Uptake of aluminium into Arabidopsis root cells measured by fluorescent lifetime imaging

BACKGROUND AND AIMS: 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. METHODS: 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. KEY RESULTS: 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). CONCLUSIONS: 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.