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J. Cell Biol.,
Volume 144, Number 3, February 8, 1999 483-496
* Department Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, P-1749-016 Lisboa, Portugal; 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.
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
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