Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria

Abstract

The plant hormone cytokinin stimulates nuclear migration followed by an asymmetric cell division in target cells of the protonema of the moss Funaria hygrometrica, leading to bud formation. The role of calcium in this developmental event was investigated by examining the effects of various calcium antagonists on the cytokinin-induced division. Calcium-free medium (buffered with EGTA), the extracellular Ca2+ antagonist La3+ (lanthanum), and the Ca2+ channel inhibitors D 600 and verapamil all block bud formation. These inhibitions are partially reversed by washing the cells or by raising the extracellular [Ca2+]. The Ca2+ ionophore A23187 partially reversed the effects of D 600 and verapamil. Bud formation is also inhibited by the intracellular Ca2+ antagonist TMB-8 (8-diethylamino)octyl 3,4,5-trimethoxybenzoate HCl), and this inhibition is partially reversed by washing or raising the extracellular [Ca2+]. The cross walls of both the filaments and bud initial cells formed during TMB-8 exposure exhibit a distorted morphology. High concentrations of TMB-8 block nuclear migration. The calmodulin inhibitor trifluoperazine stops cytokinin-induced budding more effectively than the related compound chlorpromazine. Low concentrations of these two compounds do not affect nuclear migration; however, the target cell does not enter mitosis. These results support the hypothesis that a rise in intracellular calcium mediates cytokinin-induced bud formation in Funaria. It is concluded that the proposed cytokinin-induced rise in intracellular calcium may be effected in part by the activation of calmodulin. The essential source of Ca2+ appears to be extracellular, because blocking Ca2+ uptake with Ca2+ transport inhibitors can block both nuclear migration and subsequent division.