Cytokinin stimulates dihydropyridine-sensitive calcium uptake in moss protoplasts

Ca²⁺ influx through dihydropyridine (DHP)-sensitive Ca²⁺ channels is thought to be an early event in cytokinin-induced bud formation in moss protonema because DHP antagonists inhibit bud formation in the presence of cytokinin and DHP agonists stimulate bud formation in the absence of cytokinin [Conrad, P. A. & Hepler, P. K. (1988) Plant Physiol. 86, 684-687]. In the present study, we established the presence of a DHP-sensitive Ca²⁺ transport system by measuring ⁴⁵Ca²⁺ influx into moss protoplasts. Ca²⁺ influx was stimulated by external KCI (up to 5 mM), indicating that transport is voltage-dependent. K⁺-induced Ca²⁺ influx was DHP-sensitive with >50% inhibition at 500 nM nifedipine. Ca²⁺ influx was stimulated by increasing concentrations of the DHP Ca²⁺ channel agonist Bay K8644 with half-maximal effects at 25 nM; this stimulation was seen only in the absence of K⁺, suggesting that the agonist works preferentially on polarized membranes. Ca²⁺ influx was also inhibited by phenylalkylamines (verapamil) and benzothiazepines (diltiazem). The phytohormone 6-benzylaminopurine consistently stimulated Ca²⁺ influx with a K_m value of 1 nM, whereas adenine, indoleacetic acid, and gibberellic acid had no effect on Ca²⁺ transport. The cytokinins kinetin and trans-zeatin caused a greater stimulation of Ca²⁺ influx and induced more bud formation than did 6-benzylaminopurine. These results indicate that Ca²⁺ is taken up into moss protoplasts through voltage-dependent DHP-sensitive Ca²⁺ channels on the plasma membrane and that one of the cytokinin effects in the induction of bud formation is regulation of this plasma membrane Ca²⁺ channel.