Membrane-associated calcium during pollen grain germination: a microfluorometric analysis

Summary

Pear (Pyrus communis L.) pollen was germinated and grown in hanging drop cultures containing phenothiazine drugs, trifluoperazine and chlorpromazine, potent inhibitors of the Ca²⁺-calmodulin complex. Responses for the two drugs were similar: at 1.0–2 10.0 ΜM pollen germination and tube growth were inhibited. Inhibition of tube growth was not uniform; at 10.0 ΜM growth of about half of those tubes which had germinated was inhibited while the remaining half of the population grew normally. This bimodal distribution of tube growth was noted, but to a lesser extent, at lower concentrations of the drugs as well. Microfluorometric analysis of membrane calcium using chlorotetracycline, a fluorescent chelate probe for membrane calcium, revealed that upon pollen grain hydration there was a sharp decrease in the concentration of calcium associated with membranes and that membrane calcium became localized primarily at the periphery of the newly hydrated pollen grain. Prior to germination there was a reloading of calcium onto membranes in the region of the germination aperture through which the pollen tube would emerge. The release of Ca²⁺ from membrane sites upon hydration was partially inhibited by treatment with 10 ΜM trifluoperazine. Distribution of membrane calcium in populations of the inhibitor-treated pollen grains was not bimodal however; approximately half of the population had CTC-fluorescence emissions exceeding the maximum value found in the control population. These results suggest that there is a maximum concentration of membrane-associated calcium consistent with normal pollen germination and tube growth and that phenothiazines interfere with the unloading of Ca²⁺ from membrane sites.