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** Ca²⁺ and H⁺ play crucial roles in pollen tube growth, directional elongation, and morphogenesis. The elongation of differentiated cells in pollen tubes relies on the concentration gradients of Ca²⁺ and H⁺. While lily is not ideal for molecular genetic studies due to its difficulty in establishing stable transgenic systems, tobacco offers more advanced transgenic and cell biology research opportunities. Its pollen tubes are also easily obtainable, making it a better model for studying pollen tube dynamics. Portuguese scientists like Feijó and Richard used tobacco as an experimental system to transiently express pHluorin and yellow Cameleon proteins as probes for intracellular H⁺ and Ca²⁺ ratio imaging. Using the non-invasive micro-test technique, they measured ion flow and applied Fourier decomposition and continuous wavelet analysis to study the ion concentration gradient, 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, along with a sub-tip alkalized region. Extracellular proton flow oscillations were observed at about 10–40 pmol·cm⁻²·s⁻¹, while intracellular H⁺ oscillations showed one or two peaks. A Ca²⁺ concentration of 0.2–1.0 μM was detected at the tip, with oscillation periods ranging from 1 to 4 minutes. Extracellular Ca²⁺ flow oscillations varied between 2 and 50 pmol·cm⁻²·s⁻¹. Confocal and widefield microscopy revealed distinct patterns of H⁺ and Ca²⁺ within the pollen tube, suggesting different spatial distributions and potential complementary roles in growth regulation. This study demonstrates how non-invasive micro-test technology and fluorescent protein probes can enhance the accuracy of ion dynamics research in pollen tubes. It exemplifies the integration of molecular biology techniques with physiological detection methods to explore the mechanisms underlying pollen tube growth. --- **Above**: The H⁺ flux at the tip of the tobacco pollen tube was measured using pHluorin-stained tip cells and the non-damage micro-test technique. Positive values indicate efflux, while negative values represent influx. **Keywords**: 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 challenging for molecular genetics due to the lack of efficient transgenic protocols. In contrast, tobacco is well-suited for transformation and cell biology, with accessible and easily visualized reproductive organs. Pollen tubes in tobacco are ideal for subcellular imaging with modern microscopy techniques. To date, ion homeostasis in tobacco pollen tubes has not been fully characterized. This study used two fluorescent genetic probes, pHluorin and YC3.1 yellow CaMeleon, along with direct measurement of extracellular flux via ion-sensitive vibrating probes, to investigate H⁺ and Ca²⁺ spatial and temporal patterns. A clear 0.4 pH unit acidic gradient was found to extend from the tip up to 40 µm into the tube shank. This gradient exhibited oscillations with a period of 1–4 minutes and decreased during non-growing phases. Sub-membrane and extracellular H⁺ fluxes oscillated between 10 and 40 pmol cm⁻² s⁻¹. Fourier and continuous wavelet analyses identified one or two major oscillatory components in both extracellular and intracellular H⁺ oscillations. Cytosolic Ca²⁺ was imaged using confocal microscopy, revealing a V-shaped gradient extending 40 µm from the tip, ranging from 0.2 to 1.0 µM, with oscillations of 1–4 minutes and only one dominant component. Extracellular Ca²⁺ fluxes oscillated between 2 and 50 pmol cm⁻² min⁻¹, similar to H⁺, with one or two major oscillatory peaks. Combined confocal and widefield microscopy showed that H⁺ and Ca²⁺ displayed distinct patterns and shapes inside the cell, sometimes indicating a structurally complementary role for these two second messengers 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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