P lanta (1991,184:403-409 P1 anta 9 Springer-Verlag1991 Glutathione and elicitation of the phytoalexin response in legume cell cultures Robert Edwards, Jack W. Blount, and Richard A. Dixon* Plant BiologyDivision, Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, OK 73402, USA Received 16 July 1990; accepted 14 January 1991 Abstract. Both reduced glutathione (GSH) and oxidized glutathione elicited the phytoalexin response in cell- suspension cultures of bean (Phaseolus vulyaris L.) but had no effect in those of alfalfa (Medica9o sativa L.). In bean cells, homoglutathione (HGSH) was the predomi- nant soluble thiol and treatment of cells with fungal elicitor resulted in the accumulation of HGSH but not GSH. In contrast, GSH was more abundant than HGSH in unelicited alfalfa cells, and the intracellular levels of both thiols increased in response to fungal elicitor. Treat- ment of bean or alfalfa cells with L-Oxothiazolidine-4- carboxylate, an artificial precursor for GSH biosynthesis, increased intracellular thiols in an analogous manner to that observed following treatment with fungal elicitor, but did not result in elicitation of the cultures. Differ- ences were observed in the initial metabolic fates of exogenously supplied [35S]GSH in bean and alfalfa, but our data do not yet provide a basis for explaining how GSH acts as an elicitor. We conclude that the changes observed in intracellular thiols following exposure of cells to fungal elicitor are a consequence rather than a cause of the initial elicitation signal(s). Key words: Cell culture (phytoalexin response) - Elicita- tion - Glutathione - Isoflavonoid phytoalexins - Medicago - Phytoalexin - Phaseolus (phytoalexin re- sponse) Introduction Leguminous plants contain high levels of glutathione (GSH; 7-L-glutamyl-L-cysteinyl-glycine), homogluta- thione (HGSH; 7-L-glutamyl-i-cysteinyl-alanine) or a * To whom correspondence should be addressed Abbreviations: GSH = glutathione (reduced); GSHM = mono- ethyl ester of GSH; HGSH = homoglutathione; OTC = L-2-oxo- thiazolidine-4-carboxylate; PAL = L-phenylalanine ammonia-lyase (EC 4.3.1.5). mixture of the two soluble thiol tripeptides (Klapheck 1988). The tripeptides have multiple functions, being implicated in the transport and storage of sulfur within the plant and in the detoxification of electrophilic radi- cals and adducts formed during photosynthesis (Rennen- berg 1982; Alscher 1989). A protective role of GSH in the cell is further supported by the observation that its level increases markedly during responses of plants to en- vironmental stresses such as exposure to extremes of temperature and water stress (Rennenberg 1982; Alscher 1989). The oxidized and reduced forms of GSH may also be involved in regulating plant cell division and develop- ment (Earnshaw and Johnson 1985) and protein syn- thesis (Alscher 1989). Recently, it has been proposed the GSH may be invol- ved in signal transduction during the initiation of the phytoalexin defense response in legumes by fungal elic- itors (Wingate et al. 1988). Thus, treatment of suspen- sion-cultured cells of bean (Phaseolus vulgaris L.) with GSH (0.01-1 mM) induced the appearance of hydroxy- proline-rich glycoproteins and enzymes of the phenyl- propanoid pathway such as phenylalanine ammonia- lyase (PAL; EC 4.3.1.5) and chalcone synthase (EC 2.3.1.74) (Wingate et al. 1988). Elicitation by GSH invol- ved the rapid and selective activation of transcription of defense genes in an analogous manner to that observed following treatment of bean cells with an elicitor prepara- tion from the cell walls of the anthracnose fungus Coll- etotrichum lindemuthianum (Sacc. et Magn.) Bri. et Cav. (Wingate et al. 1988). Likewise, GSH elicits the accu- mulation of the pterocarpan phytoalexin pisatin in pea epicotyls (Yamada et al. 1989) and enhances the dose- response of coumarin biosynthesis to elicitation by chitosan in parsley cells in conditioned growth medium (Conrath et al. 1989). Recent studies have shown that exposure to GSH activates the expression of a chimeric gene comprising the 5' regulatory sequence of bean chalcone synthase fused to a chloramphenicol-acetyl-transferase reporter gene in electroporated protoplasts of soybean, tobacco or alfalfa (Medicago sativa L.) (Choudhary et al. 1990a,