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

Abstract

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.

The Regulation of Actin Organization by Actin-Depolymerizing Factor in Elongating Pollen Tubes[W]

“…This finding suggests that the actin-interacting activity of NtADF1 may be modulated along a pH gradient, such as that generated by differential proton fluxes at the tip and subapical regions of elongating pollen tubes (Feijo et al, 1999) (Figure 5E). To correlate the location of the ADFrich actin mesh to intracellular H ϩ conditions, we used lily pollen tubes, in which a tip-concentrated H ϩ gradient and a subapical alkaline band in the proximity of the base of the clear zone have been observed (Feijo et al, 1999).…”

“…However, it is apparent that GFP-LlADF was prominently associated with actin filaments in a region relatively more alkaline than the apex (Feijo et al, 1999). A similar spatial relationship between the GFP-NtADF1-labeled actin mesh and intracellular H ϩ concentration likely exists in tobacco pollen tubes in which differential H ϩ fluxes around the tube tip have been observed (C. Chen, H. Wu, A.Y.…”

NAD(P)H Oscillates in Pollen Tubes and Is Correlated with Tip Growth

“…There could by many key steps that require ATP; however, we draw attention to a few events that are confined to the apical 10 mm of the pollen tube. First, we note the internal alkaline band (Feijó et al, 1999), which presumably is generated by a proton-pumping ATPase located on the plasma membrane. The pumping of H 1 creates a local gradient that likely affects the structure and activity of the nearby cortical actin fringe, as well as membrane transport activities.…”

Self-Incompatibility-Induced Programmed Cell Death in Field Poppy Pollen Involves Dramatic Acidification of the Incompatible Pollen Tube Cytosol

“…Oscillations of [pH] cyt between approximate pH values of 6.9 and 7.3 have been linked to tip growth in L. formosanum pollen tubes (Lovy-Wheeler et al, 2006). The vacuole and the apoplast have a highly acidic pH between pH 5 and pH 6 (Katsuhara et al, 1989;Feijó et al, 1999 (Bibikova et al, 1998). Root gravitropic responses stimulate small transient [pH] cyt alterations (Scott and Allen, 1999;Fasano et al, 2001;Johannes et al, 2001).…”

“…Pollen tubes, like other tip-growing cells, have [pH] cyt gradients (Gibbon and Kropf, 1994;Feijó et al, 1999). The [pH] cyt of the pollen tube shank is an approximate pH of 6.9 to 7.11 (Fricker et al, 1997;Messerli and Robinson, 1998).…”