用離子流技術和熒光蛋白提高胞內外Ca2+和H+的時空分辨率
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Ca2+和H+在花粉管生長、定向伸長及形態形成方面起重要作用，花粉管中分化細胞的伸長需要Ca2+和H+的濃度梯度的存在 。由于百合不易構建穩定的轉基因體系，因此并不適合作為分子遺傳研究的模式植物，而煙草轉基因及細胞生物學方面的研究較為深入，其花粉管也便于獲得，因此更適合作為花粉管研究的模式材料。 葡萄牙科學家Feijó和Michard等研究人員用煙草作為實驗材料，瞬時表達了pHluorin和黃色Cameleon蛋白作為探針分別用于胞內H+和Ca2+的比例成像分析，H+和Ca2+離子流通過非損傷微測技術流獲得，并應用傅立葉分解法及連續小波分析法分析花粉管伸長期間離子濃度梯度、離子流及生長速率。結果表明，煙草花粉管尖端存在一個0.4pH單位的梯度，而且花粉管上存在一個亞尖端的堿化區域。胞外質子流振蕩大約在10～40pmol·cm-2·s-1，花粉管胞內及胞內H+振蕩存在一個或兩個峰，Ca2+在花粉管尖端存在一個0.2～1.0uM的離子濃度梯度，振蕩周期為1～4min，胞外的Ca2+流振蕩大約在2～50pmol·cm-2·s-1。共聚焦及寬譜顯微觀察表明，花粉管細胞內的H+和Ca2+的模式和形狀有所差異。 這項研究應用非損傷微測技術和熒光指示蛋白使花粉管H+和Ca2+的研究精度得以提高，是分子生物學手段與生理檢測手段結合研究花粉管的一個范例。 |
 上圖： 通過pHluorin染色的花粉管尖端胞內的H+濃度和應用非損傷微測技術測定煙草花粉管尖端的H+流。正值為外流，負值為內流。 | |||
關鍵詞：花粉管(Pollen tube)，鈣信號(Calcium signaling)，質子信號(Proton signaling)，細胞分化(Cell polarization)
參考文獻：Erwan Michard. et al. Sexual Plant Reproduction, 2008, 21: 169-181
全文下載：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.
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.
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