Z = 4 Formeleinheiten pro Elementarzelle. In Tabelle 1 sind die Gitterkonstanten sowie die Dichtewerte f/Jr alle neu erhaltenen Verbindungen zusammengestellt. Zur Darstellung dieser Verbindungen wurden jeweils iiquimolare Gemenge der bin/iren Fluoride N a F bzw. AgF, MF 2 und MF 3 in Goldampullen gefiillt und diese 10-14 Tage auf Temperaturen zwischen 500-700 ~ erhitzt. Unter diesen Reaktionsbedingungen wurden

jeweils mikrokristalline Proben der beschriebenen Verbindungen erhalten. Zur pyknometrischen Dichtebestimmung wurde wasserfreies Kerosin (Siedeintervall 200-246 ~ als Absperrflfissigkeit benutzt. Eingegangen am 25. April 1985 I. Teil der Dissertation Univ. GieBen 2. Koch, J., Hebecker, Chr., John, H.: Z. Naturforsch. 37b, 1659 (1982)

Induction of Heavy Metal-Sequestering Phytochelatin by Cadmium in Cell Cultures of Rauvolfia serpentina E. Grill and M.H. Zenk Lehrstuhl fiir Pharmazeutische Biologie der Universit/it, D-8000 Mfinchen E.-L. Winnacker Genzentrum der Universitfit Miinchen, D-8033 Martinsried Plants are able to live and grow on soils with varying heavy metal content. They must have developed a specific detoxification system to cope to some degree with poisonous heavy metals. In mammals this is accomplished by metallothioneins [1]. For a number of years proteins sequestering cadmium and other heavy metals (presumed Mr 10 kD) have been isolated from the tissues of several plant species and postulated to be metallothionein-like [2]. Recently we reported that this 10 kD protein does not exist in plant cell cultures, where a set of sulfur-rich peptides with the structure (7-glu-cys),gly (n = 3-7) is responsible for the sequestration of heavy metals. The name phytochelatin (PC) was proposed for this new class of natural product, which occurs in oligomeric forms, in particular with molecular weights of 2500 and 3600 in heavy-metal-exposed higher plants. These compounds have a structure totally different from the wellcharacterized metallothioneins reported from animals and fungi [1]. In this report we describe the induction kinetics of PC in a plant cell suspension culture by exogenously applied Cd(NOa) z. Complexed Cd a + of DEAE-binding material is due to PC 432

ongoing increase in cell number. The increase of Cd2+-pc concentration is accompanied by a parallel rise of the SH content within the cells, ascribed to the metal-induced synthesis of PC. Total SH concentration rose about sixfold as compared to the control. Metabolic studies involving a s s o ~ - in the medium demonstrated that there are only negligible amounts of PC present in the untreated control tissue, while there is a massive and rapid synthesis of labeled PC on addition of Cd z + ions (data not shown). This induction of PC synthesis in cell cultures in the presence of heavy metals represents one of the relatively few clearly induceable reactions in higher plants. Because of its y-glutamic acid linkages, PC cannot be regarded as a primary gene product but is possibly synthesized by induced enzymes. Investigation of PC synthesis should give a clue to the induction and regulation mechanisms triggered by heavy metals. This work was supported by the Bundesministerium fiir Forschung und

[3]. It can be analyzed by absorbing the soluble fraction of a crude cell-free extract onto DEAE-Biogel, washing the column with 1 0 m M Tris-HC1 buffer, pH 8.6, until all uncomplexed C d 2+ is removed, subsequent elution of PC with 0.5 M NaC1, 10 m M TrisHC1 buffer, pH 8.6, and by quantitation of eluted Cd z + via atomic absorption spectroscopy. Furthermore the SH groups of PC show a positive reaction with Ellman's reagent [4] which can be used to quantitate this peptide [3] and also to monitor the total SH content of the soluble cell extract. Cells of Rauvolfia serpentina Benth. (160g packed cells 1-1) were transferred to fresh medium [5] and cultivated for 7 days. Samples were taken every day to determine both dry weight and number of SH groups in the soluble cell extract. After a growth period of 65 h, 200 g M Cd(NO3)2 was administered to one of the cultures and samples were taken at times indicated in Fig. 1. Addition of the heavy metal resulted in an immediate and linear rise in the intracellular concentration of the Cd 2 +-PC complex for approximately 2 days. However, the concomitant drop of soluble Cd 2 + in the medium reached its maximum after 4 days, due to the

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Fig. 1. A) Time course of PC induction in Rauvolfia serpentina cell suspension cultures after addition of Cd z +, monitored by the Cd z+ content of DEAE-binding material and by the SH-group concentrations of crude cell extracts. Results were corrected for unspecific Cd 2+ binding using frozen cells to which Cd z + was added as control. B) Time course of cell growth in cell suspension cultures with and without addition of Cd z +

Naturwissenschaften 72 (1985) 9 Springer-Verlag 1985

Technologie, Bonn, and by the Fonds der Chemischen Industrie. Received April 24, 1985 1. K/igi, J.H,R., Nordberg, M. : Experientia Suppl. 34 (1979) 2. Weigel, H.J., Jfiger, H.J. : Plant Physiol. 65, 480 (1980); Bartolf, M., Brennan, E., Price, C.A.: ibid. 66, 438 (1980); Rauser, W.E., Curvetto, N.R.: Nature 287, 563 (1980); Cataldo, D.A., Garland, T.R.,

Wildung, R.E.: Plant Physiol. 68, 835 (1981); Wagner, G.J., Trotter, M.M.: ibid. 69, 804 (1982); Kaneta, M., et al.: Agric. Biol. Chem. 47, 417 (1983); Wagner, G.J.: Plant Physiol. 76, 797 (1984) 3. Grill, E., Winnacker, E.-L., Zenk, M.H. : in press 4. Ellmann, G.L. : Arch. Biochem. Biophys. 82, 70 (1959) 5. Linsmaier, E.M., Skoog, F.: Physiol. Plant. 18, 100 (1965)

The Sporopollenin of the Schizaeaceous Fern

Anemia phyllitidis H. Schraudolf and R. Haag Abteilung Allgemeine Botanik der Universit~it, D-7900 Ulm In comparison with spermatophytes, the number of analyzed sporopollenins isolated from spore walls of pteridophytes is low, and their taxonomical distribution is poor. Since an ultrastructural investigation of the exine formation of the schicaeaceous fern Anemia phyllitidis L. Sw. showed differences in the course of wall deposition as compared to the pollen wall formation in higher plants [1], we analyzed the sporopollenin from spores of this species. For this purpose 2 g of ripe spores were ground at low temperature in a glassbead homogenizer and extracted according to the method described for the isolation of sporopollenin from Ambrosia pollen [2]. In general, the infrared absorption spectrum of Anemia sporopollenin resembles the spectra reported for other plants (Fig. 1). Apart from a higher resolution of the bands in the range between 1600 and 1400 cm 1, the sporopollenin preparation o f A. phyllitidis is additionally characterized by an I R band at 2 620 c m - a . This band cannot be attributed to CO s impurities, since the corresponding bands in the range of 600 c m - 1 are indetectable. The data of elemental analysis are shown in Table 1. The high residue is the consequence of an exceptionally high mineral deposition in the spore wall of A. phyllitidis. The nitrogen value corresponds with that from sporopollenin of many other plants. Surprisingly the molecular formula, arbitrarily based on C9o units, shows

neither correspondence to the spores of the few Pteridophytae so far analyzed (Selaginella krausiana (megaspore):

C9oHt24018 ; Lycopodium clavatum: C9oHt44027; Equisetum pratense: C9oH144031), nor to sporopollenin iso-

Table 1. Elemental analysis of the sporopollenin of A. phyllitidis. The molecular formular is arbitrarily based on a C9o unit which has no physiological significance Res- Elements [% d.w.] idue [%] C H O N

Molecular formula

3.85 61.99 7.00 24.9 0.59 C9oH12202v

lated from conifers [3-5]. However, more comparable to sporopollenin of lower organisms (Pediastrum duplex: C90H121028) [5] there exist close correlations to the oxidative polymer o f flcarotene (C9oH13oO3o). This at least may indicate a relation between the accumulation of ~-carotene during the late phases of spore development of A. phyllitidis and the respective exine sporopollenin formation of this fern species [1]. We thank Prof. Dr. A. R u o f f (Sektion ffir Schwingungsspektroskopie, Universit/it Ulm) for the determination of

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Naturwissenschaften 72 (1985) 9 Springer-Verlag 1985

433