Babourina Olga, Rengel Zed
School of Earth and Geographical Sciences M087, University of Western Australia, Crawley, WA, Australia.
Ann Bot. 2009 Jul;104(1):189-95. doi: 10.1093/aob/mcp098. Epub 2009 Apr 28.
Measuring the Al(3+) uptake rate across the plasma membrane of intact root cells is crucial for understanding the mechanisms and time-course of Al toxicity in plants. However, a reliable method with the sufficient spatial and temporal resolution to estimate Al(3+) uptake in intact root cells does not exist.
In the current study, fluorescent lifetime imaging (FLIM) analysis was used to quantify Al(3+) uptake in the root-cell cytoplasm in vivo. This was performed via the estimation of the fluorescence lifetime of Al-lumogallion {5-chloro-3[(2,4-dihydroxyphenyl)azo]-2-hydroxybenzenesulfonic acid} complexes and measurements of intracellular pH while exposing arabidopsis seedlings to acidic and Al(3+) stresses.
The lifetime of Al-lumogallion complexes fluorescence is pH-dependent. The primary sites for Al(3+) entry are the meristem and distal elongation zones, while Al(3+) uptake via the cortex and epidermis of the mature root zone is limited. The maximum rates of Al uptake into the cytoplasm (2-3 micromol m(-3) min(-1) for the meristematic root zone and 3-7 micromol m(-3) min(-1) for the mature zone) were observed after a 30-min exposure to 100 microM AlCl(3) (pH 4.2). Intracellular Al concentration increased to 0.4 microM Al within the first 3 h of exposure to 100 microM AlCl(3).
FLIM analysis of the fluorescence of Al-lumogallion complexes can be used to reliably quantify Al uptake in the cytoplasm of intact root cells at the initial stages of Al(3+) stress.
测量铝离子（Al(3+)）跨完整根细胞质膜的吸收速率对于理解植物铝毒的机制和时间进程至关重要。然而，目前尚无一种具有足够空间和时间分辨率来估算完整根细胞中Al(3+)吸收的可靠方法。
在本研究中，荧光寿命成像（FLIM）分析被用于在体内定量根细胞细胞质中的Al(3+)吸收。这是通过估计铝-荧光镓络合物{5-氯-3[(2,4-二羟基苯基)偶氮]-2-羟基苯磺酸}的荧光寿命并测量细胞内pH值来实现的，同时将拟南芥幼苗暴露于酸性和Al(3+)胁迫下。
铝-荧光镓络合物荧光的寿命取决于pH值。Al(3+)进入的主要部位是分生组织和远侧伸长区，而成熟根区通过皮层和表皮的Al(3+)吸收有限。在暴露于100 microM AlCl(3)（pH 4.2）30分钟后，观察到Al进入细胞质的最大速率（分生组织根区为2 - 3 micromol m(-3) min(-1)，成熟区为3 - 7 micromol m(-3) min(-1)）。在暴露于100 microM AlCl(3)的最初3小时内，细胞内Al浓度增加到0.4 microM Al。
对铝-荧光镓络合物荧光的FLIM分析可用于在Al(3+)胁迫初始阶段可靠地定量完整根细胞细胞质中的Al吸收。
