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 (Ca 2+ ) 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 subcellular 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 Ca 2+ 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 lm 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 Ca 2+ was imaged by confocal microscopy, showing a V-shaped 40 lm gradient extending from the tip, from 0.2 to 1.0 lM, which oscillates with a 1-4 min period, but with only one major oscillatory component. Extracellular Ca 2+ 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 Ca 2+ 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.

Pollen Tube Growth Regulation by Free Anions Depends on the Interaction between the Anion Channel SLAH3 and Calcium-Dependent Protein Kinases CPK2 and CPK20

“…Correlation between a Cytosolic Anion Concentration Gradient and Ca 2+ Homeostasis at the Tip On in vitro growing conditions (especially in tobacco and lily), it is well established that pollen tubes tend to display oscillations in growth rate, which are associated with changes in tip-localized [Ca 2+ ] cyt changes in various species (Heslop-Harrison et al, 1985;Holdaway-Clarke et al, 1997;Camacho et al, 2000;Lazzaro et al, 2005;Michard et al, 2008). A set of CPKs was recently demonstrated to regulate anion channel activity in guard cells (Geiger et al, 2009b(Geiger et al, , 2011Brandt et al, 2012;Scherzer et al, 2012).…”

“…The activity of the K + -inward rectifier SPIK was recently demonstrated to be CPK dependent (Zhao et al, 2013). Likewise, it is well established that intracellular H + and Ca 2+ gradients generated at the pollen tube tip are closely linked to pollen tube guidance, growth direction, and speed (Pierson et al, 1996;Holdaway-Clarke et al, 1997;Certal et al, 2008;Michard et al, 2008). Many of these physiological processes are interconnected, rendering the analysis of the Ca 2+ signaling cascades difficult.…”

“…In fact, a close regulation of ion dynamics, namely in terms of distinct patterns of extracellular fluxes and the formation of cytosolic ion gradients, is associated with pollen tube growth (Michard et al, 2009). Studies based on chemical and genetically encoded reporters showed that tip-focused cytosolic calcium ([Ca 2+ ] cyt ) and proton (H + ) gradients direct pollen tube growth (Pierson et al, 1994;Feijó et al, 1999;Michard et al, 2008). These cytosolic ion gradients are in line with the amplitude and direction of extracellular Ca 2+ fluxes and H + fluxes and it is generally accepted that the cytosolic gradients are, to a great extent, a consequence of local plasma membrane ion fluxes (Michard et al, 2009;Hepler and Winship, 2010).…”

Techniques for the analysis of protein-protein interactions in vivo

“…One reason for the popularity of FRET is that, in addition to its use for detecting PPIs, it also can be used with genetically encoded, unimolecular sensors, and it can be used in native species in vivo (Frommer et al, 2009;Uslu and Grossmann, 2016). A range of sensor molecules have been developed or optimized for use in plants, enabling the detection of calcium (Krebs et al, 2012;Wagner et al, 2015), phosphate (Mukherjee et al, 2015), zinc (Lanquar et al, 2014), and abscisic acid (Jones et al, 2014a) as well as physiochemical states of the cell such as pH (Michard et al, 2008) and membrane voltage . One great advantage of using intramolecular FRET sensors lies in the theoretically equal stoichiometry, rendering ratiometric readouts independent of sensor concentration.…”

An outlook on ion signaling and ionome of mycorrhizal symbiosis

“…Actually, it has been shown that pH changes exert a profound effect on most biological processes, including nutrient uptake, cell growth and plantmicrobe interactions (Feijó et al, 1999;Felle, 2001;michard et al, 2008). Previously, it was postulated by A.r.…”