Survey of the Taxonomic and Tissue Distribution of Microsomal Binding Sites for the Non-Host Selective Fungal Phytotoxin, Fusicoccin Christiane Meyer3, Kerstin Waldkötter3, Annegret Sprenger3, Uwe G. Schlösset, Markus Luther0, and Elmar W. Weiler3 a Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität, D-44780 Bochum, Bundesrepublik Deutschland b Pflanzenphysiologisches Institut (SAG) der Universität, D-37073 Göttingen, Bundesrepublik Deutschland c KFA Jülich, D-52428 Jülich, Bundesrepublik Deutschland Z. Naturforsch. 48c, 595-602(1993); received June 4/July 13, 1993 Vicia faba L., Fusicoccin-Binding Sites. Taxonomie Distribution, Tissue Distribution, Guard Cell Protoplasts The recent identification of the fusicoccin-binding protein (FCBP) in plasma membranes from monocotyledonous and dicotyledonous angiosperms has opened the basis for an elucida­ tion of the toxin’s mechanism(s) of action and indicated a widespread occurrence of the FCBP in plants. Results of a detailed taxonomic survey of fusicoccin-binding sites are reported. Bind­ ing sites were not found in prokaryotes, animal tissues, fungi and algae including the most direct extant ancestors of the land plants (Coleochaete). From the Psilotales (Psilophytatae) to the monocotyledonous angiosperms, all taxa analyzed possessed high-affinity microsomal fusicoccin-binding sites. A heterogeneous picture emerged for the Bryophvta. Anthoceros cri- spulus (Anthocerotae), the only hornwort available to study, lacked fusicoccin binding. Within the Hepaticae as well as the Musci, species lacking and species exhibiting toxin binding were found. The binding site thus seems to have emerged very early in the evolution of the land plants. The tissue distribution of fusicoccin-binding sites was studied in Vicia faba L. shoots. All tissues analyzed showed fusicoccin binding, although not to the same extent. On a per-cell basis, guard cells were found to contain, compared to mesophyll cells, a nine-fold higher num­ ber of binding sites. Based on cell surface area, the site density is by a factor of 32 higher in guard cells than in mesophyll cells. Tissue specific expression of the binding sites is suggested by these findings. Introduction the most immediate being the stimulation of apo- The perception of signal molecules at the plasma plastic acidification. The FCBP has been charac­ membrane of higher plants has recently been stud­ terized in detail from oat root tissue [2], broadbean ied with increased emphasis. Whereas the occur­ leaf [3, 4], Arabidopsis thaliana [5] and Corydalis rence of hormone receptors at the plasma mem­ sempervirens cell suspension cultures [6]. The puri­ brane is still a subject of debate, recent evidence fied FCBP of Commelina communis consists of two strongly suggests that higher plants perceive cer­ polypeptides of 30.5 and 31.5 kDa apparent mo­ tain signalling molecules at the plasma membrane lecular mass [7], and photoaffinity labelling experi­ ments suggest a similar situation in broadbean and (e.g. [1]). One of the best-characterized plasma membrane systems with receptor-like properties is Arabidopsis [4, 5]. These studies have revealed remarkably con­ the binding protein (FCBP) for the Fusicoccum stant properties of all the FCBPs characterized so amygdaly wilt-inducing toxin, fusicoccin (FC). The binding site is probably involved in the media­ far, suggesting a widespread occurrence and evolu­ tionary conservation of FC-binding sites. How­ tion of most, if not all, FC effects on plant cells, ever, a systematic study of FCBP distribution in the plant kingdom is not available and only a Abbreviations: FC, fusicoccin; FCBP, fusicoccin-binding single report deals with a survey of FC-induced protein; GCP, guard cell protoplast; MCP, mesophyll cell protoplast. H+/K + exchange in a limited number of species of higher and lower plants [8]. We therefore exam­ Reprint requests to Prof. Dr. Elmar W. Weiler. ined the taxonomic and tissue distribution of Verlag der Zeitschrift für Naturforschung, D-72072 Tübingen FC-binding sites. The results of this study are re­ 0939-5075/93/0700-0595 $01.30/0 ported here. 596 Ch. M eyer et al. ■ Microsomal Fusicoccin-Binding Sites Materials and Methods by the Biologische Anstalt Helgoland and shipped Higher plants in sea water. Chlamydomonas was grown as de­ scribed [11]. Chlorella, Chlorococcum, Scenedes­ V. faba cv. “Osnabrücker Markt” was grown mus and Monoraphidium were grown in N 8 Medi­ from May to September 1989 in standardized field um [12] with microelements according to Payer plots at the Botanical Garden of the Ruhr-Univer­ and Trültsch [13]. The brown and red algae as well sität Bochum. Plant development was classified as Tribonema, Apatococcus, Coleochaete, Klebsor- according to the standard nomenclature [9]. The midium, Raphidonema and Stichococcus were culti­ most relevant growth stages will be detailed in the vated as described in [14], The algae were grown to experimental section (cf. Table V). exponential phase and then harvested. Chara co- Leaf samples of angiosperms, gymnosperms and rallina was kindly provided by Prof. Dr. W. Har­ pteridophytes were, unless otherwise stated, col­ tung, Würzburg. Some algae cultures grown by lected during the growth season 1989 from the Bo­ one of us (U.S.) for our experiments were from the tanical Garden of the Ruhr-Universität. Samples collection of algae strains at the University of of healthy leaves were collected on ice and, unless Göttingen (SAG) carrying the following strain processed immediately, were frozen in liquid nitro­ designations: Raphidonema longiseta (SAG 470-1), gen and stored at -80 °C for a maximum of two Tribonema aequale (SAG 200.80), Dictyota dicho- weeks. Anemia phyllitidis was a kind gift of Prof. toma (SAG 207.80), Ectocarpus siliculosus (SAG Dr. R. Schraudolf, Ulm, and Psilotum nudum 63.81), Antithamnion sp. (SAG 95.79), Chlamy­ plants were generously made available by Prof. domonas reinhardtii (SAG 83.81), Chlorococcum Dr. H.-J. Schneider-Pötsch, Cologne. hypnosporum (SAG 213-6), Apatococcus lobatus Bryophytes (SAG 34.83), Coleochaete scutata (SAG 50.90). Mosses were, unless stated otherwise, obtained Fungi as a gift from Prof. Dr. W. Hartung, Würzburg, All fungi tested were grown as pure cultures un­ and were grown on soil substrates. The thalli were der standard conditions as specified in the ATCC thoroughly cleaned and rinsed with water prior to manuals. further processing. Sterile gametophytes of A. cri- spulus were generously provided by Prof. Dr. H. Bacteria Binding, Kiel, and were maintained on agar-solidi- fied B5 medium [10] containing 0.5% sucrose and Escherichia coli JM83 was grown in bacto tryp- no phytohormones. The thalli were grown in short tone (10 g l“1), yeast extract (5 g l-1), and NaCl days (8 h photoperiod, PAR 67 |iE m-2 s“1) at (10 g I-1) at 37 °C and Agrobacterium tumefaciens 20 °C ± 2 °C. Marchantia polymorpha thallus was C58, strain P I 145, supplied by Prof. Dr. C. I. grown sterile on agar containing 100 mg L 1 each Kado, Davis, U.S.A., was grown in bacto peptone of K2HP04, CaCl2 • 6 H20, M gS04 • 7 H20, 200 mg (10 g I"1), NaCl (5 g I“1) at 30 °C. l 1 NH4N 03 and 5 mg l“1 FeCl2, pH 3.5 (constant weak light, 4.6 (iE m -2 s-1, 24 °C ± 2 °C). Cerato- Animal tissues don, Mnium and Leptobryum species were provid­ All animal tissues analyzed were obtained fresh ed as sterile flask cultures by Prof. Dr. E. Hart­ and using standard techniques from laboratories mann, Berlin. The Scapania and Plagiochila sam­ of the Departments of Biology and Medicine, ples were obtained as sterile cultures from Prof. Ruhr-Universität Bochum. Dr. E. Becker, Saarbrücken, and Polytrichum com­ mune, Dicranium scoparium, Racomitrium canes- Protoplast preparation cens and Rhytidiadelphus squarrosus were collected Mesophyll cell protoplasts (MCP) and guard and made available by Prof. Dr. R. Mues, Saar­ cell protoplasts (GCP) were prepared exactly as brücken. described [15], When digestions were made in the Algae presence of FC (3 jiM), the following modifications Cladophora, Codium, Fucus, Laminaria, Cera- were introduced: K+ was omitted from all solu­ mium and Chondrus species were locally collected tions; the mannitol concentration was increased to Ch. M eyer et al. • Microsomal Fusicoccin-Binding Sites 597 0.4 m (step 1), 0.5 m (step 2) and 0.6 m (step 3) of tic acidification [8], Plant tissue cultures may, how­ the procedure of Key and Weiler [15]. Additional­ ever, loose certain cellular functions through dege­ ly, epidermal peels were pre-incubated in K+-free, nerative mechanisms. On the other hand, hy- 0.4 m mannitol in order to lower the amount of apoplastic K+, before being transferred to the en­ Table I. Analysis of FC-binding in prokaryotic and zyme solutions. lower eukaryotic organisms. Preparation of microsomes Taxon Number of species analyzed positive Microsomal preparations were obtained from 2 0 the various tissues using standard techniques. For Eubacteria a a detailed description of the method used for the M ycota O om ycetes l b 0 higher plants and algae, see [3]. Unicellular algae Zygomycetes l c 0 were homogenized in a mortar in the presence of Ascomycetes 19d 0 aluminum oxide; other tissues were homogenized Deuteromycetes l e 0 2 0 in a Waring blendor. Chlamydomonas was pro­ Basidiomycetes f cessed according to Spalding and Jeffrey [16], Phy- Heterokontophyta Bacillariophyceae 28 0 comyces according to [17], Saccharomyces as de­ Xanthophyceae l h 0 scribed by Gaber et al. [18], the other fungi accord­ Phaeophyceae 4' 0 ing to [19]. Bacterial membranes were prepared Rhodophyta y 0 according to Kaback [20], Microsomes from ani­ Chlorophyta mal tissues were prepared as described in [21], Chlorophyceae 10k 0 Charophyceae l 1 0 Assays for the determination of FC-hinding a Agrobacterium tumefaciens C 58, Escherichia coli The high affinity microsomal binding sites for JM 83. the toxin were probed with the radioligand [3H]-9'- b Phytophthora megasperma Drechsler.