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Sexual Plant Reproduction

, Volume 21, Issue 3, pp 169–181 | Cite as

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

  • Erwan Michard
  • Pedro Dias
  • José A. FeijóEmail author
Original Article

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.

Keywords

Pollen tube Calcium signaling Proton signaling Cell polarization 

Notes

Acknowledgments

JAF’s laboratory is supported by FCT grants POCTI/BIA-BCM/60046/2004 and POCTI/BIA-BCM/61270/2004. EM is the recipient of an FCT fellowship (SFRH/BPD 21056/2004). We thank M. Watahiki (University of Hokkaido, Japan) and J. Rothman (Memorial Sloan-Kettering Cancer Center, NY, USA) for YC3.1 and pHluorin clones and constructs, respectively. We thank Nuno Moreno and Ricardo Henriques (IMM) for technical advice and support on microscopy experiments. We thank Prof. Scott D. Russell (Norman, OK, USA) for reading and comments on the manuscript. We thank Ana Catarina Silva for tobacco farming and molecular biology help.

Supplementary material

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Instituto Gulbenkian de Ciência, Centro de Biologia do DesenvolvimentoOeirasPortugal
  2. 2.Department of Biologia VegetalUniversidade de LisboaLisboaPortugal

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