Tip-localized calcium entry fluctuates during pollen tube growth

Studies have been conducted on the dynamics of Ca2+ entry in pollen tubes using ratiometric ion imaging to measure the intracellular gradient and an ion selective vibrating electrode to detect the extracellular influx. A steep tip-focused gradient occurs in all species examined, including Lilium longiflorum, Nicotiana sylvestris, and Tradescantia virginiana. Anlaysis of Lilium pollen tubes loaded with dextran conjugated fura-2 reveals that the gradient derives from Ca2+ entry that is restricted to a small area of plasma membrane at the extreme apex of the tube dome. Since the apical membrane is continually swept to the flanks during tube elongation, either Ca2+ channels are specifically retained at the extreme apex or, as seems more likely, the Ca2+ channels which were active at the tip rapidly inactivate, as new ones are inserted during vesicle fusion. Ratiometric imaging further indicates that the high point of the gradient fluctuates in magnitude from 0.75 to above 3 microM, during measuring intervals of 60 sec, with the elevated points being correlated with an increased rate of tube growth. Independent analysis of the growth at 2- to 3-sec intervals reveals that the rates can fluctuate more than threefold; tubes longer than 700 mu m exhibit oscillations with a period of 23 sec, while tubes shorter than 700 mu m display erratic fluctuations. Inhibition of pollen tube growth caused by mild temperature shock or caffeine (1.5 to 3.0 mM) is correlated with the dissipation of the tip-focused gradient and the Ca2+ influx. Recovery from both treatments is denoted by a global swelling of the pollen tube tip, concomitant with a high transient entry of Ca2+ in the tip. The location of the highest Ca2+ domain within the tip region defines the point from which normal cylindrical elongation will proceed.