinus sylvestris/Suillus bovinus Ectomycorrhizae P. Behrmann and W. Heyser 213 Homologies in the Structural Genes Coding for Sul- phate Reducing Enzymes from Higher Plants and 152 Growth Characteristics and Elicitor-induced Re- Prokaryotes actions of Photosynthetically Active and Hetero- G. Gisselmann, Andrea Niehaus, and J. D. Schwenn trophic Cell Suspension Cultures of Lycopersicon peruvianum (Mill.) 395 Evidence for a G-Protein Regulated Adenylate Cyclase Andrea Beimen, L. Witte, and W. Barz and a Ca2+/Calmodulin Controlled Phosphodiesterase in the Phytoflagellate Chlorogonium 382 Temperature Dependency of Circadian Period in a R. Gromes and K. Zetsche Single CeMfAcetabularia) S. Berger, J. Dirk, L. von Lindern, D. Wolff, and 223 The Effect of Pressurized Gas Transport on Nutrient D. M ergenhagen Uptake Düring Hypoxia of Alder Roots W. Grosse and Doris Meyer 285 Experimental and Modelling Studies of Competition for Light in Roadside Grasses 168 Influences of Blue and Red Light on the Photosyn- W. Beyschlag, R. J. Ryel, and I. Ulimann thetic Apparatus of Chlorella kessleri - Alterations in Pigment-Protein Complexes 146 Hydroxycinnamic Acid Transferases in the Bio- Regina Grotjohann, Myung-Sook Rho, and synthesis of Acylated Betacyanins: Purification and W. Kowallik Characterization from Cell Cultures of Chenopodium rubrum and Occurrence in Some Other Members of 407 Studies on Sporopollenin Biosynthesis in Tulipa the Caryophyllales Anthers III. Incorporation of Specifically Labeled Maria Bokern, Susanne Heuer, and D. Strack 14C-Phenylalanine in Comparison to Other Precursors S. Gubatz and R. Wiermann 273 K+ and Cl" Conductance of Arabidopsis thaliana Plas• ma Membrane at Depolarized Voltages 278 Photoinhibition of Photosynthesis and its Recovery in Raffaella Cerana and Roberta Colombo Red Algae D. Hanelt, K. Huppertz, and W. Nultsch 161 Cyanobacterial Picoplankton from Lake Constance II. Classification of Isolates by Cell Morphology and 55 Immunolocalization of Cytosolic Phytochrome in the Pigment Composition Green Alga Mougeotia Anneliese Ernst, G. Sandmann, Christine Postius, Christel Hanstein, F. Grolig, and G. Wagner Susanne Brass, U. Kenter, and P. Böger 63 Okadaic Acid as a Probe to Analyse the Cell Cycle 323 Towards a Revision of the Systematics of the Genus Progression in Plant Cells Chlamydomonas (Chlorophyta) 1. Chlamydomonas S. Hasezawa and T. Nagata applanata Pringsheim H. Ettl and U. G. Schlösser 266 Travelling Pattern of Acidity in the Epidermis of Tulip Leaves Z. Hejnowicz Bot Acta 105 (1992)

457 Austropeltum glareosum gen. et sp. nov., a New 414 Quantification of Visible Structural Changes of the Liehen from Moimtain Plateaux in Tasmania and New VoVi-ATPase in the Leaf-tonoplast of Mesembry• Zealand anthemum crystallinum by Freeze-fracture Replicas

A. Henssen, H. Döring, and G. Kantvilas Prepared Düring the C3-Photosynthesis to CAM Transition

34 The Irreversible C3 to CAM Shift in Well-watered and Rebecca Klink and U. Lüttge Salt-stressed Plants of Mesembryanthemum crys- tallinum is under Strict Ontogenetic Control 343 Geosiphon pyriforme, an Endosymbiotic Consortium W. Herppich, Margaretha Herppich, and of a Fungus and a Cyanobacterium (Nostoc), Fixes Ni- D. J.vonWülert trogen M. Kluge, D. Mollenhauer, R. Mollenhauer, 387 Morphology ofHeliotropium (Boraginaceae) Dispersal and R. Kape Units H. H. Hilger 246 The Role of the Epidermis in the Control of Elongation Growth in Sterns and Coleoptiles 121 The Control of Lateral Root Development in Cultured U. Kutschera Pea Seedhngs II. Root Fasciation Induced by Auxin Inhibitors 435 Investigations of the Blue-green Fluorescence Emis• Maud A. W. Hinchee and Th. L. Rost sion of Plant Leaves M. Lang, P. Siffel, Zuzana Braunovä, and 127 The Control of Lateral Root Development in Cultured H. K. Lichtenthaler Pea Seedlings III. Spacing Intervals Maud A. W. Hinchee and Th. L. Rost 319 In Vitro Ovary Culture of some Apogon Garden Irises {Iris pseudacorus L., /. setosa Pall., /. versicolor L.) 441 Altered Pterin Patterns in Photoreceptor Mutants of Genevieve Laublin and M. Cappadocia Phycomyces blakesleeanus with Defective madl Gene N. Hohl, P. Galland, H. Senger, and A. P. Eslava 1 Editorial A. Läuchli 104 Immunological Evidence of Connexin-like Proteins in the Plasma Membrane of Vicia faba L. 97 Ferric Ion and Oxygen Reduction at the Surface of Carola Hunte, Heide Schnabl, 0. Traub, Protoplasts and Cells ofAcer pseudoplatanus K. Willecke, and Margot Schulz F. Macri, E. Braidot, E. Petrussa, M. Zancani, and A. Vianello 116 External pH Modifies the Intracellular pH and the

Mode of Photosynthetic C02-Assimilation in Photo- 362 A Comparative Study of Internal Light Environment autotrophic Cell Suspension Cultures ofChenopodium in Bifacial Leaves of Different Plants rubrum L. A. Martinez v. Remisowsky, J. H. McClendon, and W. Hüsemann, R. Callies, and D. Leibfritz L. Fukshansky

41 Changes in the Ultrastructure oiPrasiola crispa ssp. 232 Transport Processes in Vacuoles of Higher Plants antaretica under Salinity Stress E. Martinoia A. Jacob, H. Lehmann, G. 0. Krist, and Chr. Wiencke 13 Autumn Leaves of Ginkgo biloba L.: Optical Prop• erties, Pigments and Optical Brighteners 70 Developmental Changes in the Anatomy of the Sug- Ph. Matile, Barbara M.-P. Flach, and B. M. Eller arcane Stern in Relation to Phloem Unloading and Suc- rose Storage 26 Structure of Protein Bodies and Elemental Compo- Karin Ruth Jacobsen, D. G. Fisher, A. Maretzki, and sition of Phytin from Dry Germ of Maize [Zea mays L.) P. H. Moore M. Miku§, M. Bobäk, and A. Lux

400 Immunofluorescence Study of Microtubule Organ• 81 Control of Nitrate Reductase and Nitrite Reductase ization in Some Polarized Cell Types of Selected Gene Expression by Light, Nitrate and a Plastidic Brown Algae Factor Chr. I. Katsaros H. Mohr, A. Neininger, and B. Seith

355 The Specific Mineral Metabolism of Selected Plant 133 Somatic Hybridization of Dihaploid Potato Protoplasts Species and its Ecological Implications as a Tool for Potato Breeding H. Kinzel and Ilse Lechner C. Möllers and G. Wenzel

174 y-Glutamylcysteinylserine - A New Homologue of Glu- 421 Bafilomycin Inhibits Vacuolar pH Regulation in a tathione in Plants of the Family Poaceae Fresh Water Charophyte, Chara corallina S. Klapheck, B. Chrost, J. Starke, and Y. Okazaki, M. Tazawa, Y. Moriyama, and H. Zimmermann N. Iwasaki Bot. Acta 105 (1992)

206 Enzymatic Pathways for the Consumption of Carbonyl 190 Adenosine 5'-Phosphosulfate Sulfotransferase from Sulphide (COS) by Higher Plants Norway Spruce: Biochemical and Physiological Prop• G. Protoschill-Krebs and J. Kesselmeier erties Marianne Suter, A. Tschanz, and Chr. Brunold 253 Confocal pH Topography in Plant Cells - Acidic Layers in the Peripheral Cytoplasm and the Apoplast 219 Secondary Carotenoids of Eremosphaera viridis De W. Koos Bary (Chlorophyceae) Under Nitrogen Deficiency B. Vechtel, U. Kahmann, and H. G. Büppel 3 Osmotic Biosensors. How to Use a Characean In• ternode for Measuring the Alcohol Content of Beer 90 Immunolocalization and Western Blot Analysis of M. Rüdinger, P. Hierling, and E. Steudle Nitrogenase in Oscillatoria limosa Düring a Light- dark Cycle 449 Lichenizing Rhizomorphs and Thallus Development M. Villbrandt, L. J. Stal, B. Bergman, and in the Squamulose Liehen Aspicilia crespiana Rico W. E. Krumbein ined. (Lecanorales, Ascomycetes) W. B. Sanders and V. J. Rico 306 Effects of Exogenously Supplied Ammonium on Root Development of Scots Pine (Pinus sylvestris L.) 18 Produktion of the Phytoalexin Glyceollin I by Soybean Seedlings Roots in Response to Symbiotic and Pathogenic In- P. Vollbrecht and Helga I. Kasemir fection Petra E. Schmidt, M. Parniske, and D. Werner 331 Nuclear DNA Polymorphisms Among Strains of Microseris bigelovii (Asteraceae: Lactuceae) Ampli- 367 In Vitro and in Vivo Phosphorylation of Stomatal fied from Arbitrary Primers Phosphoenolpyruvate Carboxylase from Vicia faba L. A. W. von Heusden and K. Bachmann Heide Schnabl, M. Denecke, and M. Schulz 47 Isolation and Characterization of the Putative Photo- 345 Leaf Surface Microflora May Significantly Affect reeeptor for Phototaxis in Amoebae of the Cellular Studies on Foliar Uptake of Chemicals Slime Mold, Dictyostelium diseoideum L. Schreiber and J. Schönherr H.-P. Vornlocher and D.-P. Hader

111 Immunofluorescent Localization of a Connexin 26- 300 Lipophilic Phenolics from the Leaves of Empetrum like Protein at the Surface of Mesophyll Protoplasts nigrum - Chemical Structures and Exudate Loca• from Vicia faba L. and Helianthus annuus L. lization Margot Schulz, 0. Traub, Mona Knop, K. Willecke, E. Wollenweber, M. Dörr, R. Stelzer, and and Heide Schnabl F. J. Arriaga-Giner

375 The Complex-Heterozygotes ofOenothera grandiflora A 7 Mitgliederliste L'Her. A 1 Mitteilungen des Vorstandes der DBG E. Schumacher, E. Steiner, and W. Stubbe A 5 Mitteilungen des Vorstandes der DBG 180 Changes in Sulfur Metabolism Düring Needle Devel• A21 Mitteilungen des Vorstandes der DBG opment of Norway Spruce R. Schupp and H. Rennenberg A23 Mitteilungen des Vorstandes der DBG A27 Mitteilungen des Vorstandes der DBG 197 Enzymatic Characteristics of UDP-sulfoquinovose: Diacylglycerol Sulfoquinovosyltransferase from Chlo- roplast Envelopes U. Seifert and E. Heinz Author's Index

A Grotjohann, Regina 168 Lörz.H. 140 Schulz, Margot 104,111, Gubatz, S. 407 Lüttge, U. 260,414 367 Arriaga-Giner, F. J. 300 Lux, A. 26 Schumacher, E. 375 H Schupp, R. 180 B Hader, D.-P. 47 M Schwenn,J.D. 213 Bachmann, K. 331,337 Hanelt, D. 278 Macri, F. 97 Seifert, U. 197 Barz.W. 152 Hanstein, Christel 55 Maretzki, A. 70 Seith, B. 81 Bauer, W. 370 Hasezawa, S. 63 Martinez v. Remisowsky, A. Senger, H. 441 Behre, Barbara 260 Heinz, E. 197 362 Sififel,P. 435 Behrmann, P. 427 Hejnowicz, Z. 266 Martinoia, E. 232 Stadler, R. 370 Beiman, Andrea 152 Henssen, A. 457 Matile, Ph. 13 Stal,L.J. 90 Berger, S. 382 Herppich, Margaretha 34 Matthys-Rochon, E. 313 Starke, J. 174 Bergman, B. 90 Herppich, W. 34 McClendon.J.H. 362 Steiner, E. 375 Bernadello, L. 292 Heuer, Susanne 146 Mergenhagen, D. 382 Stelzer, R. 300 Beyschlag, W. 285 Heyser,W. 427 Meyer, Doris 223 Steudle, E. 3 Bobäk,M. 26 Hierling, P. 3 Mikus.M. 26 Strack, D. 146 Böger, P. 161 Hilger.H.H. 387 Mohr.H. 81 Stubbe,W. 375 Bokern, Maria 146 Hinchee, Maud A. W. Mollenhauer, D. 343 Suter, Marianne 190 Braidot, E. 97 121,127 Mollenhauer, R. 343 Brass, Susanne 161 Hohl,N. 441 Möllers, C. 133 T Braunovä, Zuzana 435 Hunte, Carola 104 Moore, P. H. 70 Tazama, M. 421 Brunold, Chr. 190 Huppertz, K. 278 Moriyama, Y. 421 Traub.O. 104,111 C Hüsemann.W. 116 Tschanz,A. 190 Callies.R. 116 N Cappadocia, M. 319 Nagata, T. 63 U Cerana, Raffaella 273 Iwasaki, N. 421 Neininger, A. 81 Chrost,B. 174 Niehaus, Andrea 213 Ulimann, I. 285 Colombo, Roberta 273 J Nultsch, W. 278 Jacob, A. 41 V D Jacobsen, Karin Ruth 70 0 van Heusden, A. W. 331 Denecke, M. 367 Jahne, A. 140 Vechtel, B. 219 Dirk, J. 382 Okazaki.Y. 421 Vianello, A. 97 Döring, H. 457 K Villbrandt, M. 90 Dörr, M. 300 Kahmann, U. 219 P Vollbrecht, P. 306 Dumas, C. 313 Kantvilas, G. 457 Parniske, M. 18 von Lindern, L. 382 Kape, R. 343 Petrussa, E. 97 vonWillert,D. J. 34 E Kasemir, Helga I. 306 Postius, Christine 161 Vornlocher, H.-P. 47 Eller, B. M. 13 Katsaros, Chr. 400 Protoschill-Krebs, G. 206 W Ernst, Anneliese 161 Kenter, U. 161 R Wagner, G. 55 Eslava, A. P. 441 Kesselmeier, J. 206 Ratajczak, R. 260 Wenzel, G. 133 Ettl, H. 323 Kinzel, H. 355 Rennenberg, H. 180 Werner, D. 18 Kirst.G.O. 41 Rho, Myung-Sook 168 Wiencke, Chr. 41 F Klapheck, S. 174 Rico,V.J. 449 Wiermann, R. 407 Feierabend, J. 227 Klink, Rebecca 414 Roos.W. 253 Wild,A. 348 Fisher, D. G. 70 Kluge, M. 343 Rost, Th. L. 121,127 Willecke, K. 104,111 Flach, Barbara M.-P. 13 Knop, Mona 111 Rüdinger, M. 3 Witte, L. 152 Flammersfeld, U. 348 Kowalik, W. 168 Ruppel,H.G. 219 Wolff,D. 382 Fretz.A. 140 Krumbein, W. E. 90 Ryel,R.J. 285 Wollenweber, E. 300 Fukshansky, L. 362 Kutschera, U. 246 S Z G L Sanders, W. B. 449 Zancani, M. 97 Gaillard,A. 313 Lang, M. 435 Sandmann, G. 161 Zenk,M.H. 370 Galetto, L. 292 Laublin, Genevieve 319 Schlösser, G. 323 Zetsche, K. 395 Galland, P. 441 Läuchli, A. 1 Schmidt, Petra E. 18 Zimmermann, H. 174 Gisselmann, G. 213 Lechner, Ilse 355 Schnabl, Heide 104,111, Grolig, F. 55 Lehmann, H. 41 367 Gromes, R. 395 Leibfritz, D. 116 Schönherr, J. 345 Grosse, W. 223 Lichtenthaler, H. K. 435 Schreiber, L. 345 Index of Names of

A Caryophyllaceae 355,359 Cladoniaceae 467 Abromeitiella brevifolia 292 Caryophyllales 146 Cochranea species 387 - lorentziana 292 Catalpa bignonioides 362 Coelogyne ovalis 304 Acacia neovernicosa 304 Catharanthus roseus 237 Combretaceae 304 Acer pseudoplatanus 97,119, 227, - species 237 Combretumfarinosum 295 236 Cayratiajaponica 295 - species 304 Acetabularia acetabulum 382,422 Ceballosiafruticosa 387 Conophilus americana 228 - mediterranea 382 - species 387 Crassulaceae 360 - species 382 Celosia argentea 147 Crepis capülaris 355 Adiantum species 59 Ceratopteris species 127 Crocus sativus 320 Agrostis gigantea 178 Cerinthe species 387 Cucumis sativus 126 Aizoaceae 149 Chara australis 235 Cucurbitaceae 355 Algae 41,55,161,168,219,278, - corallina 3,421 Curcurbita species 127 323, 395,400 Characeae 3,421 Cuscuta europaea 228 Alnus glutinosa 223 Chenopodiaceae 149,359 - n?/7e*a 228 Alpinia speciosa 304 Chenopodium rubrum 116,146 Amanita muscaria 146 Chlamydomonas acidophüa 324 Cyanobacteria 90,161, 213, 343 Amaranthaceae 149,355 - aggregata 323 D Amaranthus lividus 355 - akinetos 323 Anabaena species 95 - applanata 323 Dacrydium biforme 459 - variabilis 95 - biconvexa 329 Daucus carota 146 Anacystis nidulans 161,214 - bipartita 329 Delesseria sanguinea 278 Ankistrodesmus braunii 221 - callosa 324 Deuterocohnia longipetala 292 Annonaceae 304 - culleus 324 Dictyostelium discoideum 47 Aphanacapsa species 214 - debaryana 324 - species 398 Arabidopsis thaliana 214,273, 395 - dysosmos 324 Dictyota dichotoma 284,404 Ascomycetes 449,457 - gloeophila 327 Dioscoreaceae 304 Aspergillus niger 155 - grandistigma 327 Discodermia calyx 67 Dolichos species 125 - oryzae 408 - humicola 323 Donatia novaezelandiae 463 Asperugo species 391 - insana 329 Dracophyllum milliganii 459 Aspicilia cinerea 450 - isogama 327 Drosera arcturi 463 - contorta 449 - komarekii 329 Drymonia serrulata 295 - crespiana 449 - kuteinikowii 327 Dunaliella bardawil 219 Astasia longa 228 - mantonii 329 - species 41 - species 227 - microscopica 327 Dyckiafloribunda 292 Asteraceae 304,355 - moewusii 324 - ragonesei 292 Asteraceae: Lactuceae 331, 337 - multitaeniata 324 Atriplex species 238 - noctigama 324 E Austropeltum glareosum 457 - oblonga 324 Ectocarpus siliculosus 400 Avena sativa 247 - perpusilla 327 Elodea canadensis 435 - species 55 - peterfii 324 - otewsa 118 - pitschmannii 324 Elymus repens 285 B - planoconvexß 329 Empetraceae 300 Basidiomycetes 427 - reinhardiy 323 Empetrum nigrum 300 Berberis stolonifera 370 - species 382,395 Endocarpon pusillum 449 Beta species 141 - sphaeroides 324 Enterobacteria 213 - vulgaris 147 - zeöra 324 Epifagus virginiana 227 Boraginaceae 387 Chlamydophyceae 329 Eremosphaera viridis 219 Boraginaceae-Heliotropioideae 387 Chlorella kessleri 168 Eritricheae 391 Boraginoideae 391 - species 41,190 Eritrichum canum 387 Boraginoideae-Cynoglosseae 391 Chlorococcales 221 Escherichia coli 213 Boraginoideae-Eritricheae 391 Chlorogonium elongatum 395 Eschscholtzia californica 374 Botryococcus braunii 221 Chlorophyceae 219 Eucalyptus pilularis 427 Bradyrhizobiumjaponicum 18 Chlorophyta 323 Euglena gracilis 227 Bromeliaceae 292 Chondrus crispus 278, 284 - species 190,227,382 Bromelioideae 292 Chorellafusca 397 Bryophyllumfedtschenkoi 367 Ciadia aggregata 463 Bulbophyllum gymnopus 304 - fuliginosa 463 F Byronia dioica 355 - inflata 463 Fao/Ms sylvatica 13 - moniliformis 463 Ficuspumila 126 C - retipora 463 Flemingia chappa 304 Calluna vulgaris 300 - sullvanii 463 Flourensia oolepis 304 Campsis radicans 295 Cladina confusa 463 Freesia species 320 Carpha curvata 459 - southlandica 463 Fritschiella tuberosa 221 VIII Bot Acta 105 (1992)

Fungi 343,345,427,441 K Oreobolus oligocephalus 463 Fusarium oxysporum 152 Kalanchoe daigremontiana 236, Oscillatoria limosa 90 260,415 - rubescens 161 G Oxalidaceae 355 Galium aparine 84 L Ctea/is stricta 355 Geosiphon pyriforme 343 Lampranthus sociorum 146 Geum urbanum 355 Larrea nitida 304 P Ginkgo biloba 13 Lathrea clandestina 228 Peltigera species 458 Gladioulus species 320 Lecanorales 449 Penicillum cyclopium 253 Globodera rostochiensis 133 Leguminosae 304 Phaseolus vulgaris 158,195 Gloecapsa species 460 Lemna minor 195 Philophorus species 467 Glomus species 343 Leptospermum scoparium 459 Phormidium persicinum 165 Glycine max 18 Lichens 449,457 Phycomyces blakesleeanus 441 Gomphrena globosa 147 Liliaceae 407 Phycorys rubens 278 Gonyaulax species 382 Lithospermeae 387 Phyllophora truncata 278 Gossypium hirsutum 253 Loroglossum hircinum 304 Phytophthora megasperma 18 - species 238 Lupinus polyphyllus 240 Picea aötes 180,190, 345, 348,435 Graminaceae 360 Lychnis viscaria 355 Pilophorus species 463 Gymnoschoenus species 463 Lycopersicon esculentum 153 P/hms contorta 306 Gypsophila species 359 - peruvianum 152 - radiata 306 - sylvestris 306,427 H M Pisolithus tinctorius 427 Haematococcus lacustris 221 Marchantia species 227 Pisonia species 427 - species 219 Melampyrum pratense 228 Pz'sMm sativum 121,127,195 Hakea species 128 Melilotus alba 233 Pitcairnioideae 292 Halichondria okadai 63 Membranoptera alata 278 Plantago maritima 239 Halobacteria 174 Menispermaceae 370 - raedza 239 Halopterisfilicina 400 Mesambryanthemaceae 34 - species 238 Helianthus annuus 111 Mesambryanthemum Platymonas subcordiformis 44 Heiichrysum species 463 crystallinum 34, 264,414 Pleurophascum grandiglobum 463 Heliophytum species 387 Mesotaenium caldariorum 60 Poaceae 174 Heliotropioideae 387 - species 57 Polycarpus dacrydioides 459 Heliotropium arborescens 388 Micarea austroternaria 463 Polygonaceae 359 - digynum 388 Microseris bigelovii 331,337 Polyneura hilliae 278 - erosum 388 - elegans 333,338 Pontederia species 130 - indicum 388 Mirabilisjalapa 147 Porphyra purpurea 44 - maris-mortui 388 Mougeotia scalaris 56 - umbilicalis 41 - messerschmidioides 388 - species 55 Potentilla recta 355 - pectinatum 388 Mühria species 467 Prasiola crispa 41 - peruvanium 388 Protaceae 126 - species 387 N Puccinellia distans 285 - supinum 387,388 Narcissus pseudonarcissus 227 Pi/ya spathacea 292 - zeylanicum 388 Neophyllis melacarpa 463 Pycnothelia caliginosa 463 Hevea brasiliensis 235 Neopongiococcum species 221 Hordeum distichum 31 Nephrops norvegicus 109 R - species 272 Neurospora crassa 234 Rhizobium leguminosarum 18,91 - vulgare 31,140,247 - species 422 - meleloti 18, 215 Hyoscyamus niger 318 Nicotiana species 141 Rhodospirillum rubrum 91,215 - tabacum 64,81,214,435 Richia curtisiae 463 I Nitellaflexilis 3 Rochelia species 387 Iresine lindenii 147 - species 248,271 Rosaceae 355 Iris ensata 319 Nostoc punctiforme 343 - germanica 319 Nyctaginaceae 149 S - hollandica 319 Saccharomyces cerevisiae 214 - pseudacorus 319 0 Saccharum qfficinarum 70 - pumila Oenothera biennis 375 - species 177 - setosa 319 - e/ata 377 Salmonella typhimurium 214 - versicolor 319 - grandiflora 375 Scenedesmus species 172 Isophysis tasmanica 459 - nutans 375 Sclerocaryopsis species 391 Ixorhea tschudiana 391 - suaveolens 377 Setariafaberi 178 - villosa 375 Sinapis alba 81,215 J - «;op 375 Sinorhizobiumfredü 18 Jatrorrhiza palmata 370 Orchidaceae 304 Siphula decumbens 459,463 Orc/ws militaris 304 - foliacea 463 Bot Acta 105 (1992)

- fragilis 463 T V - jamesii 463 Tamus communis 304 Vallisneria spiralis 435 Solanaceae 152 Tetracystis species 329 Viciafaba 104,111,121,274,367 Solanum nigrum 111 Tetraselmis species 41 - sativa 24 - tuberosum .133,158 - subcordiformis 44 Viminaria juncea 126 . Solenopsora holophaea 458 Thalictrum species 374 Vitis riparia 362 Sorghum species 177 Tiaridium species 387 Volvocales 221,329 - tricolor 240 Tilia americana 362 Volvox species 395 Sphacelaria rigidula 400 Tillandsioideae 292 - tribuloides 400 Trentepohlia aurea 219 Z Spinacia oleracea 81,198 Trichoderma reesi 155 Zea diploperennis 29 Spirogyra species 61 Trichodesmium thiebautii 94 - mays 26,56,206,247,313,360, Stachys sieboldi 233 Triticum aestivum 31,84,174,206, 422 Stereocaulaceae 457 247 - species 177 Stereocaulon sorediiferum 467 Tulipa gesneriana 266 Zingiberaceae 304 - species 463 - species 407 Zuccagnia punctata 304 Suillus bovinus 427 Tussilagofarfara 355 Zygnemataceae 57 Synechococcus elongatus 161 Zygomycetes 441 - leopoliensis 161 U Zygophaceae 304 - rubescens 161 Ulotrichales 221 - species 214 Ulva rotundata 279 Synechocystis species 161,214 Umbilicaria species 453 Uvaria angolensis 304 Subject Index

A tracer experiments 427 Calcium Absorbance spectra Apoplastic transport plant metabolism 355 cyanobacterial picoplankton of oflanthanum nitrate 427 Calmodulin Lake Constance 161 ofsulphorhodamineG 427 Stimulation of Phosphodiesterase Absorption coefficient through fungal sheath 427 activity 395 of plant leaves 362 Apothecial development Caloxanthin 161 Acidity pattern ofAustropeltum glareosum 457 CalyculinA 63 in tulip leaves 266 Ascocarp ontogeny CAM 34,260,414 Action spectra of Austropeltum glareosum 457 deinduction 34 gravitropic equilibrium of fungi Astaxanthin esters 219 induction 34,414

441 Atmospheric trace gases 206 V0VrATPaseinleaftonoplast 414 photoinhibition of photosynthesis ATPase activity cAMP

in red algae 278 inhibited by bafilomycin At 421 concentration in algal cells 395 phototaxis 47 Authentic strains Canadine 372 Acylated betacyanins ofChlamydomonas 323 Canopy photosynthesis biosynthesis 146 Auxin antagonists roadside grasses 285 purification from cell cultures effect on root development 121 Canthaxanthin 219 ofChenopodium rubrum 146 p-chlorophenoxyisobutyric acid Carbohydrate transport 232 Acyltransferase 121 Carbonic anhydrase from cell cultures of Auxin(s) 246 metabolism of COS 206 Chenopodium rubrum 146 Auxin inhibitors Carbonyl sulfide (COS) Adenosine 5'-phosphosulfate 3,3a-dihydro-2-(p-methoxy- metabolism in plants 206 sulfotransferase in Picea abies phenyl)-8//-pyrazolo[5,1 -a]iso- ß-Carotene 161 190 indol-8-one 121,127 Carotenoid(s) 161 Adenylate cyclase efffect on root fasciation 121 Carotenoid esters activity Stimulation by guanosine- 2,3,5-triiiodobenzoic acid 121 in senescent Ginkgo leaves 13 (0-3-thio)triphosphate 395 Cation transport 237 G-protein regulation in Cell(s) Chlorogonium 395 B of Acer pseudoplatanus 97 Air pollutants Bafilomycin Aj iron reduction 97 effect on thylakoid membranes inhibitor of vacuolar pH regulation oxygen reduction 97 348 421 Cell cultures Beer Alcohol content of beer 3 of Chenopodium rubrum 146 Algae alcohol content 3 of Hordeum vulgare 140 brown osmotic biosensor for alcohol content oi Lycopersicon peruvianum 152 3 polarized cells 400 routine maintenance 140 green 41,55,219,323,395 Berberine alkaloids 372 Cell cycle Berberrubine 372 cAMP concentration in cells 395 changes in microtubules 63 Biopterin 441 red effect of calyculin A 63 Biosynthesis photoinhibiton of photo• effect of okadaic acid 63 acylated betacyanins 146 synthesis 278 Progression in plant cells 63 4,4'-bisjatrorrhizine 370 recovery of photosynthesis 278 Cell morphology ofsporopollenin 407 Alkaloid demethylation cyanobacterial picoplanton of 4,4'-Bisjatrorrhizine 370 peroxidase-catalyzed 370 Lake Constance 161 biosynthesis 370 Alkaloid dimerization Cell Suspension cultures Blue light peroxidase-catalyzed 370 ofLycopersicon peruvianum 152 effect on photosynthetic apparatus Amaranthin Cell viability ofChlorella 168 esterification 146 after cryopreservation 140 Blue light adaptation Amino acid transport 234 of Hordeum vulgare 140 oiChlorella 168 (R)-(l -amino-2-phenylethyl) Cell wall Blue-green fluorescence phosphonic acid inhibition of autolysin, of Chlamydomonas 323 of plant leaves 435 nodulation 18 extensibility 246 Boundary tissue Ammonium accumulation formation 313 of Austropeltum glareosum 457 effects on root development 306 thickness 247 Buds Ammonium toxicity 306 ultrastructure 247 Amoebae Cell wall-bound phenolics of Norway spruce 180,190 of cellular slime mold 47 in cell Suspension cultures of Androgenesis 313 Lycopersicon peruvianum 152 Anion transport 235 C Cellufluor 427 Antennae chlorophyUs Celosianins I and II 146 C3-Photosynthesis increased oxidation speed 348 transition to CAM 34,414 Chalcone(s) Apoplast 427 Ca2+/calmodulin 2,4-dihydroxy- 300 acidic layers 253 control of Phosphodiesterase 2',4-dihydroxy- 301 pH oscillations in tulip 266 activity 395 2',4'-dihydroxy-4'-methoxy- 302 Caffeoylshikimic acid 152 Bot. Acta 105 (1992)

2' ,4' -dihydroxy-6' -methoxy- Grassulacean acid metabolism; Elicitation dihydro- 302 see CAM of cell Suspension cultures of ofEmpetrum nigrum 300 Cryopreservation Lycopersicon peruvianum 152 2' -hydroxy-4' ,6' -dimethoxy- of embryogenic Suspension Elongation growth dihydro- 302 cultures 140 roleofepidermis 246 2'-hydroxy-4'-methoxy- 301 ofHordeum vulgare 140 Embryogenic cell suspensions 4'-hydroxy-2'-methoxy- 302 Cryoprotectants, effects of 140 cryopreservation 140 Characean internode Cyclic adenosine-3' ,5' - of Hordeum vulgare 140 alcohol content of beer 3 monophosphate, see cAMP Endosymbiosis Cheilanthifoline 372 cys-gene homologies 213 fungus and eyanobacterium 343 Chlorogenic acid 152 Cytochromes 348 Enzymology 146 p-Chlorophenoxyisobutyric acid 121 Cytogenetic analysis Epicuticular wax 345 Chlorophyll breakdown ofOenothera grandiflora 375 Epidermis 246,266 in senescent Ginkgo leaves 13 role in elongation growth 246 Chlorophyll fluorescence D of tulip leaves, travelling of plant leaves 435 Deep-freezing acidity pattern 266 Chloroplast(s) 197, 227,435 of embyrogenic Suspension Epiphytic fiuigi contribution to leaf cultures 140 effect on foliar uptake of fluorescence 435 of Hordeum vulgare 140 chemicals 345 Chloroplast movement Dehydrocheilanthifoline 372 Ethyleneglycol-bis(2-aminoethyl- in Mougeotia 55 Dehydrocorygovanine 372 ether)-N,N,N' ,N' -tetraacetate, Chromoplasts 227 Dehydroscoulerine 372 see EGTA Circadian rhythm Developmental control, of CAM 34 Evolution oxygen production ofAcetabularia Diacylglycerol selectivity ofOenothera species 375 382 in chloroplasts 197 External pH Cl" conduetance Diagnosis effect on intracellular pH 116 oiArabidopsis thaliana plasma of Austropeltum glareosum 457 Exudate localization membrane 273 of Chlamydomonas applanata in Empetrum nigrum 300 Classification 323 of cyanobacterial picoplankton Dihydrochalcones, see also chalcones F from Lake Constance 161 ofEmpetrum nigrum 300 Fe-Protein 90

C02-assimilation Dihydroflavokawin-B 302 Ferredoxin: sulfite reductase 213 effect of external pH 116 3,3a-Dihydro-2-(p-methoxyphenyl)- Ferulicacid 152 in photoautotrophic cell cultures of 8/7-pyrazolo[5,1 -a]isoindol- Feruloylamaranthin 146 Chenopodium rubrum 116 8-one 121,127 Feruloyltyramine 152 Columbamine 372 9,10-Dihydrophenanthrenes Flavine adenine dinucleotide (FAD) Comparative physiology 4,5-dmydroxy-2,3-dimethoxy- 304 441 internal hght environment of ofEmpetrum nigrum 300 Flavins 441 leaves 362 5-hydroxy-2,3,4-trimethoxy- 302 Flavonoids mineral metabolism 355 Dinitrogenase 90 role in plant development 18 Complex-heterozygotes Dinitrogenase reductase 90 Flowers ofOenothera grandiflora 375 Dispersal of Heliotropium 387 Confocal pH topography ofMicroseris bigelovii 331,337 Fluorescence of plant cells 253 Dispersal units of senescent Ginkgo leaves 13 Conifers 306,345 ofHeliotropium 387 Fluorescence spectra 161 effect of ammonium on roots 306 Diurnal cycle cyanobacterial picoplankton of effect of surface microflora on nitrogenase activity in Lake Constance 161 foliar uptake 345 Oscillatoria 90 Flushing Connexin(s) 104,111 DNA polymorphisms, nuclear of Norway spruce 180 Connexin-like proteins 104,111 ofMicroseris bigelovii 331,337 Foüar senescence immunological detection in of Ginkgo biloba 13 Viciafaba 104 E Foüar uptake of chemicals in mesophyll chloroplasts of Echinenone 219 effect of leaf microflora 345 Heliathus annuus 111 Ecophysiology Freeze fracturing in mesophyll chloroplasts of mineral metaboHsm of plants 355 leaftonoplast 414 Viciafaba 111 Ectomycorrhizae Fruits in plasma membrane of apoplastic transport through fungal ofHeliotropium 387 Vicia faba 104 sheath 427 Fungal sheath Corygovanine 372 ofPinus sylvestris/Suillus of ectomycorhizal roots 427 p-Coumaric acid 152 bovinus 427 of Pinus sylvestris/Suillus p-Coumaroylamaranthin 146 EGTA bovinus 427 p-Coujnaroylsliikimic acid 152 inhibition of Phosphodiesterase p-Coumaroyltryamine 152 activity 395 Bot. Acta 105(1992)

G biosynthesis of acylated beta• Leafage

G-Protein cyanins 146 andC3toCAMshift 34 regulation of adenylate cyclase in Caryophyllales 146 Leaf epidermis in Chlorogonium 395 in cell cultures of Chenopodium blue-green fluorescence 435 Gel eletrophoresis rubrum 146 Leafexudates protein-pigment complex of 1 -O-Hydroxycinnamoyl-ß-glucose: ofEmpetrum nigrum 300 Dictyostelium discoideum 47 amaranthin O-hydroxycinnamoyl- Leaf optics Gene expression 81, 227 transferase 146 comparative study 362 nitrate reductase, control 81 Hypothallus 449 Leaf surface microflora in non-green plants 227 Hypoxia effect on foliar uptake of Genotypes nutrient uptake of roots 223 chemicals 345 of Iris 319 Leafyellowing Ginkgofluor 15 I in Ginkgo biloba 13 Glandulär cells Immature inflorescences Leukoplasts 227 ofEmpetrum nigrum 300 oflris 319 Lichen(s) 449,457 y-Glutamylcysteinylserine Immunoblots occurrence ofAustropeltum new homologue of glutathione detection of phytochrome in glareosum 457 174 Mougeotia 55 Liehen development in Poaceae plants 174 Immunofluorescence 55,111,400 role of rhizomorphs 449 in Triticum aestivum 174 brown algae 400 Lichenization Glutathione 174 localization of connexin-like of rhizomorphs 449 in Picea abies 180,190 proteins 111 Light and growth 246 Glutathione reductase 174 locahzation ofMougeotia Light competition Glyceollin I phytochrome 55 ofroadside grasses 285 content in root exudate 18 microtubule Organization in Light gradients content in root hairs 18 polarized cells 400 of plant leaves 362 Gravitropic equilibrium of fungi Immunolocalization Light propagation action spectra 441 nitrogenase activity in Oscillatoria in plant tissue 362 Groenlandicine 372 90 Lignification Growth characteristics Intracellular pH 116, 253 and sugar transfer 70 cell Suspension cultures of effect of external pH 116 of sugarcane stem 70 Lycopersicon peruvianum 152 Intramembraneous particles Lipid bodies Guanosine-(0-3-thio)-triphosphate increased size 414 content of secondary carotenoids Stimulation of adenylate cyclase Iodoacetamide 133 219 activity 395 Ion Channels 239 Lipid inhibition Guard cells Ion currents of sulfoo^iinovosyltransferase ofViciafaba 367 ofArabidopsis thaliana plasma activity 197 membrane 273 Lipophilic phenolics Iron reduction ofEmpetrum nigrum leaves 300 H in Acer pseudoplatanus 97 Lucifer yellow 313 H+-ATPase Isoamaranthin Lutein 168 selective reconstitution of tono- esterification 146 Lutein ester 219 plast 260 Isoenzymes 133 Hartig net analysis in potato hybrids 133 M distribution of sulphorhodamine G madl gene mutants 427 ofPhycomyces blakesleeanus 441 Head and stalk structure J Jatrorrhizine 370 Magnesium VoVrATPase 414 plant metabolism 355 Heterologous hybridization 213 precursor of 4,4'-bisjatrorrhizine 370 Magnesium shift Heterotrophic cell Suspension chloroplast enzyme activities 197 cultures Malate formation of Lycopersicon peruvianum K effect of external pH 116 152 + K conductance in photoautotrophic cell cultures Hill reaction ofArabidopsis thaliana plasma of Chenopodium rubrum 116 in Chlorella 168 membrane 273 Mericarpids Homoglutathione 174 Km values ofHeliotropium 387 Hummingbird poUination 292 chloroplast enzyme activities 197 Mesophyll protoplasts Hybridization ofHelianthus annuus 111 heterologous 213 L ofVicia faba 111 ofOenothera grandiflora 375 Lanthanum nitrate presence of connexin-like proteins ofpotatos 133 apoplastic distribution 427 111 p-Hydroxybenzaldehyde 152 Lateral root development Metabolie inhibitors Hydroxycinnamic acid(s) 146 control 121,127 iodoacetamide 133 Hydroxycinnamic acid transferases spacing intervals 127 rhodamin-6-G 133 Bot. Acta 105(1992) XIII

Methylglucose immunolocalization in Oscillatoria pH effects foliar uptake 345 90 on root development 306 Metribuzin Nodule initiation pH mapping tolerance of potato hydrids 133 inhibited by (R)-(l-amino-2- of Gossypium hirsutum cells 253 Microinjection phenylethyl)phosphonic acid 18 pH optimum of DNA to microspores 313 Norflurazon of UDP-sulfoquinovose: diacyl- Microspores inhibition of secondary carotenoid glycerol sutfoo^iinovosyltrans- microinjection of DNA 313 synthesis 219 ferase 197 Microtubule(s) 63,400 Norway spruce 180,190 pH regulation 253,421 changes in cell cycle enzyme activity 190 of Chara vacuoles 421 Progression 63 needle development 180 Phenotypic correlations cytoskeleton in brown algae 400 Nostoxanthin 161 ofMicroseris bigelovii 337 Organization 63,400 Novel forest decline 345, 348 Phenylalanine Mineral metabolism Nutrient uptake 223,355 labeled, incorporation into ecological implications 355 and mineral metabolism 355 sporopollenin 407 MoFe-Protein 90 of roots under hypoxia 223 specifically labeled 407 Molecular phylogeny Phloem unloading ofMicroseris bigelovii 331 0 and developmental changes in Monoclonal antibody Z-3B1 55 Okadaic acid anatomy 70 Morphogenesis inhibition of protein phosphatases sugarcane stem 70 in lichens 449 63 3' -Phosphoadenylyl sulfate Morphology probe for cell cycle progression 63 reductase 213 of Heliotropium dispersal units Ontogenetic diferentiation 227 Phosphodiesterase 387 Ontogeny activity inhibition by EGTA 395 Multicellular pseudoplasmodia (slugs) of Heliotropium 387 activity inhibition by trifluoperazin 47 Open-top Chambers 348 395 Multiflux theory Optical brighteners activity Stimulation by calmodulin hght gradients in leaves 362 in senescent Ginkgo leaves 13 395 Multispecies canopy model Optical properties control by Ca2+/calmodulin 395 roadside grasses 285 of senescent Ginkgo leaves 13 Phosphoenolpyruvate carboxylase Organellar DNA 227 206, 367 Osmotic biosensors 3 metabolism of COS 206 N Osmotic cells 3 phosphorylation 367 NADH-dependent peroxidases 97 Osmotic stress Phosphorylation Nandinine 372 effect on Prasiola crispa 41 of phosphoenolpyruvate Near-UV/blue-light photoreceptor ultrastructural changes in Prasiola carboxylase 367 441 crispa 41 Photoautotrophic cell cultures Nectar Ovary(ies), in vitro culture 319 116,152 removal 292 Oxidation speed of Chenopodium rubrum 116 secretion pattern 292 increased, of antennae Chlorophylls of Lycopersicon peruvianum 152 sugar content 292 348 Photoautotrophy Needle development Oxygen production cell Suspension cultures of of Picea abies 180,190 ofAcetabularia 382 Lycopersicon peruvianum 152 Neopterin 441 Oxygen reduction Photobehavioral mutants Neoxanthin 168 in Acer pseudoplatanus 97 ofPhycomyces blakesleeanus 441 Nicotine Ozone 348 Photoinhibition 278 inhibition of secondary of photosynthesis in red algae 278 carotenoid synthesis 219 P Photomixotrophic cell Suspension Nitrate 81,306 P-700 348 cultures effect on nitrate reductase gene Parasitic plants 227 of Lycopersicon peruvianum 152 expression 81 Patch-clamp investigation Photoreceptor(s) plant response 306 ofArabidopsis thaliana plasma offungi 441 Nitrate reductase membrane 273 Photoreceptor pigment control of gene expression 81 Pentachlorophenol ofDictyostelium discoideum 47 Nitrite reductase foliar uptake 345 Photosynthesis control of gene expression 81 Peripheral cytoplasm in red algae 278 Nitrogen deficiency acidic layers 253 Photosynthetic apparatus secondary carotenoids of Peroxidase(s) 97,370 ofChlorella 168 Chlorophyceae 219 from Berberis stolonifera 370 Phototaxis Nitrogen fixation catalysis of protoberberine alkaloid action spectra 47 by Geosiphon pyriforme 343 demethylation 370 of multicellular pseudoplasmodia Nitrogenase 90,343 catalysis of protoberberine alkaloid (slugs) 47 diurnal cycle in Oscillatoria 90 dimerization 370 Phototropism 441 of Geosiphon pyriforme 343 NADH-dependent 97 Phycobilisomes XIV Bot. Acta 105 (1992)

cyanobacterial picoplankton of Protein phosphatases 1 and 2A spacing intervals 127 Lake Constance 161 inhibition by calyculin A 63 of Pisum sativum 121,127 Phylogenetic adaptation 227 inhibition by okadaic acid 63 Root exudate Phytoalexins 18 Protein phosphorylation content of glyceollin I 18 Phytochrome(s) 55,81 and cell cycle progression 63 Root fasciation control of nitrate reductase gene inhibited by okadaic acid 63 effect of auxin inhibitors 121 expression 81 Prothallus 449 of Pisum sativum 121 localization in Mougeotia 55 Protoberberine alkaloids 370 Root hairs Phytoflagellate peroxidase-catalyzed de- content of glyceollin I 18 cellularcAMPlevel 395 methylation 370 Root primordial mass 121 Picoplankton peroxidase-catalyzed dimerization cyanobacterial, from Lake 370 S Constance 161 Proton efflux Salt stress 34,41 Pigment(s) 13,161,168,219 variable in tulip leaves 266 and CAM induction 34 ofEremosphaera viridis 219 Proton pumps 232 ultrastructural changes in in senescent Ginkgo leaves 13 Protoplast(s) Prasiola crispa 41 Pigment composition of Acer pseudoplatanus 97 Scattering coefficient cyanobacterial picoplankton of iron reduction 97 of plant leaves 362 Lake Constance 161 oxygen reduction 97 Scoulerine 372 Pigment-protein complexes Protoplast fusion Seasonal changes ofChlorella 168 of dihaploid potato clones 133 sulfur metabolism of Picea abies Plant cells, confocal pH topography Protoporphyrin 180,190 253 and phototaxis ofDictyostelium Secondary carotenoids Plant cuticle 345 discoideum 47 of Chlorophyceae under nitrogen Plant leaves, blue-green fluorescence Pseudopodetium deficiency 219 emission 435 of Austropeltum glareosum 457 Secondary metabolite transport 240 Plant regeneration Pterins 441 Shoot/root ratio oilris 313,319 6,7-dimethyl- 441 of Pinus sylvestris 306 of transgenic maize 313 Pycnidial development Slime molds 47 Plant tissue, light propagation 362 of Austropeltum glareosum 457 Slugs 47 Plasma membrane(s) 104, 273 Solubilization 260 ofArabidopsis thaliana 273 R Solute transport 3 presence of connexin-like Radioimmunoassay Somatic hydridization, for potato proteins 104 glyceollin I content in soybean roots breeding 133 ofViciafaba 104 18 Soybean roots PlastidicDNA 227 Reconstitution content of glyceollin I 18 Plastidic factor selective, of tonoplast H+-ATPase infected 18 control of nitrate reductase gene 260 Spectral analysis expression 81 Recovery Single cells ofAcetabularia 382 Plastidic RNA-polymerase 227 of photosynthesis in red algae 278 Sporopollenin Plastids Red light biosynthesis 407 defective 227 effect on photosynthetic apparatus degradation 407 non-green 227 ofChlorella 168 incorporation of 14C-labels non-photosynthetic 227 Red light adaptation 407 31P-NMR spectroscopy ofChlorella 168 Squamules in vivo 116 Reflectance formation 449 of photoautotrophic cell cultures of senescent Ginkgo leaves 13 Starch granules of Chenopodium rubrum 116 Rhizomorphs in Prasiola crispa 41 Polarized cells of squamulose lichens 449 Stern anatomy immunofluorescence study of Rhodamin-6-G 133 developmental changes on phloem microtubules 400 Riboflavin 441 unloading 70 microtubule Organization 400 Ribulose-1,5-bisphosphate carboxy• developmental changes on sucrose Pollen 407 lase, metabolism of COS 206 storage 70 Populations Roadside grasses sugarcane 70 ofMicroseris bigelovii 331, canopy photosynthesis 285 Suberization 337 light competition 285 andsugartransfer 70 Potassium Root(s) 18,121,127, 223, 306 of sugarcane stem 70 plant metabolism 355 Root development 121,127, 306 Substrate induction Potato breeding effect of auxin inhibitors 121,127 nitrate reductase gene ex• somatic hydridization 133 effect of exogenous ammonium pression 81 Pressurized gas transport 306 Sugar storage effect on nutrient upatke of roots lateral 121,127 and developmental changes in under hypoxia 223 effect of auxin inhibitors 121,127 anatomy 70 Principal components ofPisum sativum 121,127 in sugarcane stem 70 ofMicroseris bigelovii 337 Bot Acta 105 (1992)

Sulfate assimilation Tissue tension 246 V

in higher plants 213 Tobacco BY-2 cells V0VrATPase in Picea abies 180,190 cell cycle progression 63 head and stalk structure 414 Sulfate reduction Tonoplast 232,260,414 structural changes 414 in higher plants 213 freeze fracturing 414 Vacuolar ATP-dependent FT pump in Picea abies 180 selective reconstitution of inhibited by bafilomycin Ai 421 in prokaryotes 213 FT-ATPase 260 Vacuolar pH regulation

Sulfite reductase 213 Tracer experiments inhibited by bafilomycin At 421 Sulfur metabolism of Picea abies labeled Phenylalanine 407 Vacuolar proton pump

seasonal variations 180,190 sporopollenin biosynthesis 407 effect of bafilomycin At 421 Sulfur nutrition Transformation Vacuoles 41,232 in Picea abies 180 by DNA microinjection 313 of higher plants, transport pro• Sulphorhodamine G Transport processes 232 cesses 232 apoplastic distribution 427 amino acids 234 of Prasiola crispa under salt Symbiosis anions 235 stress 41 Glycine max/Bradyrhizobium carbohydrates 232 Vanillin 152 japonicum 18 cations 237 Vegetation Syn-mericarpids secondary metabolites 240 global sink for carbonyl sulfide ofHeliotropium 387 vacuoles of higher plants 232 206 Systematics Travelling wave revised of Chlamydomonas 323 acidity pattern in tulip leaves 266 Violaxanthin 168 Tree roots T nutrient uptake under W Temperature compensation hypoxia 223 Water relations 3 oxygen production ofAcetabularia Trifluoperazin Western blot analysis 90,104 382 inhibition of Phosphodiesterase localization of connexin-like Temperature dependence 278,382 activity 395 proteins in Vicia faba 104 oxygen production ofAcetabularia 2,3,5-Triiiodobenzoicacid 121 localization of nitrogenase in 382 Tulip leaf Oscillatoria 90 of photosynthesis in red algae 278 travelling acidity pattern 266 Wetlandtrees 223 Tetrahydrogroenlandicine 372 Turgor pressure 246 X Tetrahydroprotoberberines 372 Xanthophylls 161 Tetrahydrothahfaurine 372 U Xanthopterin 441 Thalifaurine 372 UDP-sutfocpiinovose: diacylglycerol Thallus morphology sulfoo^iinovosyltransferase 197 Z of Austropeltum glareosum 457 Unicellular cyanobacteria Zeaxanthin 161 Thiols from Lake Constance 161 in Picea abies 180 Untrastructural changes Thylakoid membranes 348,435 of Prasiola crispa under contribution to leaf fluorescence salt stress 41 435 Uvangoletin 302 effect of air pollutants 348 XVI Bot. Acta 105(1992) Production of the Phytoalexin Glyceollin I by Soybean Roots in Response to Symbiotic and Pathogenic Infection

Petra E. Schmidt, M. Parniske, andD. Werner Fachbereich Biologie der Philipps-Universität Marburg, Marburg, FRG

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

Abstract Key words

The amount of the phytoalexin glyceollin I Flavonoids, nodule initiation, Glycine max, in root exudate and root hairs of individual seedlings of Bradyrhizobium japonicum, Phytophthora megasperma Glycine max (L. Merr. cv. Preston) was analysed using a f. sp. glycinea, root exudate, root hairs. radioimmunoassay. Bradyrhizobium japonicum 110spc4, which is able to form nitrogen fixing nodules with this Abbreviations and Symbols plant, caused an increase of up to 50-fold in glyceollin I levels in root exudate relative to uninfected control APEP: (R)-(l-amino-2-phenylethyl)phosphonic seedlings. Maximum glyceollin I levels were reached acid within 10 h of incubation. Elevated glyceollin I levels were cfu: colony forming units also observed after incubation of soybean roots in sterile CHS: chalcone synthase bacterial supernatant, a Suspension of autoclaved MES: 5 mM morpholinethanesulfonic acid, bacteria or the supernatant from broken cells of Brady• 1 mM CaCl2, pH 6.2 rhizobium japonicum. Increased glyceollin I production is PAL: phenylalanine-ammonia-lyase not due to the process of active root hair penetration by rtm: root tip mark the microsymbiont since living bacterial cells are not XMM: xylose minimal medium necessary for the induction. The observed glyceollin I production in response to Bradyrhizobium japonicum is several times lower than that after pathogenic infection. Infection with zoospores of the phytopathogenic oomycete, Phytophthora megasperma f. sp. glycinea 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 re• sults support the idea that phytoalexins are implicated in determination of compatibility in pathogenic interactions. Crude cell extracts of different symbiotic bacteria {Bradyrhizobium japonicum 110s/?c4, Rhizobium meliloti 2011, Rhizobium leguminosarum PRE 8, Sinorhizobium fredii HH 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 Glycine max. 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.

Bot. Acta 105 (1992) 18-25 © Georg Thieme Verlag Stuttgart • New York Glyceollin I Accumulation in Soybean Rhizosphere Bot. Acta 105 (1992)

Introduction roots, an effect that was also found with synthetic auxin transport inhibitors (Hirsch et al., 1989). The symbiosis between the soybean Glycine max and the Gram-negative soil bacterium Brady• Taken together, these findings suggest that rhizobium japonicum (110spc4) results in the formation of flavonoids might be involved in meristem induction in both root nodules in which the bacteria actively fix atmospheric developmental processes, nodule and lateral root mor- nitrogen. It is well established that the early stages of the phogenesis. If this is true, then one should expect an rhizobia-legume interaction can be subdivided into two alteration of local flavonoid concentration preceeding fundamentally different physiological processes (Long, meristem formation. It has already been shown that the 1989): on the one hand, rhizobia infect root hairs and flavonoid composition of root hairs undergoes changes thereby resemble plant pathogens invading host tissues during the symbiotic infection process (Parniske et al., (Djordjevic et al., 1987). On the other hand, rhizobia in- 1988). In this paper we restricted our measurement to duce changes in the developmental program of root cells so glyceollin I concentrations in root hairs and root exudate that they become meristematic (Rolfe and Gresshoff, 1988). and report a significant glyceollin I induction by Brady• rhizobium japonicum 110spc4. A possible physiological One group of secondary plant products, the role for the symbiotic glyceollin I production is discussed. flavonoids, has been implicated both in plant defense re- actions as well as in plant developmental processes. The Materials and Methods isoflavonoid glyceollin I is an antibiotic phytoalexin found Chemicals in Glycine max. Phytoalexins are considered to play an im• portant role in pathogenic interactions as an early plant de• The PAL inhibitor (RMl-amino-2-phenylethyl)- fense response (Hahn et al., 1985; Ebel and Grisebach, phosphonic acid a generous gift of Dr. E. K. Baylis, Ciba-Geigy, 1988). A well investigated plant-pathogen system is root Manchester, USA. The antiserum against glyceollin I was a gift and stem rot of soybean caused by Phytophthora mega• from Prof. Dr. H. Grisebach, Freiburg, Germany. Anti-rabbit- solid-phase was obtained from Fa. Laboserv, Gießen, Germany. sperma f. sp. glycinea. Two lines of evidence indicate that Perlite was obtained from the Deutsche Perlite GmbH, Dortmund, glyceollin I is an important early defensive barrier erected Germany and Vermiculite from the Deutsche Vermiculite by the plant against the invading pathogen and involved in Dämmstoff GmbH, Sprockhövel (Haßlinghausen), Germany. determination of cultivar/race specific compatibility. Firstly, a detailed analysis of the distribution of fungal hy- Growth of seedlings phae in plant tissue and the spatial and temporal pattern of glyceollin I accumulation was performed by Hahn et al. Soybean seeds {Glycine max cv. Preston, Pioneer (1985). They found that limitation of fungal growth in the Hi-Bred Intern. Inc., Iowa, USA, Glycine max cv. Maple Arrow, incompatible interaction is correlated with a rapid Agriculture Canada Forage section, Ottawa, Ontario, Canada, Glycine max cv. Kenwood, Iowa State University, Arnes, USA) accumulation of glyceollin I at the infection site. Secondly, were surface sterilized for 10 min in 30% H202, washed treatments that reduce the amount of phytoalexin present 7-10times with sterile H20 and soaked for 6-8hours in sterile in resistant plants cause a corresponding increase in H20. Seedlings were grown in Vermiculite/Perlite (1:1) for 2 days sensitivity to pathogens. For example, Waldmüller and at 25 °C, 13Wm"2, 75% humidity and a day night regime of Grisebach (1987) found that inhibition of glyceollin produc• 14:10h. tion with the PAL inhibitor APEP enables a normally in• compatible race of Phytophthora megasperma f.sp. Bacteria and culture condition glycinea to infect soybean roots. By using indirect im- Bradyrhizobium japonicum 110spc4 (Regens• munofluorescence of hyphae in cryotome cross-sections of burger and Hennecke, 1983), Rhizobium meliloti 2011 (Casse et roots, they could show that the growth pattern of the in• al, 1979), Rhizobium leguminosarum PRE 8 (Dr. Lie, Agricultural compatible race 1 of Phytophthora megasperma f.sp. University of Wageningen, Netherlands) and Sinorhizobium fredii glycinea changed to that of the compatible race 3 under HH 103 (Dowdle and Bohlool, 1985) were grown at 28 °C on a conditions where APEP caused loss of resistance against rotary shaker in 20E-Medium (Werner et al., 1975) or xylose Phytophthora megasperma f. sp. glycinea race 1. 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 In contrast to their well documented pyridoxine HCl, 1 x 10"4 M mesoinositol, 2 x 10'5 thiamine-di- function in plant defense, less attention has been paid to HCl,2x 10"6MCa-D-pantothenate,3x 10"6MD(+)-biotin, 1 x 10"5M another aspect of flavonoid physiology, namely their nicotinic acid. possible role in plant development. Flavonoids can function as modulators of polar auxin transport (Jacobs Bacterial inoculum and Rubery, 1988). The isoflavonoid glyceollin acts at low concentrations synergistically with auxin to stimulate Bacteria, grown in appropriate medium to mid log phase (max. 109 cfu ml"1), were centrifüged and resuspended adventitious root formation on mung bean (Yoshikawa et in 5 mM MES (pH 6.2) plus 1 mM CaCl2 and adjusted to the desired al., 1986). Root primordia formation is also stimulated. cell densities. Bacteria were killed by autoclaving a buffer washed Therefore, glyceollin appears to enhance early processes of Suspension (8 x 1010 cfu ml"1) for 15 min at 120 °C. This treatment root initiation rather than simply promoting development resulted in 100% killing as confirmed by plating aliquots on 20E of already existing primordia. It is tempting to speculate agar plates. A cell free bacterial exudate was obtained from a cell that the induction of root formation by glyceollin is due to Suspension of Bradyrhizobium japonicum in buffer (8 x 1010 cfu ml"1), the ability of flavonoids to influence lateral auxin transport. stirred at room temperature for lh. Cells were removed by Recently, Hirsch et al. (1990) found that certain flavonoids centrifugation and the supernatant was filtered through a 0.2 um elicit the formation of nodule-like structures on alfalfa 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- Results moved by centrifiigation. The extracts were adjusted to an equiv- Bradyrhizobium japonicum alent concentration of 2 x 1010 cfu ml"1 with MES buffer. 110spc4 induced production of glyceollin I

Culture andzoospore induction of Phytophthora megasperma / sp. glycinea Effect of inoculum cell density on glyceollin I production Phytophthora megasperma f. sp. glycinea race 1 was obtained from T. Waldmüller and J. Ebel (Freiburg) and The initial studies focussed on the effect of cultured on Lima-Bean-Agar (Difco) in petri dishes at 25 °C in the Bradyrhizobium japonicum on phytoalexin concentration dark. Zoospores were obtained from 6 day old cultures according in roots and root exudate of Glycine max. When buffer- to Eye et al. (1978) with the exception that agar plates with washed cells ofBradyrhizobium japonicum were applied to Phytophthora megasperma f. sp. glycinea mycelia were washed

1 1 soybean roots, we observed that glyceollin I accumulated with mineral Solution (0.58g l" Ca(N03)2, 0.15g l" MgS04 x

1 1 during the symbiotic interaction. Phytoalexin levels in root 7 H20,1.15 g l" KH2P04,0.06 g l" KCl) instead of distilled water. exudate increased with increasing infection titers (Fig. 1). Inoculation procedure Beginning with inoculation titers of 1010 cfii/ml, Brady• rhizobium japonicum induced significant amounts of Roots of 2 day old soybean seedlings were trans• glyceollin I in the root exudate which were up to 50 fold ferred to 2.3 ml test tubes containing either buffer, the respective higher than those of control seedlings (Fig. 1), whereas no bacteria-derived inducing agents or a Suspension of Phytophthora glyceollin I accumulated in root hairs. megasperma f. sp. glycinea zoospores (2.5 x 104/plant). After an in• cubation time of 20 h (except where otherwise indicated) under the conditions described above, the volume of the liquid in the test To exclude the possibility that the induction tubes was made up to 2.3 ml with buffer (compensation for differ• was caused by traces of medium-derived yeast extract ent transpiratory losses) and aliquots were removed for which might contain fungal elicitors of phytoalexin quantification of glyceollin I. The root hairs were harvested by accumulation, we incubated the seedlings with bacteria scraping them with a sharpened spatula into an Eppendorf-cup. grown in yeast-free xylose minimal medium. These bacteria also induced elevated glyceollin I production. The Quantification of Glyceollin I effects were independent of the bacterial growth phase Freeze-dried root hairs were extracted with 1 ml (data not shown). methanol, 0.5 ml of the extract was evaporated to dryness and re- dissolved in 10% methanol. An aliquot of the root exudate was Kinetics of glyceollin I accumulation adjusted to 10 % methanol. Glyceollin I concentrations in root hair during symbiotic interaction extracts and root exudate were determined by a modification of the radioimmunoassay of Hahn et al. (1985), in which anti-rabbit- The kinetics of glyceollin I accumulation in solid-phase was used to precipitate Anti-Glyceollin I. Appropriate response to inoculation with Bradyrhizobium japonicum is dilutions were assayed in duplicate. shown in Fig. 2. At every time point analysed, the major Proportion of the glyceollin I produced appeared in the root Application of PAL inhibitor APEP during exudate (Fig. 2). Maximum glyceollin I levels were reached seedling infection after 10 h of incubation and this level was maintained for The effect of the PAL inhibitor APEP on nodule up to 43 h. formation was assayed on plants growing in growth pouches (Northrup, King Seed Co., Minneapolis, MN, USA). Examples and c 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 trans• ferred 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 re• moved 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 1— 9 9 10 i and number of the nodules were determined. 2X10 5X10 10 3X10 inoculum density (cfu/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. Glyceollin I Accumulation in Soybean Rhizosphere Bot. Acta 105 (1992)

C7> 800 c M in root hairs 250 • in root hairs 700 i i in root exudate O in root exudate 600 I 500

400 300 1 200

100

0 i

0 10 20 30 40 Fig. 3 Glyceollin I (pmol/seedling) in root hairs and root exudate of Glycine incubation time (h) max cv. Preston after 20 h of incubation in different preparations of a Bradyrhizobium japonicum UOspcA cell Suspension. 1 = Bacteria grown Fig. 2 Glyceollin I (pmol/seedling) in root hairs and root exudate of in 20E medium, washed and adjusted to 8 x 1010 cfu/ml with MES buffer. 2 Glycine max cv. Preston at different times after exposure to a Sus• = same as 1, but cells were autoclaved for 15 min prior to application pension of 3.9 x 1010 cfu/ml Bradyrhizobium japonicum UOspcA cells, to the seedlings. 3 = same as 1, but cells were removed by centrifugation grown in 20E medium, washed and resuspended in MES buffer. Each point and the supernatant, filtered through a 0.2 jum pore size filter, was used represents the mean of data from 8 seedlings. The MES control for the plant assay. Each column represents the mean of data from seedlings contained on average 5 pmol glyceollin I/seedling in root hairs 8-10 seedlings. The MES control seedlings contained on average and 20 pmol glyceollin I/seedling in the root exudate. Bars indicate SEM 3 pmol glyceollin I/seedling in root hairs and 10 pmol glyceollin 1/ values. seedling in the root exudate. Bars indicate SEM values.

Glyceollin I induction by living cells, dead Glyceollin Iproduction by soybean roots cells and cell-free exudate of during phytopathogenic interactions Bradyrhizobium japonicum 110spc4 Symbiotic and pathogenic interactions Glyceollin I production by soybean roots were compared with respect to glyceollin I accumulation after incubation with Bradyrhizobium japonicum might be which they induced in the rhizosphere of soybean. Since due to the infection of epidermal root cells by rhizobia. there are no well characterised soybean root pathogenic Alternatively, it may be a response to bacterial elicitors, in- bacterial strains available, zoospores of Phytophthora dependent of root hair penetration. In order to assess the megasperma f.sp. glycinea, a soybean root pathogenic probability of these alternatives, we tested dead bacteria, oomycete, were tested for their effect on glyceollin I pro• which are unable to infect the root, for their inducing duction by soybean roots. This oomycete has commonly capability.

As shown in Fig. 3, autoclaved rhizobia in• 60 duced a glyceollin I response that was fully comparable cr> •• in root hairs with that obtained by living bacteria. Both the magnitude c I I in root exudate of the response as well as the distribution pattern of T3 50 phytoalexin between root hairs and exudate were similar.

Glyceollin I induction by rhizobidl strains

Bradyrhizobium japonicum induces the production of low but significant amounts of glyceollin I by Glycine max. These glyceollin I levels remain low even at high inoculum densities, indicating that the microsymbiont were strong differences between the two soybean is capable of suppressing or preventing this plant defense nodulating rhizobia analysed in this study. Disrupted response. Such a reduction in the plant defense response Bradyrhizobium japonicum cells failed to induce any more could be a prerequisite for a successful infection. We were glyceollin I accumulation than whole cells (compare data interested whether rhizobia that are unable to nodulate from Fig. 5 with Figs. 1,2, 3). As seen in Fig. 5, a crude ex• soybean differ from Bradyrhizobium japonicum with re- tract of Sinorhizobium fredii cells induced glyceollin I con• spect to the amounts of glyceollin I/seedling they induce. centrations in soybean root exudate which are in the same When soybean roots were infected with suspensions of order of magnitude as those found after compatible in• Rhizobium meliloti (unable to nodulate Glycine max), fection with zoospores of the root pathogen Phytophtora highly variable responses were observed. These ranged megasperma f. sp. glycinea race 1. from no response up to a production of 3 nmol/seedling in the root hairs. It was determined that the Variation was not No correlation could be found between the due to the length of the incubation time (data not shown). ability of the rhizobial strains to infect soybeans and the However, the glyceollin I concentration seemed to be de- glyceollin I levels induced by cell preparations from these pendent on the batch of bacterial cells used. In order to strains. analyse the factors responsible for the Variation, we tested crude cell extracts for their inducing capacity. Inter- Absolute glyceollin I concentrations showed estingly, incubation in such a preparation of Rhizobium considerable variations between different tests i.e. differ• meliloti cells always led to very high glyceollin I concentra• ent batches of seedlings. This Variation in phytoalexin re• tions in the nmol-range. sponse between different batches of seedlings was also observed by other workers (e.g. Bonhoff and Grisebach, Since it was found that the use of cell ex• 1988; Hahn et al., 1985). Consequently each figure con- tracts led to more reproducible reactions by plant roots, tains data derived from the same test. these conditions were chosen to test two additional rhizobial strains (Fig. 5). Sinorhizobium fredii, another Inhibition of nodulation by Glycine max nodulating bacterium, and Rhizobium the PAL-inhibitorAPEP leguminosarum, a bacterium which is unable to nodulate Glycine max, were used. The two strains tested that are Having observed low but significant in• unable to nodulate soybean, induced different glyceollin I duction of the phytoalexin glyceollin I after infection with levels. The glyceollin I levels increased significantly after Bradyrhizobium japonicum, we were interested to assess incubation with the supernatant from a crude Rhizobium the physiological significance of this in the early stages of meliloti cell extract, whereas no enhancement could be the symbiotic interaction. Our approach was to analyse the observed after incubation with a comparable preparation effect of an inhibitor of flavonoid synthesis on nodule from Rhizobium leguminosarum cells. In addition, there formation. Glyceollin I Accumulation in Soybean Rhizosphere Bot Acta 105 (1992)

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 re• duced 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 Glycine max 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 0 0.01 0.1 1 seedlings were incubated with living cells of Brady• rhizobium japonicum 110spc4, a bacterium that is able to APEP (mM) form effective nodules on Glycine max roots, a significant production of glyceollin I was observed. Typically, most of Fig. 6 Effect of the PAL inhibitor APEP on the number of nodules the glyceollin I produced during the symbiotic interaction formed above the root tip mark after infection of Glycine max cv. Preston accumulated in the root exudate, whereas a smaller with Bradyrhizobium japonicum 1 lOspcA (5.2 x 103 cfu/plant). The pri- portion was found in root hairs. The elicitor of this reaction mary roots were incubated for 20 h in 2.3 ml test tubes containing in• hibitor and bacteria, then transferred to growth pouches. The position of appears to be a soluble, heat stable factor of a size < the root tip was marked. After 11 days, position and number of the nodules 0.2/im since autoclaved cells as well as filtered cell-free were determined. Each point represents the mean of data from 12-16 bacterial exudate or a crude cell extract had the same in- plants. Bars indicate SEM values. 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.

Table 1 Effect of preculture of Bradyrhizobium japonicum 110spc4 in For the pathogenic interaction between isoflavonoid-containing medium on the inhibition of nodulation by APEP. Glycine max and Phytophthora megasperma f.sp.

Nodule number2 above rtm glycinea, evidence has accumulated that the production of Preculture1 after incubation of roots in glyceollin plays an important role in resistance of the host of bacteria | MES APEP plant towards incompatible races of Phytophthora megasperma f.sp. glycinea (Waldmüller and Grisebach, control 8.5a 1.5b 1987; Hahn et al., 1985). Our data confirm this idea since isoflavonoid 6.5a 1.0b during the first 20 h of the incompatible interaction 1 Bradyrhizobium japonicum was precultured in 20E medium containing either {Glycine max cv. Maple Arrow - Phytophthora 10 uM each of daidzein and coumestrol (isoflavonoid) or 0.02 % (v/v) dimethylform- megasperma f. sp. glycinea race 1), much higher amounts amide (control). Plants were infected with 8 x 103 cfu/plant. All other conditions of phytoalexins accumulated than during the compatible were the same as described in Fig. 6. 2 Values with different indices differ with a significance of < 5 % (t-test). interaction {Glycine max cv. Kenwood-Phytophthora megasperma f. sp. glycinea race 1).

In contrast to pathogenic Systems, where A key enzyme in flavonoid synthesis is the incompatibility is correlated with glyceollin I levels, no phenylalanine-ammonia-lyase (PAL). A specific inhibitor of connection could be found between the ability of a this enzyme is (R)-(l-amino-2-phenylethyl)phosphonic rhizobial strain to form nodules on Glycine max and the acid (APEP) (Laber et al., 1986). The effect of the PAL in• amount of glyceollin I produced in root hairs or exudate. hibitor on nodulation was tested with seedlings growing in Unexpectedly, the highest glyceollin I levels, induced by growth pouches. In this experiment, the position of the root crude bacterial cell extract, were obtained with Sino• tip mark (rtm) corresponded to the time of removal of the rhizobium fredii (able to nodulate Glycine max), whereas root from the inhibition Solution. Starting with APEP con• the lowest levels were obtained with Rhizobium legu• centrations of 330 JUM, the number of nodules over the rtm minosarum (unable to nodulate Glycine max) (Fig. 5). was found to be drastically reduced (Fig. 6). On APEP However, there was a strong Variation observed amongst treated plants, a burst of nodulation was typically observed the strains that were unable to nodulate Glycine max, since directly below the rtm (data not shown) indicating that Rhizobium meliloti induced higher levels of glyceollin I nodulation was possible as soon as the inhibitor was re- than Rhizobium leguminosarum. The comparison of the moved. The highest APEP concentration applied in this ex• four rhizobial strains, with respect to their glyceollin I in- periment (ImM) inhibits neither bacterial nor plant ducing capacity, showed no correlation between their abil• growth (data not shown). These observations show that the ity to infect Glycine max and the glyceollin I levels induced reduced nodule initiation in the presence of APEP is not by crude cell extracts from these strains. Certainly, these due to a non-specific toxic effect. The nodulation of APEP- conclusions have to be prefaced with the remark that these treated seedlings could not be increased by using rhizobia results were obtained by the use of crude cell extracts since Bot Acta 105 (1992) Petra E. Schmidt, M. Parniske, andD. Werner it was impossible to obtain reproducible plant reactions ceptor. These flavonoids disturb polar auxin transport in a with living Rhizobium meliloti cells. It is possible that these manner similar to that of synthetic auxin transport in• conditions do not reflect the in vivo Situation. hibitors and therefore may have an influence on auxin dis• tribution and local concentrations. In that connection it is In this study we observed that Brady• an interesting finding that enhanced expression of a bean rhizobium japonicum \\0spc4 induces a small but signifi• CHS8-GUS gene fusion was found in root areas of trans- cant glyceollin I exudation by Glycine max roots. Estabrook genic tobacco where the formation of lateral roots had be• and Sengupta-Gopalan (1991) analysed mRNA levels for en initiated (Schmid et al., 1990). CHS is a key enzyme of PAL and CHS in roots of Glycine max after symbiotic in• flavonoid synthesis. fection with Bradyrhizobium japonicum USDA 110. They used these key enzymes of the phenylpropanoid and Since both the initiation of lateral roots and flavonoid biosynthetic pathway to investigate if an in• that of symbiotic root nodules (Rolfe and Gresshoff, 1988) duction of the pathway, leading to symbiotic signal require the induction of meristematic activity, the oc- molecules and phytoalexins like glyceollin I, had taken currence of similar physiological processes during the place and were able to show significant increases in the ontogenesis of the two root-derived plant organs seems quantity of specific mRNAs even on the first day after in• possible. The close ontogenic relationship between nodules oculation. This finding is consistent with the glyceollin I and lateral roots is also evidenced by the Observation that exudation observed in our study. some Rhizobium meliloti strains stimulate both normal nodules and "hybrid" structures intermediate between a Flavonoid accumulation in the root exudate nodule and a lateral root (Dudley et al., 1987). The finding of leguminous plants in response to symbiotic infection was that auxin transport inhibitors, as well as certain also described by Recourt et al. (1991). They reported that flavonoids, elicit nodule-like structures on alfalfa, which inoculation with the homologous symbiont Rhizobium closely resemble Rhizobium-induced legume nodules leguminosarum bv. viciae results in an increase of nod (Hirsch et al., 1989,1990), is evidence for the critical role gene inducing flavonoids in root exudate of Vicia sativa of auxin during nodule development. subsp. nigra. The glyceollin I levels induced in soybean The increased glyceollin I levels, induced roots infected with the compatible microsymbiont Brady• by the symbiotic microorganism, are in line with the pre- rhizobium japonicum 110spc4 were low when compared to viously observed increase in flavonoid synthesis (Parniske those characteristic of a pathogenic interaction. In both et al., 1988). Nevertheless, the physiological significance of types of pathogenic interactions, compatible and in• glyceollin I exudation by soybean roots is still unclear. An compatible, the levels of glyceollin I production were increase in phenylpropanoid derivative concentration several times higher than those observed as a result of the seems to be indispensible for a successful symbiotic inter• symbiotic interaction. It is likely that at these low concen• action since its inhibition leads to a significant reduction in trations glyceollin I has plant hormone characteristics and nodule formation. That PAL inhibition in alfalfa roots leads plays a role in nodule induction rather than in plant de• to a reduced synthesis and release of flavonoids was re• fense. Evidence for glyceollin I as a plant hormone comes ported by Maxwell and Phillips (1990). We observed that in• from the experiments of Yoshikawa et al. (1986) who hibition of flavonoid and glyceollin I synthesis with the PAL observed an increased meristem induction by mung bean inhibitor APEP, drastically reduced induction of nodule roots in response to glyceollin I/auxin treatments. Our re• formation. The negative effect of PAL inhibition on nodule sults indicate that the plant is in need of PAL products formation could not be explained by an absence of nod during the early stages of nodule formation. Experiments gene inducers since nodule formation was still inhibited are in progress to analyse whether this is due to the in- when the bacteria were grown in an isoflavonoid-(= nod volvement of flavonoids in meristem induction. gene inducers) containing medium (Tab. 1). The effective inhibitor concentrations were the same as described by Acknowledgement Waldmüller and Grisebach (1987). They used a similar assay design and observed a 46 % inhibition of glyceollin I We thank the laboratory of the late Prof. Dr. H. production with 330 pM APEP and a 67% reduction with Grisebach for the supply of glyceollin antibody. We thank Dr. E. K. 1 mM APEP. Based on their data, we conclude that PAL and Baylis for the gift of APEP. We thank the Deutsche Forschungs• glyceollin I synthesis were also inhibited in our ex- gemeinschaft (Bonn) for continuing support in the SFB 305 periment. The reduction in nodule formation caused by "Ökophysiologie, Verarbeitung von Umweltsignalen". 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- Glyceollin I Accumulation in Soybean Rhizosphere Bot. Acta 105 (1992)

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