Survey of the Plant Kingdom for the Ability to Bind Heavy Metals through Phytochelatins Walter Gekeler**, Erwin Grill, Ernst-Ludwig Winnacker*, and Meinhart H. Zenk Lehrstuhl für Pharmazeutische Biologie der Universität München, Karlstraße 29, D-8000 München 2, Bundesrepublik Deutschland * Laboratorium für Molekulare Biologie — Genzentrum der Ludwig-Maximilians-Universität, Am Klopferspitz, D-8033 Martinsried, Bundesrepublik Deutschland Z. Naturforsch. 44c, 361 — 369 (1989); received January 25/March 14, 1989 Dedicated to Professor Achim Trebst on the occasion of his 60th birthday Phytochelatins, Glutathione, homo-Glutathione, homo-Phytochelatins, Heavy-Metal Binding Peptides, Cell Cultures Differentiated plants and suspension cultures of the taxonomic divisions Bryophyta, Pteridophyta, and Spermatophyta have been investigated as to their ability to detoxify heavy metals like Cd2+ through the formation of (y-Glu —Cys)„—Giy peptides, the phytochelatins. Over 200 individual plants have been checked and there was not a single exception observed. Only in the order Fabales several species mainly of the tribus Fabaceae form upon exposure to Cd2+ ions peptides of the general structure (y-Glu — Cys),, —ß-Ala, the homo-phytochelatins. The existence of glutathione and homo-glutathione within a given species determines whether phytochelatins or their homo-derivatives are formed. The ability to form phytochelatins for metal homeostasis and metal detoxification is a principal feature of plant metabolism. Introduction bind heavy metals. They are composed only of All living cells are confronted with the dilemma 3 amino acids, namely L-cysteine, L-glutamic acid that on one side they need certain amounts of free and glycine. Glutamic acid is linked to each cysteine heavy metal ions (such as Zn2+, Cu2+, Ni2+, etc.) for by a y-peptide linkage. They are not primary gene their normal metabolic function, and on the other products. The general structure of this set of peptides side they have to protect themselves from an intra­ is (y-Glu—Cys)„—Giy (n = 2—11). They are called cellular excess of these metal ions which would lead phytochelatins (PC’s) [1, 3]. In a few members of the to cell death. This dilemma can only be overcome by Fabales (Leguminosae), phytochelatins are substi­ a stringent regulation of free metal ion concentration tuted by a peptide family containing a ß-alanine car- within the cells, which can be regulated in several boxy terminus instead of the glycine. These peptides ways such as: metal-binding to cell walls, reduced were termed homo-phytochelatins (y-Glu—Cys)„— transport across cell membrane, active efflux, com- ß-Ala (n = 2—7) [4]. Phytochelatin induction has partmentalization and chelation [1]. The mechanism been found to occur in some fungi [5, 6 ], in algae [7] which has been studied most closely in recent years is and in some differentiated as well as tissue-cultured chelation. Heavy metals in vertebrates and certain higher plants [1], There remained, however, always fungi are detoxified by sulfur-rich, 6.5 kDa proteins the question as to the ubiquity of this metal-binding devoid of aromatic amino acids, the metallothioneins process in the plant kingdom. Therefore, we under­ [2]. Plant cells, on the other side, after heavy metal took the task to screen a large number of plant exposure, synthesize small, sulfur-rich peptides that species belonging to the divisions Bryophyta, Pteridophyta and Spermatophyta. Abbreviations: GSH, glutathione; h-GSH, homo-gluta- thione; PCn, phytochelatin with n-(y-Glu —Cys) units; h-PCn, homo-phytochelatin with n-(y-Glu —Cys) units; Materials and Methods dwt, dry weight. Biological material ** Present address: Lever GmbH, Rhenanicastraße 78—102, D-6800 Mannheim 81, Bundesrepublik Plant cell cultures were provided by our cell cul­ Deutschland. ture laboratory. If not otherwise stated, the cultures Reprint requests to Prof. M. H. Zenk. were grown at 23 ± 1.5 °C at 70% humidity, 600 lux Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen incandescent light and 1 0 0 rpm on gyratory shakers. 0341-0382/89/0500-0361 $01.30/0 The media used were: LS = Linsmaier and Skoog 362 Walter Gekeler et al. ■ The Ability to Bind Heavy Metals through Phytochelatins [8 ]; 4X = Gamborg et al. medium [9] containing as rate of 2 mix min-1. The mixture was passed hormones 0.5 mg/1 indoleacetic acid, 0.5 mg/1 naph- through a reaction loop (5 ml volume) which corre­ thaleneacetic acid, 2 mg/1 2,4-dichlorophenoxyacetic sponds to a reaction time of 1.25 min and sub­ acid, 0.2 mg/1 kinetin; DAX = Gamborg et al. sequently the absorption at 410 nm was recorded and medium [9] containing as hormone 2 mg/1 2,4-di- the peak area automatically integrated [15]. Gluta­ chlorophenoxyacetic acid; MS = Murashige and thione was used as standard. The nmol peptides were Skoog [10]. related to dry weight of the plant sample or protein Seeds of plants were obtained from the botanical content. One ng of phytochelatin complex could still gardens of Cologne, Bayreuth and Munich. Seed­ be quantitated and resolved into individual PC lings were placed on styrofoam rafts so that their species by this method. This method yielded at least roots were exposed to Hoagland-nutrient solution 90% of cadmium PC present in the sample. [11] which was aerated (0.5 1 air x h - 1 x r'-nutrient medium) at 27 °C at 1200 lux. Exposure to C d(N 0 3 ) 2 Results was done for 4—12 days at a concentration of usually The heavy-metal binding peptides from lower plants 20—50 |o ,m . The moss Marchantia polymorpha was cultivated under sterile conditions in Knop solution It has previously been shown that algae are capa­ [12] solidified with 1.2% agar. Dnfrrentiated plants ble of forming phytochelatins in response to heavy of several Pteridophyta were exposed to heavy met­ metal stress and that in contrast to previous reports als in the same way, however, the Hoagland-nutrient not proteins are responsible for this detoxification solution [ 1 1 ] was diluted to Yw strength. but rather the peptides of the (y-Glu—Cys)„—Gly type [7]. In order to extend our knowledge about the ubiquity of the heavy-metal binding phytochelatin Analytical procedures system in the plant kingdom, plants of the division Heavy metals were determined by atomic absorp­ Bryophyta and Pteridophyta were tested. As shown tion spectroscopy (Perkin-Elmer PE-1100B) with in Table I, a member of the mosses, Marchantia flame mode. Samples (50 mg) were digested with polymorpha, when grown in axenic culture was capa­ concentrated H 2 S 0 4 or H N 0 3 (50 ^1) and analyzed ble of forming PC 2 and PC3. The HPLC profile is after appropriate dilution (to 1.5 ml). Protein was given in Fig. 1A. No PC synthesis was detected in determined according to [13], SH-groups according the control sample. In the division of Pteridophyta, to [14], Phytochelatins were assayed as follows [7, however, where particularly slow growing plants are 15]: Tissue was frozen with liquid nitrogen, ground, found, none of the plants grown in the field or under and to 400 mg powder was added 0.4 ml 1 n NaOH greenhouse conditions yielded PC synthesis upon ex­ containing 0.4 mg NaBH4. The sample was sonicated posure to Cd2+ in their native soil, except the water (Branson, 3x5 sec, setting 4) and subsequently cell fern Azolla filiculoides, which could be directly debris centrifuged off. The supernatant was transfer­ transferred to the heavy metal solution. There was red into an Eppendorf vial, acidified with 100 fil the possibility that the heavy metal was adsorbed to 3.6 n HC1 and put on ice for 15 min. Precipitated soil particles and could not reach the roots. The protein was removed by centrifugation. 20—250 (il of availability of heavy metals was achieved by satura­ this clear supernatant were injected into an HPLC tion of the soil particles with Al3+ first, as described system (Spectra-Physics, as described in [7]). by Fischer [16]. Plants growing in their natural envi­ Nucleosil (10 C-18) or LiChrosorb (RP-18; 7 n) ronment, treated with a mixture of 1 mM Al3+, 1 mM 4 x 250 mm columns were used for separation of the Zn2+ and 100 piM Cd2+ in aqueous solution, reacted peptides. Elution was achieved by a gradient using clearly by PC induction after a period of 4 days 0.05% H 3PO 4 in 0—20% acetonitrile-H 2 0 . Detec­ (Table I). Ms. B. Rittgen in our laboratory suc­ tion was for pure phytochelatins at 2 2 0 nm, crude ceeded also to establish a cell culture of Equisetum extracts were analyzed by -SH specific detection giganteum, the first cell culture reported for a using Elman’s reagent (DTNB) [14]. Post-column member of the Equisetales. It could be shown that derivatization was accomplished by mixing to the these suspension cultures reacted immediately and to eluate of the separation column DTNB reagent the same extent as cultures of higher plants towards (75 |i m DTNB in 50 m M K_2 P 0 4 buffer pH 8 ) at a exposure to 100 |xm Cd2+ with the synthesis of PC’s Walter Gekeler et al. ■ The Ability to Bind Heavy Metals through Phytochelatins 363 Table I. Phytochelatin synthesis in some members of Sporophyta. Species Order PC2a PC, PC4 PCs PC, Total [nmol/mg dwt] PCh Marchantia polymorpha Marchantiales 0.5 0.2 0.0 0.0 0.0 1.7 Selaginella viticulosa* Selaginellales 0.2 0.1 0.1 0.0 0.0 0.9 Lycopodium clavatum* Lycopodiales 0.2 0.0 0.0 0.0 0.0 0.5 Equisetum giganteum* Equisetales 0.0 0.5 0.2 0.0 0.0 2.5 Equisetum giganteum S Equisetales 0.5 0.2 0.2 0.1 0.1 3.3 Azolla filiculoides Hydropteridales 0.2 0.1 0.0 0.0 0.0 0.7 * Plants in natural substrate irrigated with 1 m M A l3+, 1 m M Zn2+ and 100 |j.m Cd2+.