Inhibition of Cytokinin-Regulated Responses by Calmodulin- Binding

Two classes of compous which bind to calnodulin in a calciumdependent manner (neuroleptic drugs and local anaesthetics) were used to investigate the possible involvement of a calcium-dependent regulator protein in the action of the plant hormones cytokinins. The cytokinin-induced synthesis of betacyanin in Amarandus tricolr seedlings was used as one test system. The calmodulin antagonists inhibited betacyanin synthesis with the following order of potency: fluphenazine > trifluoperazine = pimozide > chlorpromazine > dibucaine > penfluridol > haloperidol > tetracaine, over a concentration range (ICs,) of 0.1 to 0.6 minmoolar. Red lht and fusicoccin increase betacyanin accumulation and are synergistic with cytokinins. These red lightand fusicoccin-dependent inductions were inhibited by lower concentrations of the drugs than cytokiin-dependent induction, and the order of potency of the drugs was not precisely the same. The results are compatible with the hypothesis that cytokinins may act, at least in part, by nging ion fluxes, with the additional involvement of a cainoduln The second cytokinin-regulated response studied was growth in soybean callus culture. In this system, inhibition was observed with lower concentrations of drugs than in cytokinin-dependent betacyanin synthesis with an order of inhibitory potency of pimozide = trifluoperazine > penfluridol > chlorpromazine > haloperidol > tetracaine. The effective concentration range (IC5o) was from 0.07 to 0.5 milmolar. Inhibition of betacyanin synthesis by 0.15 mflimolar trifluoperazine and of soybean callus growth by 2 milimolar tetracaine were both reversible. Cytokinins can be shown to regulate the induction in the dark of betacyanin accumulation in Amaranthus tricolor half-seedlings. Inhibition ofsynthesis ofmembrane components during a seedling pretreatment involving heat shock and aging, and during induction of the half-seedlings shows that new membrane synthesis, and possibly increased membrane synthesis, are required for the potentiation of the cytokinin response (10). Some idea of the identity of the membrane function involved has been gained from the following experiments. Increased proton extrusion has been shown in Amaranthus cotyledons and protoplasts (3) and in leaf parts and cotyledons of other plants (22) in response to the synthetic cytokinin BA. Betacyanin accumulation is stimulated 50 to 100% by K+ in the exogenous medium, the best induction being given by K+/Na+ mixture with phosphate as the anion (6). Cytokinin and fusicoccin (a phytotoxin which enhances H+ extrusion and K+ uptake [23]) are synergistic in increasing betacyanin accumulation (5) as are cytokinin and red light (8). These results led to the conclusion that cytokinins affect ion fluxes, particularly ' Supported by grants from the Australian Research Grants Committee and the Flinders University Research Budget. KV. This conclusion is supported by inhibitory effects of uncouplers used for destroying proton fluxes and of inhibitors of membrane ATPases (5). Previous work on the Amaranthus system has, in addition, given evidence of Ca2" involvement in betacyanin accumulation (see Discussion). Hence, in view of the widespread involvement of calmodulin in many Ca2"-regulated events (see references in Ref. #9), the present study was undertaken to see if certain drugs which inhibit calmodulin-dependent processes have any effect on the cytokinin-regulation of betacyanin synthesis. Preliminary experiments on the use of trifluoperazine m this system have been reported (9). The second cytokinin-regulated response (control of cell division in cell cultures) was included in this study to explore the generality of the drug effects on cytokinin action. MATERIALS AND METHODS Chemicals. Benzyladenine, dibucaine, chlorpromazine, tetracaine, and L-tyrosine were from Sigma. Generous gifts of chemicals were received from the following companies: E.R. Squibb and Sons (fluphenazine dihydrochloride), Searle Laboratories (haloperidol), Janssen Pharmaceutica Pty. Ltd. (pimozide and penfluridol), Smith, Kline, and French Laboratories (trifluoperazine). Standard Assay Conditions for Betacyanin Accunulation. Methods have been described elsewhere (7) for the germination of seeds ofAmaranthus tricolor and the pretreatment of half-seedlings (ie. cotyledons plus the top 5 mm of the hypocotyl). Aging of half-seedlings in water (1.5 h at 40°C followed by 1.5 h at 25°C) was found to maximize subsequent cytokinin-induction. The basic induction-medium was 5 mM L-tyrosine (betacyanin precursor) in 10 mm K2HPO4-NaH2PO4 (pH 6.8) ± 0.5 ,UM BA (synthetic cytokinin), and the incubation was at 25°C for 24 h in the dark. Betacyanin was extracted in 3.33 mm acetic acid and measured at 537 nm (7). Soybean Callus Assay. The procedure of Miller (24) was used for the growth of soybean callus material and the assay of cytokinin-dependent growth. Cultures were maintained on standard medium with the addition of 0.5 pM BA and 30 ,UM IAA.