Plant Cell Culture

A series of structurally related substituted phenethylamines shows extreme toxicity toward wild-type callus tissue cultures of tobacco (Nicotiana tabacum), soybean (Glycine max), corn (Zea mays), and sunflower (Helianthus annuus L.), but tobacco crown gall cultures are resistant to the compounds. The essential components that result in toxicity of the phenethylamines include one aromatic hydroxyl and one primary aliphatic amino group. A series of attenuated crown gall cultures, generated by transformation of tobacco with various modified Agrobacterium strains, has been used to demonstrate that the resistance of crown galls to the phenethylamines is due to the expression in these tissues of isopentenyl transferase, a first step in cytokinin biosynthesis. The toxicity of the compounds to tissue cultures is very rapid, but can be overcome by prior exposure of the calli to exogenous cytokinin. Because of the relationships we have observed between cytokinins and these compounds, we propose that the substituted phenethylamines may represent a class of chemicals that can be used as specific probes to further an understanding of cytokinin metabolism in plant tissues. to metabolic regulatory events in an as yet unrevealed way (3). Unfortunately, the complex interactions of the various plant hormones with one another frequently complicate the development ofa clear understanding ofthe underlying mechanisms of hormone activity. Because a number of seemingly unrelated studies have implicated the catecholamines in metabolic situations characterized by rapid hormone responses, we investigated a series of the compounds for growth altering characteristics on plant tissue cultures. In screening for cytokinin-active compounds, we initially observed that octopamine, a substituted phenethylamine, was exceedingly toxic to wild-type tobacco callus, but had no apparent effect on crown gall tissues. We explored this effect further by observing the effect ofvarious related compounds on a variety ofcell culture types, including a series of altered or attentuated crown galls. The results of these studies suggest that octopamine and the related compounds are antagonists of cytokinin metabolism in plant tissues, and may possibly be useful probes for further analysis of the details of cytokinin metabolism in plants. MATERIALS AND METHODS A wide variety of substituted phenethylamines have been identified in plants, including norepinephrine (3, 12), tyramine (9, 24), dopamine (13, 19), adrenaline and dopa (1 1), N-methyl tyramine (22), and octopamine (16, 26). Although these compounds have been extensively studied in insects, mollusks, and crustaceans for their neurotransmitter properties (6, 18), their functions in plants remain relatively obscure. Several studies relate phenethylamine presence and activity to the actions of plant hormones, although the mechanisms have not been defined. Catecholamines enhance the induction of lettuce hypocotyl elongation by gibberellin, and such enhancement is subject to competitive inhibition by the known antiauxin, trans-cinnamic acid (20). However, because auxin does not by itself produce the gibberellin stimulation, the relationship between the catecholamines, gibberellin, and antiauxins is apparently quite complex. Work of Elstner et al. (10) has shown that endogenous dopamine in sugar beet leaves functions as a cofactor of monovalent oxygen reduction, leading to the release of the plant hormone, ethylene. The pathway for ethylene biosynthesis in plants may, therefore, be influenced by phenethylamine levels and activities, as it is influenced by the activities of other plant hormones such as auxins (15). A further involvement ofcatecholamines in plant organs of movement such as tendrils, which are likely subject to rapid hormonal regulation, again relates these compounds Plant Material Tobacco (Nicotiana tabacuum cv H425), cotton (Gossypium hirsutum cv DPL6 1), corn (Zea mays var A188), soybean (Glycine max cv Hardin, Williams, Peking, and Essex), and sunflower (Helianthus annuus L.) seeds were sterilized with commercial bleach (4-6% NaOCl) diluted 1:5 with water for 10 min, washed three times with sterile distilled water, and germinated on filter paper.