A set of novel heavy-metal complexing peptides was isolated from plant cell suspension cultures; the structure of the peptides was established as (gamma-glutamic acid-cysteine)n-glycine (n = 3 to 7). These peptides appear upon induction of plant cells with heavy metals and represent the principal metal-binding activities in the cells. The name phytochelatin is proposed for this new class of natural products.

To deter pathogenic microorganisms and herbivores, plants have developed an inducible chemical defense system. It is known that the induced synthesis of low molecular weight compounds can be provoked by exposing cultured cells to fungal cell wall fragments. In this study we show that endogenous jasmonic acid and its methyl ester accumulate rapidly and transiently after treatment of plant cell suspension cultures of RauvolJ' canescens and Eschscholtia caisfornica with a yeast elicitor. Thirty-six plant species tested in cell suspension culture could be elicited with respect to the accumulation of secondary metabolites by exogenously supplied methyl jasmonate. Addition of methyl jasmonate initiates de novo transcription of genes, such as phenylalanine ammonia lyase, that are known to be involved in the chemical defense mechanisms of plants. These data demonstrate the integral role of jasmonic acid and its derivatives in the intracellular signal cascade that begins with interaction of an elicitor molecule with the plant cell surface and results, ultimately, in the accumulation of secondary compounds.

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An enzyme has been discovered and characterized from Silene cucubalus cell suspension cultures that catalyzes the transfer of the y-glutamylcysteine dipeptide moiety of glutathione to an acceptor glutathione molecule or a growing chain of [Glu(-Cys)],-Gly oligomers, thus synthesizing phytochelatins, the metal-binding peptides of higher plants and select fungi. The enzyme was named y-glutamylcysteine dipeptidyl transpeptidase and given the trivial name phytochelatin synthase. The primary reaction catalyzed is [Glu(-Cys)]-Gly + [Glu(-Cys)],,-Gly -* [Glu(-Cys)],k+-Gly + Gly. The enzyme is isoelectric near pH 4.8 and has temperature and pH optima at 35'C and 7.9, respectively. Phytochelatin synthase is constitutively present in cell cultures of various plant species and its formation is not noticeably induced by heavy metal ions in the growth medium. The enzyme (Mr 95,000) seems to be composed of four subunits, the dimer (Mr 50,000) being also catalytically active. Cd2+ is by far the best metal activator of the enzyme followed by Ag+, Bi3+, Pb2+, Zn2+, Cu2+, Hg2+, and Au+. The Km for glutathione is 6.7 mM. The enzyme activity seems to be self-regulated in that the product of the reaction (the phytochelatins) chelates the enzyme-activating metal, thus terminating the enzyme reaction. The molar ratio of the y-glutamylcysteine dipeptide in phytochelatin to Cd2+ in the newly formed complex was 2:1.

Phytochelatins are a class of heavy-metalbinding peptides previously isolated from cell suspension cultures of several dicotyledonous and monocotyledonous plants. These peptides consist of repetitive y-glutamylcysteine units with a carboxyl-terminal glycine and range from 5 to 17 amino acids in length. In the present paper we show that all plants tested synthesized phytochelatins upon exposure to heavy metal ions. No evidence for the occurrence of metallothionein-like proteins was found. All data so far obtained indicate that phytochelatins are involved in detoxification and homeostasis of heavy metals in plants and thus serve functions analogous to those of metallothioneins in animals and some fungi. Phytochelatins are induced by a wide range of metal anions and cations. Phytochelatin synthesis in suspension cultures was inhibited by buthionine sulfoximine, a specific inhibitor of y-glutamylcysteine synthetase (EC 6.3.2.2). This finding and kinetic studies of phytochelatin induction point to a synthesis from glutathione or its precursor, r-glutamylcys-