Tobacco pollen tubes as cellular models for ion dynamics: Improved spatial and temporal resolution of extracellular flux and free cytosolic concentration of calcium and protons using pHluorin and YC3.1 CaMeleon

Abstract

The presence of both calcium (Ca2+) and proton (H+) apical gradients is necessary for polarized cell elongation to occur in pollen tubes. So far, most of these studies have been carried out in lily pollen tubes, using chemical probes. Yet, lily is a refractory model for molecular genetics, with no easy protocol available for the construction of stable transgenic lines. Tobacco, however, is well suited for both transformation and cell biology, with sexual organs that are accessible, easy to handle and visualize. Pollen tubes are in an ideal size range for sub-cellular imaging analyses using modern microscopy techniques. Ion homeostasis in tobacco pollen tubes has not been precisely characterized so far. Here, we characterize the H+ and Ca2+ spatial and temporal patterns in tobacco pollen tubes by the use of two fluorescent genetic probes, pHluorin and the YC3.1 yellow CaMeleon, and direct measurement of extracellular flux by ion-sensitive vibrating probes. A distinct 0.4 pH unit acidic gradient was found to stretch from the tip up to 40 μm into the tube shank. This gradient intensity displayed 1-4 min period oscillations and is reduced in the non-growing phase of an oscillatory cycle. Furthermore, sub-membrane and sub-apical alkaline domains were detected. Extracellular H+ fluxes oscillated between 10 and 40 pmol cm -2 s-1. Fourier and continuous wavelet analyses showed tubes with one or two major oscillatory components in both extra and intracellular H+ oscillations. Cytosolic Ca2+ was imaged by confocal microscopy, showing a V-shaped 40 μm gradient extending from the tip, from 0.2 to 1.0 μM, which oscillates with a 1-4 min period, but with only one major oscillatory component. Extracellular Ca2+ fluxes oscillate in most pollen tubes, between 2 and 50 pmol cm-2 min -1 and, like in H+, with one or two major oscillatory peaks. A combination of confocal and widefield microscopy showed that H + and Ca2+ displayed different patterns and shapes inside the cell, sometimes suggesting a structurally complementary role for these 2 second messengers in the growth process. These data suggest that fluxes at the apex of the pollen tube are directly responsible for establishment and maintenance of the gradient. © 2008 Springer-Verlag.