In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts

The focus of this study is to investigate the regulatory role of K+ influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 pm after a 6 h incubation. A decrease in external K+ concentration from 1 mM to 35 muM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K+ concentration from I mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K+ influx is associated with pollen germination and tube growth, regulation of the inward K+ channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K+ currents were first identified in Arabidopsis pollen protoplasts. The inward K+ currents were insensitive to changes in cytoplasmic Ca2+ but were inhibited by a high concentration of external Ca2+. A decrease of external Ca2+ concentration from 10 mM (control) to 1 mM had no significant effect on the inward K+ currents, while an increase of external Ca2+ concentration from 10 mM to 50 mM inhibited the inward K+ currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K+ currents. The physiological importance of potassium influx mediated by the inward K+-channels during Arabidopsis pollen germination and tube growth is discussed.