Calmodulin-Binding Drugs Affect Responses to Cytokinin, Auxin,

Trifluoperazine, a phenothiazine trnizer and tetracalne, a local anesthetic, have been found to Inhibit a variety of plant hormone responses at concentratons compatible with their known Inhibition of Ca'-calmoduln-dependent enzyme actvities. Among these responses are cytokinindependent betacyanin synthesis and increase In fresh weight InAmarahw tcolor cotyedons, auxin-dependent increase in length of wheat coleoptile segments and gibberellk aciddepndent induction of a-amylase synthesis in barley alern layers. The reversbility of some of these inhibitory effects has been demonstrated, indicating that, up to a point, a generalzed membrane destruction can be rled out. The evidence, taken in conjunction with numerous examples from the literature showing calcium involvement in the action of all of the plant hormones, support a unifying theory of hormone action. The importance of calcium in plant membranes has long been recognized, initially in a structural role related to membrane integrity (17) and in such processes as the mechanism of uptake ofmonovalent ions (see references in 9). More recently, membrane storage of calcium and membrane control of calcium transport have emerged as key processes necessary to trigger initial events in many biological responses which may depend on changes in cytoplasmic calcium concentration (1, 2, 5). Epel (14) proposed that many cellular or developmental processes may be triggered by some event such as the early changes in cation permeability, particularly the increased Ca2" concentration in the cytoplasm, seen in the fertilization of the sea urchin egg. One example given was the photoinduction of developmental programs in plants. Trewavas (27-29) has taken this fertilization system as a model to propose a unifying theory for developmental control in plants, depending on a membrane being the controlling system, with alterations in cytoplasmic ion balance leading to subsequent changes. Changes in ion transport were proposed as key events in cytokinin-dependent cell enlargement (15, 16, 21, 22, 25). Comparison with effects of the phytotoxin fusicoccin led Marre (23) to suggest that the growth effect of cytokinins (as well as effects of IAA on cell enlargement, ABA-induced inhibition of stomatal opening and of seed germination, and some aspects of GA3-induced seed germination) depends on the activation of the fusicoccin-sensitive H+/K+-exchange mechanism of ion transport, but that the activation may be an indirect one through a complex series of events possibly involving the synthesis of some short lived protein(s). In Amaranthus tricolor cotyledons, fusicoccin was found to be syn1 This work was supported by grants from the Australian Research Grants Committee and the Finders University Research Budget. ergistic with BA in induction of betacyanin synthesis, thus leading to a modification of Marre's proposal, with expansion to a general theory of cytokinin action (8, 10). Recent work has followed the line that calcium interactions with cytokinins may mean that the calcium-dependent regulator protein calmodulin is involved. It was found that drugs which bind to calmodulin and inhibit its action in other systems do indeed inhibit betacyanin accumulation, whether induced by cytokinin, fusicoccin, or red light, and also another cytokinin-dependent response, cell division in soybean callus tissues (13). This general theory of cytokinin action has now been integrated with the idea of a unifying theory of plant hormone action as referred to above (14, 23, 27, 28) and these drugs have been used to test this unifying theory in another cytokinin-dependent response, and in classical responses to auxin and to GA3. The drugs that have been used in this study are trifluoperazine and tetracaine, representatives of the two classes of calmodulin-binding compounds, neuroleptic drugs (31) and local anesthetics (26, 30), respectively. The hormone responses chosen were cell expansion growth in excised Amaranthus cotyledons (cytokinin-dependent), cell expansion growth in wheat coleoptiles (auxin-dependent), and e-amylase induction in barley aleurone layers (gibberellic aciddependent). MATERIALS AND METHODS Chemicals. BA, IAA, GA3, and TC2 HCI were from Sigma. TFP HCI was a gift from Smith Kline and French Laboratories; it was made up as a 10 mm stock aqueous solution, adjusted to pH 6.1 with 1 N NaOH. Cell Expansion Growth and Betacyanin Accumulation in Amarwudws Cotyledons. Amaranthus tricolor seeds (1 g) were germinated in the dark at 25 ± 1 °C for 96 h under standard conditions (1 1). The roots were removed and the remaining cotyledons plus hypocotyls (half-seedlings) were placed in 20 ml distilled H20 in a 15-cm Petri dish and were transferred to 40 ± 1°C in the dark for 1.5 h and a further 1.5 h at 25°C with the lid off. This treatment had been found to potentiate subsequent betacyanin induction by BA (11 and references therein). After aging, the cotyledons were pinched off from the hypocotyl and placed on filter papers covered by incubation medium. Each treatment comprised 30 cotyledons/9-cm Petri dish with 5 ml buffer (10 mm KMHPO4-NaH2PO4, pH 6.8), +5 pm BA, with and without drug additions. All treatments were in triplicate. After 24-h incubation at 25°C in the dark, increases in fresh weight and levels of betacyanin accumulation (12) were measured. Cell Expansion Growth in Wheat Coleoptiles. Dry wheat seeds (Triticum aestivum L. var Olympic) were sown on saturated but thoroughly drained vermiculite and grown for 72 h in the dark at 2Abbreviations: IC50, concentration giving 50% inhibition; TC, tetracaine; TFP, trifluoperazine. 219 www.plantphysiol.org on January 13, 018 Published by Downloaded from Copyright © 1983 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 72, 1983