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© The Rockefeller University Press, 0021-9525/1999//483 $5.00
The Journal of Cell Biology, Volume 144, Number 3, , 1999 483-496

Regular Articles

Growing Pollen Tubes Possess a Constitutive Alkaline Band in the Clear Zone and a Growth-dependent Acidic Tip



J.A. Feijó*,{ddagger}, J. Sainhas{ddagger}, G.R. Hackett§, J.G. Kunkel§, and P.K. Hepler§

* Department Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, P-1749-016 Lisboa, Portugal; {ddagger} Gulbenkian Institute of Science, P-2780-156 Oeiras, Portugal; and § Biology Department and Plant Biology Graduate Program, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003

Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 µM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

Key Words: pollen tube growth • pH • proton flux • ratiometric ion imaging • vibrating probe

Abbreviations used in this paper: ADF, actin-depolymerizing factor; BCECF, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; CCD, charge-coupled device; pHc, cytosolic pH.

J.A. Feijó received personal fellowships from the Fulbright Foundation, Luso-American Foundation for Development, Calouste Gulbenkian Foundation, and Junta Nacional de Investigação Cientifica e Tecnologica. This project was supported by a grant from the National Science Foundation (MCB-96-01087) to P.K. Hepler.


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