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

2020-05-18 17:09:45

Sex Plant Reproduction	Tobacco pollen tube as a model for ion dynamics research
	Enhance the temporal and spatial resolution of intracellular and extracellular Ca ²⁺ and H ⁺ by ion current technique and fluorescent protein

Ca ²⁺ and H ⁺ play an important role in pollen tube growth, oriented elongation and morphogenesis. The elongation of differentiated cells in pollen tubes requires the concentration gradient of Ca ²⁺ and H ⁺ . Because lily is not easy to construct a stable transgenic system, it is not suitable as a model plant for molecular genetic research. However, tobacco transgenic and cell biology research is more in-depth, and its pollen tube is also easy to obtain, so it is more suitable as a model for pollen tube research. material.

Portuguese scientists such as FeijÃ³ and Richard used tobacco as an experimental material to transiently express pHluorin and yellow Cameleon proteins as probes for intracellular H ⁺ and Ca ²⁺ ratio imaging analysis. H ⁺ and Ca ²⁺ ions flow through The non-damage micro-test technique flow was obtained, and the Fourier decomposition method and continuous wavelet analysis were used to analyze the ion concentration gradient, ion current and growth rate during the elongation of the pollen tube. The results showed that there was a gradient of 0.4 pH unit at the tip of the tobacco pollen tube, and there was a sub-tip alkalized area on the pollen tube. The extracellular proton flow oscillation is about 10~40pmolÂ·cm ^-2 Â·s ^-1 , there is one or two peaks in the intracellular and intracellular H ⁺ oscillation of pollen tube, and there is a 0.2 ~ 1.0uM Ca ²⁺ at the tip of the pollen tube. The ion concentration gradient has an oscillation period of 1 to 4 minutes, and the extracellular Ca ²⁺ flow oscillation is about 2 to 50 pmolÂ·cm ^-2 Â·s ^-1 . Confocal and broad-spectrum microscopic observations showed that the patterns and shapes of H ⁺ and Ca ²⁺ in pollen tube cells were different.

This study uses non-invasive micro-test technology and fluorescent indicator proteins to improve the research accuracy of pollen tubes H ⁺ and Ca ²⁺ . It is an example of the combination of molecular biology methods and physiological detection methods to study pollen tubes.

Above :

The H ⁺ flux at the tip of the tobacco pollen tube was determined by the intracellular H ⁺ concentration of the tip of the pollen tube stained with pHluorin and using a non-damage micro-test technique. Positive values â€‹â€‹are outflows and negative values â€‹â€‹are inflows.

Key words : Pollen tube, Calcium signaling, Proton signaling, Cell polarization

References : Erwan Michard. et al. Sexual Plant Reproduction, 2008, 21: 169-181

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Abstract The presence of both calcium (Ca ²⁺ ) and proton (H ⁺ ) apical gradients is necessary for polarized cells 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 characterized 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 u Nit acidic gradient was found to stretch from the tip up to 40 lm into the tube shank. This gradient intensity displayed 1â€“4 min period oscillations and is reduced in the non-growing phase of an oscillatory cycle. further, sub-membrane and sub - extracellular H+ fluxes oscillated between 10 and 40 pmol cm ^-2 s ^-1 . Fourier and continuous wavelet groups showed tubes with one or two major
The 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 1â€“4 min period, And 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.

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