Cytokinin stimulates dihydropyridine-sensitive calcium uptake in moss protoplasts.

Abstract

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,

Phytochrome signal transduction pathways are regulated by reciprocal control mechanisms.

“…Similar results were observed with nifedipine, which blocks animal and plant L-type calcium channels (Schroeder and Thuleau 1991;Schumaker and Gizinski 1993). At a concentration of 60 | XM it could totally inhibit the light induction of cab and fnr, whereas chs induction was again superinduced (Fig.…”

Hyperaccumulation of Cadmium and Zinc in Thlaspi caerulescens and Arabidopsis halleri at the Leaf Cellular Level

“…It has been shown that verapamil at micromolar concentrations inhibited voltage-dependent Ca and Cd influx in animal cells (Hinkle et al, 1987), but the effects reported in plant studies seem inconsistent (Pineros and Tester, 1997): Ca influx into protoplasts from Physcomitrella patens (Schumaker and Gizinski, 1993), carrot (Daucus carota; Graziana et al, 1988), Amaranthus tricolor (Rengel and Elliot, 1992), and tobacco (Nicotiana tabacum; Volotovski et al, 1998) and into plasma membrane vesicles isolated from oat (Avena sativa) roots (Gonzalez et al, 1999) was reduced upon exposure to verapamil, although up to 100 m verapamil did not block the calcium influx into plasma membrane vesicles isolated from wheat roots or oat seedlings (Huang et al, 1994;Babourina et al, 2000). Furthermore, in aquatic plants, it was found to inhibit Ca uptake but to increase Cd uptake (Karez et al, 1990;Tripathi et al, 1995).…”

1,4-Dihydropyridine Binding Sites in Moss Plasma Membranes

“…While There is strong evidence for three calcium antagonist receptor sites on the L-type voltage-dependent calcium channels in animal cells: one for DHPs, one for phenylalkylamines, and one for benzothiazepines (42). We investigated the ability of these molecules to compete for [ ]azidopine binding in a manner qualitatively similar to their ability to inhibit calcium influx into moss protoplasts (21). Inhibition is most likely due to a direct competition for the azidopine-binding site by the DHPs and due to an allosteric inhibition of azidopine binding by verapamil and diltiazem (30 -33).…”

“…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).…”

“…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).…”