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 4-Ca2+ 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 Km 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. an adenine-type cytokinin (8), and subsequently, a small initial cell is formed (6). Asymmetric division of the initial cell leads to bud formation and a change from the twodimensional filamentous protonemata to the three-dimensional "leafy" gametophore.Cytokinin applied to caulonema cells causes profuse premature bud formation (6), and localized increases in Ca2+ precede this cytokinin-induced cell division (9-13). Wholeplant studies indicate that cytokinin-modulated Ca2+ entry takes place via dihydropyridine (DHP)-sensitive channels on the plasma membrane (13). DHPs are organic compounds that modulate Ca2+ movement through voltage-dependent Ca2+ channels in the plasma membrane of animal cells (14) by fluorescent tubes. Petri dishes containing appropriately supplemented basal medium overlaid with sterile cellophane were inoculated with spore suspensions. To prepare spore suspensions, mature sporophytes were harvested, and capsules were sterilized by soaking in 3% (wt/vol) sodium hypochlorite/0.1% Triton X-100 for 10 min followed by five rinses in sterile distilled water. Capsules were opened with sterile forceps, and the spores were dispersed in sterile distilled water; plates were inoculated with 1 ml of spore suspension (104 viable spores per ml).To isolate protoplasts, protonemata were incubated with 2% (wt/vol) driselase (laminarinase, xylanase and cellulase,

The role of calcium as an activator and regulator of many biological processes is linked to the ability of the cell to rapidly change its cytoplasmic calcium levels. Calcium acts as an intracellular messenger in hormone-induced bud formation during the development of the moss Physcomitrella patens. Calcium transport and ligand binding studies have implicated plasma membrane-localized 1, 4-dihydropyridine (DHP)-sensitive calcium channels in this increase in cellular calcium. To understand the regulation of the moss calcium channel, we investigated the involvement of GTP binding regulatory proteins (G proteins). Guanosine 5'-(gamma-thio)triphosphate (GTPgammaS), a nonhydrolyzable GTP analog that locks G proteins into their active state, stimulated DHP binding to high affinity receptors in the moss plasma membrane. DHP binding was measured as the ability of the DHP agonist Bay K8644 or the DHP antagonist nifedipine to compete with the DHP arylazide [3H]azidopine for binding to moss plasma membranes. G protein stimulation of binding was seen when competition was carried out with either nifedipine or Bay K8644. G proteins regulated the rates of association and dissociation of bound [3H]azidopine, and stimulation was dependent on GTPgammaS concentration. Guanosine 5'-(beta-thio)diphosphate, a GDP analog that locks G proteins into their inactivated state, did not affect the dose dependence of either the agonist or the antagonist. These results suggest that G proteins may act via a membrane-delimited pathway to regulate calcium channels in the moss plasma membrane.

An increase in cytoplasmic calcium is an early event in hormone (cytokinin)-induced vegetative bud formation in the moss Physcomitrella patens. Whole cell and calcium transport studies have implicated 1,4-dihydropyridine-sensitive calcium channels in this increase in cellular calcium. Controlled changes in cellular calcium concentrations have been identified as important components of signal transduction pathways in plants. Cytoplasmic calcium concentrations are highly regulated; levels are modulated by coordinating passive fluxes and active transport across organellar and plasma membranes (1-4). Cytoplasmic calcium levels have been shown to increase in response to a variety of stimuli including light (5, 6) and hormones (7,8), and small fluctuations in cellular calcium may modulate processes as diverse as secretory activity in the barley aleurone (7), pollen tube growth (9), and phase transition in mitosis (10). Studies examining the effect of calcium channel inhibitors on physiological processes (11-14) have suggested a role for calcium channels in stimulus-induced increases in cytoplasmic calcium levels; however, little is known about the biochemical or molecular properties of these transport systems.Calcium acts as an intracellular messenger in hormone (cytokinin)-induced vegetative bud formation during the development of the filamentous protonemata (the young gametophore) in the moss Physcomitrella patens (15-18). Formation of vegetative buds is an integral part of the moss life cycle leading to the development of the mature gametophore which is essential for subsequent sexual reproduction. Cytokinin applied to moss cells causes profuse premature bud formation (19). Localized increases in calcium take place after addition of cytokinin but precede the cytokinin-induced cell division (15, 18). In moss cells not stimulated by cytokinin, cytoplasmic calcium levels (250 nM) are three orders of magnitude lower than levels in the external medium (0.1-1.0 mM) (18). After addition of cytokinin, cytoplasmic calcium levels increase to 750 nM (18). Whole plant studies indicate that cytokinin-modulated calcium entry takes place via dihydropyridine (DHP) 1 -sensitive channels (20). In moss protonemata, application of DHP calcium channel agonists in the absence of cytokinin stimulates bud initial formation, whereas DHP calcium channel antagonists block cytokinin-induced bud formation (20). We have previously characterized calcium influx into isolated moss protoplasts and have established that the transport activity of the moss calcium channel shares common characteristics with L-type calcium channels in animal cells. Calcium transport in moss is voltagedependent, stimulated by DHP agonists, and inhibited by DHP antagonists, phenylalkylamines, and benzothiazepines (21). A novel feature of the transport activity of this channel is hormonal modulation by cytokinin (21).Cytokinin-induced bud formation in moss is a simple, highly ordered developmental process and is one of few plant responses that allows direct study of stimul...