Aluminum inhibits the H(+)-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots

Abstract

Although aluminum (AL) toxicity has been widely studied in monocotyledonous crop plants, the mechanism of Al impact on economically important dicotyledonous plants is poorly understood. Here, we report the spatial pattern of Al-induced root growth inhibition, which is closely associated with inhibition of H(+)-ATPase activity coupled with decreased surface negativity of plasma membrane (PM) vesicles isolated from apical 5-mm root segments of squash (Cucurbita pepo L. cv Tetsukabuto) plants. High-sensitivity growth measurements indicated that the central elongation zone, located 2 to 4 mm from the tip, was preferentially inhibited where high Al accumulation was found. The highest positive shifts (depolarization) in zeta potential of the isolated PM vesicles from 0- to 5-mm regions of Al-treated roots were corresponded to pronounced inhibition of H(+)-ATPase activity. The depolarization of PM vesicles isolated from Al-treated roots in response to added Al in vitro was less than that of control roots, suggesting, particularly in the first 5-mm root apex, a tight Al binding to PM target sites or irreversible alteration of PM properties upon Al treatment to intact plants. In line with these data, immunolocalization of H(+)-ATPase revealed decreases in tissue-specific H(+)-ATPase in the epidermal and cortex cells (2--3 mm from tip) following Al treatments. Our report provides the first circumstantial evidence for a zone-specific depolarization of PM surface potential coupled with inhibition of H(+)-ATPase activity. These effects may indicate a direct Al interaction with H(+)-ATPase from the cytoplasmic side of the PM.

Figure 1

Effect of Al (50 μm) on the segmental elongation rate (sets A–A", B–B", C, and D) of squash roots. Sets A, A′, and A" were for control roots at 0, 3, and 6 h, respectively, marked with India ink at 2-mm intervals at 0 h. Sets B, B′, and B" represent roots treated with 50 μm Al for the same time points. Arrows denote the 2- and 4-mm positions after 3 h (A′ and B′) and subsequent alteration to their position after 6 h (A" and B"). Bars in A" and B" denote a distance of from the root tip 10 mm after 6 h. C, Elongation rate of 2-mm root regions in control and Al treatments after 3 and 6 h. D, Increment in total elongation of the apex (10 mm) during 0 to 3 and 3 to 6 h. Values in C and D are mean ± se of six replicates and representative of three independent experiments.

Figure 2

Effect of Al (50 μm) on the Al accumulation at 2-mm specific segments (A) and in the total root (10-mm) apex (B) of squash plants after 3- and 6-h Al treatments. Al was not detected in control root apices. The Al contents were determined either in three replicates, each comprising three 10-mm root apices for the total apex or in five replicates each comprising five 2-mm segments of the same distance from the tip (DFT) position. Values are mean ± se and representative of at least two independent experiments.

Figure 3

Effect of a range of Al concentrations (0, 20, 50, and 100 μm) and treatment durations (3, 6, 12, and 24 h) on the H⁺-ATPase activity (A) and zeta potential (B) of PM vesicles isolated from whole-root fractions of squash. The plants were grown in HS (1/5) adjusted to pH 4.5 for 5 d from germination. Al treatments were performed in the same solution without P and the plants were cultured in −P solution for at least 12 h prior to treatments. Values are mean ± se of three replicates and representative of two independent experiments. Al was absent in the electrophoresis medium.

Figure 4

Effect of Al (50 μm) in vitro on the zeta potential of PM vesicles isolated from whole-root fractions of squash. The plants were grown in HS (1/5) adjusted to pH 4.5 for 5 d from germination and cultured in −P solution for at least 12 h prior to isolation without Al. The control PM vesicles were subjected to a range of Al concentrations (A) and treatment duration (B) in vitro. In a parallel experiment, the PM vesicles isolated after 0, 3, 6, 12, and 24 h (C) 50 μm Al treatments in vivo (●) then they were subjected again to 50 μm Al in vitro for 10 min (▾). Values are mean ± se of at least two independent experiments.

Figure 5

Effect of Al (50 μm) treatment duration in vivo (0, 3, and 6 h) on the H⁺-ATPase activity (A) and zeta potential (B) of PM vesicles isolated from specific 5-mm root segment fractions of squash. The plants were grown in HS (one-fifth) adjusted to pH 4.5 for 5 d from germination. Al treatments were performed in the same solution without P, and the plants were cultured in −P solution for at least 12 h prior to treatments. The 5-mm DFT segments were made out of approximately 600 individual plants, and the isolated PM vesicles were pooled to increase the precision of measurement. Values are mean ± se of three replicates and representative of two independent experiments.

Figure 6

Visualization of Al-induced alteration to the PM property and Al binding capacity of the isolated PM vesicles from squash root apex (0–5 mm) using the Morin assay. A, Calibration (standard) showing a range of Al concentration and the Al-induced fluorescence of control PM vesicles (top); after 50 μm Al treatment in vivo (middle); further addition of 50 μm in vitro (10 min) to the same PM vesicles (bottom). B, Quantitative evaluation of the Morin fluorescence based on the pixel intensity corresponding to images presented in A. Results of standard (top) and after in vivo and in vitro treatments (bottom). For details, see “Materials and Methods.”

Figure 7

Analysis of the specificity of H⁺-ATPase antibody against the isolated PM proteins from squash root apex (10 mm). CB staining (A) and western blot (B). Localization of H⁺-ATPase in intact squash root apices (C–F"). Confocal images (for details, see text) of negative control of roots incubated without primary antibody (C). The plants were labeled with the antibody after 0- (D–D"), 3- (E–E"), and 6-h (F–F") Al (50 μm) treatments. The images are from the 2- to 3-mm DFT of epidermal cells (D–F), cortex (D′, E′, and F") and 7- to 8-mm DFT of epidermal cells (D", E", and F"). Note the decrease in the intensity of H⁺-ATPase upon time after Al treatments compared with control counterparts along the root apex. Bar = 40 μm.