Symplasm

Disruption of the Cytoskeleton

The cytoskeleton is a network of filamentous protein polymers that permeates the cytoplasm, providing structural stability and motility for macromolecules and organelles (67). In plants, there are two major families of proteins: actin and tubulin (67). Actin binds and hydrolyzes the nucleotide, ATP, during polymerization to form microfilaments. Proteins a- and �-tubulin bind and hydrolyze guanosine triphosphate (GTP) during polymerization to form microtubules.

Actin filaments are important in cytoplasmic streaming in giant algal cells. With an alga (Vaucheria longicaulis Hoppaugh), Alessa and Oliveira (137) demonstrated that cytoplasmic streaming of chloroplasts and mitochondria (mediated by microfilaments) decreased within 30 s of aluminum exposure and completely ceased within 3 min. Using suspension-cultured soybean cells, Grabski and Schindler (138) demonstrated that aluminum rapidly increased rigidity of the transvacuolar actin network, and they proposed that the cytoskeleton is the primary target of aluminum toxicity in plants. Grabski et al. (139) hypothesized that phosphorylated sites on myosin or other actin-binding proteins could bind aluminum, preventing access to phosphatases and resulting in a stabilized actin network. Alternatively, they hypothesized that a calcium-dependent phosphatase could be inhibited directly by aluminum. Interestingly, aluminum toxicity in wheat causes increased expression of a gene encoding for a fimbrinlike (actin-binding) protein involved in maintenance of cytoskeletal function (140). They speculated that the increased tension of cytoskeletal actin by aluminum (138) could involve cross-linking of actin filaments by fimbrins, leading to increased fimbrin gene expression.

Aluminum could disrupt microtubule assembly and disassembly through inhibition of GTP hydrolysis and reduced sensitivity to regulatory signals from Ca²⁺. When magnesium concentrations were below 1.0mM, MacDonald et al. (141) demonstrated in vitro that 4 x 10^-10 M Al could replace Mg²⁺ in polymerization of tubulin. Disappearance of microtubules was observed sometimes in cells of the EZ of aluminum-treated (3 h, 50 µM Al) wheat roots (61). In outer cortical cells of the DTZ of aluminum-sensitive corn roots, microtubules disappeared within 1 h of exposure to 90 µM Al (142). Treatment of corn roots with 50 µM Al for 3 h resulted in random or obliquely oriented microtubules in inner cortical cells compared to the transverse orientation of those from control roots (57). In addition, a 1 h pretreatment with aluminum prevented auxin-induced reorientation of microtubules in inner cortical cells of corn, and Blancafor et al. (57) proposed that aluminum induced greater stabilization of microtubules. Microfilaments seemed to be less sensitive to aluminum toxicity, with random arrays detectable in the inner cortical cells after 6 h (57).

Disturbance of Calcium Homeostasis

Siegel and Haug (143) proposed that the primary biochemical injury due to aluminum was caused by aluminum complexes with calmodulin (a calcium-dependent, regulatory protein). Similarly, Rengel (144) proposed that aluminum is the primary environmental signal, with Ca²⁺ as the secondary messenger that triggers aluminum-toxic events in plant cells. Using a fluorescent calcium-binding dye, Fura 2, Lindberg and Strid (145) showed that exposure of wheat root protoplasts to 50µM Al caused a transient and oscillating increase in cytoplasmic Ca²⁺ concentration. Similarly, using a cytosolic calcium indicator dye, Fluo-3, in intact wheat apical cells, Zhang and Rengel (146) showed an increase in cytoplasmic Ca²⁺ after 1 h treatment with 50µM Al. Using Fluo-3 and an indicator of membrane-bound Ca²⁺, chlorotetracycline (CTC), Nichol and Oliveira (147) found increased calcium concentration in the zone of elongation of an aluminum-sensitive barley cultivar. Since aluminum is known to block calcium channels that allow calcium to move into the cytoplasm, Nichol and Oliveira (147) suggested that Ca²⁺ was released from intracellular storage sites. Interestingly, aluminum-induced callose formation, a rapid marker of aluminum toxicity, is always preceded by elevated cytoplasmic Ca²⁺ (67).

In contrast, Jones et al. (148) used the fluorescent dye, Indo-1, and showed a rapid reduction in cytosolic Ca²⁺ in suspension cultures of tobacco (Nicotiana tabacum L.) cells. They (148) attributed this effect to blockage of calcium channels in the plasma membrane by aluminum.

Interaction with Phytohormones

The spatial separation between the most aluminum-sensitive site, the DTZ, and the root region that exhibits reduced cell elongation, the EZ, indicates that a signaling pathway is involved. Perhaps, the phytohormones, auxin (IAA) or cytokinin, are involved in the transduction of an aluminum-stress signal.

Auxin

Corn roots were observed to curve away from unilaterally applied aluminum (149). Similar results were found for snapbean roots that curved away from an agar surface containing aluminum (52). Hasenstein and Evans (150) showed that aluminum inhibited basipetal transport of indoleacetic acid (IAA), perhaps resulting in the tropic root response. Kollmeier et al. (151) confirmed this result, showing that exogenous 3H-IAA application to the meristematic zone of corn roots with aluminum application to the DTZ resulted in decreased basipetal transport of auxin to the EZ. They also showed that exogenous IAA application to the EZ partially ameliorated the aluminum-induced (Al applied to DTZ) inhibition of root elongation. Kollmeier et al. (151) hypothesized that aluminum inhibition of auxin transport mediated the aluminum signal between the DTZ and EZ. Sivaguru et al. (74) speculated that aluminum-induced callose in plasmodesmata could be a primary factor in aluminum inhibition of root growth through disturbance of auxin transport.

Cytokinin

Bean root elongation was inhibited after 360 min of exposure to 6.5 µM Al (152). Ethylene evolution as well as the level of zeatin (a cytokinin) from root tips increased after 5 min of aluminum exposure. Massot et al. (152) suggested a role for cytokinin and ethylene in transduction of aluminum- induced stress signal.

Oxidative Stress

Aluminum is redox inactive and is not able to initiate oxidation of lipids or proteins on its own. Yet, lipid peroxidation has been observed in barley roots after 3 h incubation with aluminum (100 µM AICI3, pH, 4.3) (153). Similarly, in pea roots, increase of lipid peroxidation and inhibition of root elongation occurred after 4 h of exposure to 10 µM aluminium (154). Sakihama and Yamasaki (153) proposal that aluminum stabilizes the oxidized form of phenolics (normally unstable), resulting in phenoxyl radicals that initiate lipid peroxidation. Alternatively, aluminum could increase formation of reactive oxygen species (ROS). Cell defense against ROS includes the enzymes, superoxide dismutase (SOD) and glutathione peroxidase (PX), which reduce ROS (153). If levels of these enzymes are not sufficient, then ROS could lead to oxidation of lipids, proteins, and DNA, and even cell death. In corn, 24 h of exposure to aluminum increased activities of SOD and PX, and increased protein oxidation in the aluminum-sensitive genotype (155).

Another possibility proposed by Ikegawa et al. (156) is aluminum-enhanced, Fe(II)-medicated peroxidation of lipids as a cause of cell death. Exposure of tobacco suspension cultures to aluminum alone for 24 h resulted in aluminum accumulation but no significant cell death (156). Addition of Fe(II) (a redox active metal) to cells with accumulated aluminum after 12 h resulted in enhanced lipid peroxidation and cell death. Lipid peroxidation does not appear to be the mechanism involved in reduction of root elongation (154). In pea roots, treatment with an antioxidant prevented aluminum-enhanced lipid peroxidation, reduced callose formation, but did not prevent aluminuminduced inhibition of root elongation (154).

Interestingly, three of four cDNA up-regulated by aluminum stress in Arabidopsis thaliana encoded genes were induced also by oxidative stress (157). Similarly, the vast majority of isolated cDNAs, whose expression increased in response to aluminum toxicity in sugarcane (Saccharum officinarum L.), showed greater expression in response to oxidative stress (158). These results indicate that oxidative stress is an important component of the plant’s response to aluminum toxicity. Overexpression of a tobacco gene encoding for glutathione S-transferase (parB) in Arabidopsis thaliana conferred a degree of aluminum resistance as well as resistance to oxidative stress induced by diamide, providing genetic evidence of a linkage between aluminum stress and oxidative stress in plants (159).

Binding to Internal Membranes in Chloroplasts

As discussed earlier, one long-term effect of aluminum toxicity is the suppression of photosynthetic activity (79,90). Photosynthetic ¹⁴CO₂ fixation of isolated spinach (Spinacia oleracea L.) chloroplasts was inhibited by 10µM Al at pH 7 (160). Hampp and Schnabel (160) attributed this effect to damage of the membrane system. Aluminum exposure of wheat for 14 days decreased the maximum photochemical yield F_v/F_m of photosystem II, (ratio of variable fluorescence over maximum fluorescence, as measured by a fluorometer) (161). Moustakas and Ouzounidou (161) attributed this effect to loss of Ca²⁺, Mg²⁺, and K⁺ from chloroplasts. Seventy days of aluminum exposure decreased F_v/F₀, or the ratio of variable fluorescence over initial fluorescence (162). Pereira et al. (162) speculated that this decrease was an indicator of aluminum-induced structural damage in the thylakoids. In the cyanobacterium, Anabaena cylindrica Lemm., aluminum was found to degrade thylakoid membranes (163).

Binding to Nuclei

Aluminum entered soybean root cells and was associated with nuclei only after 30 min of exposure to 1.45 µM Al (164). In corn root tips, high chromatin fragmentation and loss of plasma membrane integrity occurred after 48 h exposure to 36 µM Al (155). However, Al³⁺ binding to DNA is very weak and cannot compete with phosphate, ATP, or other organic ligands such as citrate (47,48). Martin (47) stated that the observed association of aluminum with nuclear chromatin must be due to its complexation to other ligands and not to DNA.