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用离子流技术和荧光蛋白提高胞内外Ca2+和H+的时空分辨率

2020.7.20

关键词：花粉管(Pollen tube)，钙信号(Calcium signaling)，质子信号(Proton signaling)，细胞分化(Cell polarization)

参考文献：Erwan Michard. et al. Sexual Plant Reproduction, 2008, 21: 169-181

全文下载：http://www.xuyue.net/xylt/attachment.php?aid=175

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 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 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 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 Ca²⁺ 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 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.

Ca²⁺和H⁺在花粉管生长、定向伸长及形态形成方面起重要作用，花粉管中分化细胞的伸长需要Ca²⁺和H⁺的浓度梯度的存在。由于百合不易构建稳定的转基因体系，因此并不适合作为分子遗传研究的模式植物，而烟草转基因及细胞生物学方面的研究较为深入，其花粉管也便于获得，因此更适合作为花粉管研究的模式材料。

葡萄牙科学家Feijó和Michard等研究人员用烟草作为实验材料，瞬时表达了pHluorin和黄色Cameleon蛋白作为探针分别用于胞内H⁺和Ca²⁺的比例成像分析，H⁺和Ca²⁺离子流通过非损伤微测技术流获得，并应用傅立叶分解法及连续小波分析法分析花粉管伸长期间离子浓度梯度、离子流及生长速率。结果表明，烟草花粉管尖端存在一个0.4pH单位的梯度，而且花粉管上存在一个亚尖端的碱化区域。胞外质子流振荡大约在10～40pmol·cm^-2·s^-1，花粉管胞内及胞内H⁺振荡存在一个或两个峰，Ca²⁺在花粉管尖端存在一个0.2～1.0uM的离子浓度梯度，振荡周期为1～4min，胞外的Ca²⁺流振荡大约在2～50pmol·cm^-2·s^-1。共聚焦及宽谱显微观察表明，花粉管细胞内的H⁺和Ca²⁺的模式和形状有所差异。