Enhance the temporal and spatial resolution of intracellular and extracellular Ca2+ and H+ by ion current technique and fluorescent protein

**Sex Plant Reproduction** **Tobacco Pollen Tube as a Model for Ion Dynamics Research** Enhancing the temporal and spatial resolution of intracellular and extracellular Ca²⁺ and H⁺ using ion current techniques and fluorescent proteins. --- A horizontal line separates the title from the main content. Calcium (Ca²⁺) and hydrogen (H⁺) ions play crucial roles in pollen tube growth, directional elongation, and morphogenesis. The elongation of differentiated cells in pollen tubes depends on the concentration gradients of these ions. While lily is often used in such studies, it is challenging to establish a stable transgenic system, making it less suitable for molecular genetic research. In contrast, tobacco has well-developed transgenic systems and its pollen tubes are easily accessible, making it a more practical model for studying pollen tube dynamics. Portuguese scientists like Feijó and Richard have used tobacco as an experimental model to transiently express pHluorin and yellow Cameleon proteins as probes for intracellular H⁺ and Ca²⁺ ratio imaging. Using the non-damage micro-test technique, they measured the flow of H⁺ and Ca²⁺ ions and applied Fourier decomposition and continuous wavelet analysis to study the ion concentration gradient, ion current, and growth rate during pollen tube elongation. Their findings revealed a 0.4 pH unit gradient at the tip of the tobacco pollen tube, with a sub-tip alkalized region. Extracellular proton flow oscillations were observed at around 10–40 pmol·cm⁻²·s⁻¹, while intracellular H⁺ oscillations had one or two peaks. A Ca²⁺ concentration of 0.2–1.0 μM was detected at the tip, with oscillation periods of 1–4 minutes. Extracellular Ca²⁺ flow oscillations ranged from 2–50 pmol·cm⁻²·s⁻¹. Confocal and widefield microscopy showed that H⁺ and Ca²⁺ patterns within the pollen tube cells differed significantly. This study highlights the use of non-invasive micro-test technology and fluorescent indicator proteins to improve the accuracy of research on H⁺ and Ca²⁺ in pollen tubes. It represents a successful integration of molecular biology methods with physiological detection techniques in the study of pollen tube function. --- **Above**: The H⁺ flux at the tip of the tobacco pollen tube was determined by measuring the intracellular H⁺ concentration using pHluorin and a non-damage micro-test technique. Positive values indicate outflow, while negative values indicate inflow. **Key words**: Pollen tube, Calcium signaling, Proton signaling, Cell polarization **References**: Erwan Michard et al., *Sexual Plant Reproduction*, 2008, 21: 169–181 **Full text download**: [http://?aid=175](http://?aid=175) **Abstract**: The presence of both calcium (Ca²⁺) and proton (H⁺) apical gradients is essential for polarized cell elongation in pollen tubes. Most previous studies have been conducted in lily pollen tubes using chemical probes, but lily is not ideal for molecular genetics due to difficulties in creating stable transgenic lines. Tobacco, however, is well-suited for transformation and cell biology, with sexual organs that are easy to handle and visualize. Pollen tubes are also an ideal size for subcellular imaging using modern microscopy techniques. Despite this, ion homeostasis in tobacco pollen tubes has not been well characterized. This study uses two fluorescent genetic probes, pHluorin and YC3.1 yellow CaMeleon, along with direct measurement of extracellular ion fluxes via ion-sensitive vibrating probes. A distinct 0.4 pH unit acidic gradient was found to extend from the tip up to 40 µm into the tube. This gradient displayed oscillations with a period of 1–4 minutes and decreased during the non-growing phase. Sub-membrane and extracellular H⁺ fluxes oscillated between 10 and 40 pmol cm⁻² s⁻¹. Fourier and continuous wavelet analyses revealed one or two major oscillatory components in both extracellular and intracellular H⁺ oscillations. Cytosolic Ca²⁺ was imaged using confocal microscopy, showing a V-shaped gradient extending from the tip, ranging from 0.2 to 1.0 µM, with oscillations of 1–4 minutes and one dominant oscillatory component. Extracellular Ca²⁺ fluxes oscillated between 2 and 50 pmol cm⁻² min⁻¹, similar to H⁺, with one or two major peaks. Combining confocal and widefield microscopy showed that H⁺ and Ca²⁺ displayed different spatial patterns inside the cell, suggesting a potentially complementary role in the growth process. These results suggest that ion fluxes at the pollen tube apex directly contribute to the establishment and maintenance of the gradient.

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