TIP-LOCALIZED CALCIUM-ENTRY FLUCTUATES DURING POLLEN-TUBE GROWTH

Studies have been conducted on the dynamics of Ca2+ entry in pollen tu bes using ratiometric ion imaging to measure the intracellular gradien t 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 v irginiana. Analysis of Lilium pollen tubes loaded with dextran conjuga ted fura-2 reveals that the gradient derives from Ca2+ entry that is r estricted to a small area of plasma membrane at the extreme apex of th e tube dome. Since the apical membrane is continually swept to the han ks during tube elongation, either Ca2+ channels are specifically retai ned at the extreme apex or, as seems more likely, the Ca2+ channels wh ich were active at the tip vapidly inactivate, as new ones are inserte d during vesicle fusion. Ratiometric imaging further indicates that th e high point of the gradient fluctuates in magnitude from 0.75 to abov e 3 mu M, during measuring intervals of 60 sec, with the elevated poin ts being correlated with an increased rate of tube growth. Independent analysis of the growth at 2- to 3-sec intervals reveals that the rate s can fluctuate more than threefold; tubes longer than 700 mu m exhibi t oscillations with a period of 23 sec, while tubes shorter than 700 m u m display erratic fluctuations. Inhibition of pollen tube growth cau sed by mild temperature shock or caffeine (1.5 to 3.0 mM) is correlate d 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 t he tip. The location of the highest Ca2+ domain within the tip region defines the point from which normal cylindrical elongation will procee d. (C) 1996 Academic Press, Inc.