Plasma Membrane

Binding to Phospholipids

Biological membranes are composed of phospholipids that contain a phosphate group (67), and aluminum can bind to this negatively charged group. Using electron paramagnetic resonance spectroscopy, Vierstra and Haug (111) demonstrated that 100mM Al at pH 4 decreased fluidity in membrane lipids of a thermophilic microorganism (Thermoplasma acidophilum Darland, Brock, Samsonoff and Conti). Using physiologically significant concentrations of aluminum, Deleers et al. (112) showed that 25 µM Al increased rigidity of membrane vesicles as indicated by the increased temperature required to maintain a specific polarization value. In addition, aluminum at30 µM could induce phase separation of phosphatidylserine (PS; a negatively charged phospholipid) vesicles, as shown by leakage of a fluorescent compound (113).

Phosphatidylcholine (PC) is the most abundant phospholipid in plasma membranes of eukaryotes, and Akeson et al. (114) showed that in vitro, Al³⁺ has a 560-fold greater affinity for the surface of PC than Ca²⁺. Further, Jones and Kochian (102) found that lipids with net negatively charged head groups such as phosphatidyl inositol (PI) had a much greater affinity for aluminum than PC with its net neutral head group. Interestingly, Delhaize et al. (115) found that expression of a wheat cDNA (TaPSS1) encoding for phosphatidylserine synthase (PSS) increased in response to excess aluminum in roots. Overexpression of this cDNA conferred aluminum resistance in one strain of yeast (Saccharomyces cerevisiae) but not in another. In addition, a disruption mutant of the endogenous yeast CHO1 gene that encodes for PSS was sensitive to aluminum (115).

Aluminum reduced membrane permeability to water as shown by a plasmometric method on root disks of red oak (116). To remove the confounding effect of aluminum binding to cell walls, Lee et al. (117) used protoplasts of red beet (Beta vulgaris L.). Within 1 min of exposure to 0.5mM Al, volumetric expansion of red beet cells was reduced under hypotonic conditions, and Lee et al. (117) hypothesized that aluminum could bridge neighboring negatively charged sites on the plasma membrane, stabilizing the membrane.

Binding of Al³⁺ to the exterior of phospholipids reduces the surface negative charge of membranes. Kinraide et al. (27) proposed that accumulation of aluminum at the negatively charged cell surface plays a role in rhizotoxicity and that amelioration of aluminum toxicity by cations is due to reduced negativity of the cell-surface electrical potential by charge screening or cation binding. Kinraide et al. (27) found a good correlation between the reduction in relative root length of an aluminum- sensitive wheat cultivar with aluminum activity as calculated at the membrane surface, but not in the bulk external solution. Ahn et al. (118) measured the zeta potential (an estimate of surface potential) of plasma membrane vesicles from squash (Cucurbita pepo L.) roots and showed that aluminum exposure resulted in a less negative surface potential. Measuring uptake of radioisotopes by barley roots, Nichol et al. (119) showed that influx of cations (K⁺, NH₄⁺, and Ca²⁺) decreased whereas influx of anions (NO₃^-, HPO₄^2-) increased in the presence of aluminum. They speculated that binding of Al³⁺ to the exterior of a plasma membrane forms a positively charged layer that retards movement of cations to the membrane surface and increases movement of anions to the surface. In contrast, Silva et al. (120) demonstrated that Mg²⁺ was 100-fold more effective than Ca²⁺ in alleviating aluminum-induced inhibition of soybean taproot elongation. They (120) suggested that such an effect could not be explained by changes in membrane surface potential and proposed that the protective effects of Mg could be due to alleviation of aluminum binding to G-protein.

Interference with Proteins Involved in Transport

In addition to phospholipids, biological membranes are composed of proteins, many of which are involved in transport functions across the membrane (5,67). Aluminum is reported to interfere with the uptake of many nutrients, perhaps through interactions with cross-membrane transporters or channels.

H⁺-ATPases

Transmembrane electric potential (Vm) is the difference in electric potential between the external environment and the symplasm; typically, the interior of the cell is negatively charged with respect to the outside (67). The potential depends on transient fluxes of H⁺ through membrane-bound H⁺- ATPases, as well as fluxes of K⁺ and other cations through membrane transporters. Measurements of net H⁺ flux using either a microelectrode or vibrating probe demonstrated that net inward currents of H⁺ occurred between 0 to 3 mm from root tips of wheat (60,121). Exposure of roots of an aluminum-sensitive wheat cultivar to 10 µM Al for 1 to 3 h inhibited H⁺ influx; however, there was no obligatory association between inhibition of H⁺ influx and inhibition of root elongation (60). Ryan et al. (60) speculated that the H⁺ influx near the root apex could be due to cotransport of H⁺ with unloaded sugars and amino acids into the cytoplasm, or a membrane more permeable to H⁺.

Conducting an in vitro enzyme test, Jones and Kochian (102) found little effect of aluminum on H⁺-ATPase activity. Similarly, Tu and Brouillette (122) found no effect of aluminum on plasma membrane-bound ATPase activity in the presence of free ATP; however, exposure of Mg²⁺-ATP to 18 µM Al competitively inhibited hydrolysis of ATP. Based on immunolocalization, H⁺-ATPases in epidermal and cortical cells (2 to 3 mm from tip) of squash roots decreased after 3 h of exposure to 50 µM Al (118). Similarly, 2 days of exposure to≥75 µM Al decreased activity of plasma membrane- bound ATPases in 1-cm root tips of five wheat cultivars (123). Since H⁺-ATPases generate the proton motive force that drives secondary transporters and channels (5,67), a decrease in activity of this membrane-bound enzyme could result in an overall decrease in nutrient uptake.

Potassium Channels

Uptake of K⁺ by pea roots was depressed by aluminum (124). Similarly, exposure of mature root cells (≥10 mm from root tip) of an aluminum-sensitive wheat cultivar to 5µM Al inhibited K⁺ influx (121). In addition, Reid et al. (105) showed partial inhibition of Rb
(analog for K⁺) uptake by>50µM Al in giant algal (Chara corallina) cells, and they attributed this effect to partial blocking by aluminum of K⁺ channels. Using the patch-clamp technique on isolated plasma membranes or whole cells from an aluminum-tolerant corn cultivar, Pineros and Kochian (125) showed that instantaneous outward K⁺ channels were blocked by 12µM Al, whereas inward K⁺ channels were inhibited by 400µM Al.

A strong dysfunction in K⁺ fluxes between guard cells and epidermal cells was observed in beech (Betula spp.) seedlings exposed to excess aluminum for 2 months (83). Measuring currents of inside-out membrane patches from fava bean (Vicia faba L.) guard cells, Liu and Luan (41) demonstrated that the K⁺ inward rectifying channel (KIRC) was inhibited by 50 µM Al when exposed on the inward-facing side of the membrane. They (41) proposed that calcium channels conduct Al³⁺ across the plasma membrane because, verapamil, a Ca²⁺ channel blocker, prevented aluminum- induced inhibition of KIRC in the whole cell configuration. In addition, Liu and Luan (41) expressed the gene, KAT1, which encodes for a KIRC, in Xenopus oocytes, injected aluminum into the cytoplasm, and observed inhibition of the KAT1 current.

Calcium Channels

Uptake by roots and translocation of 45Ca to shoots was decreased in wheat by 100 µM Al (126). Similar results occurred with 4-week-old Norway spruce seedlings, in which uptake of ⁴⁵Ca was reduced by 77 to 92% by 100 to 800 µM Al (3). Net Ca²⁺ influx was highest between 0 and 2mm from the root apex of wheat, based on a calcium-selective vibrating microelectrode (127). Addition of 20 µM Al to roots of an aluminum-sensitive wheat cultivar resulted in a dramatic decrease in Ca²⁺ influx, and this effect was attributed to blockage by aluminum of a putative calcium channel (128). However, Ryan and Kochian (107) did not find an obligatory relationship between inhibition of calcium uptake and reduction of root growth in wheat. Similarly, in Chara corallina cells, aluminum inhibited calcium influx by less than 50% at 100 µM Al, and Reid et al. (105) thought it unlikely that such a small degree of inhibition would be sufficient to inhibit growth so rapidly.

Magnesium Transporters

Exposure of annual ryegrass (Lolium multiflorum Lam.) to 6.6 µM Al competitively inhibited net Mg²⁺ uptake (129). Interestingly, McDiarmid and Gardner (130) isolated two yeast genes, ALR1 and ALR2, that encode proteins homologous to bacterial Mg²⁺ and Co₂
transport systems. Overexpression of these genes conferred increased tolerance to Al³⁺, indicating that aluminum toxicity in yeast is related to reduced Mg²⁺ influx (130).

Nitrate Uptake

In white clover, 3 weeks of exposure to 50 µM Al inhibited nitrate uptake as measured by nitrogen content in plants (131). In all regions of soybean roots, ¹⁵NO₃^- influxes were reduced within 30 min of exposure to 80 µM Al (132). In corn, 30 min of exposure to 100 µM Al decreased NO₃^- uptake as measured by NO3-N depletion in solution, but aluminum-induced inhibition of root elongation was not attributed to inhibition of nitrate uptake (133). Aluminum treatment for 3 days followed by measurement of ¹⁵NO₃^- uptake in the final hour decreased ¹⁵NO₃^- uptake in soybean at≥44 µM Al but increased ¹⁵NO₃^- uptake at aluminum levels below 10 µM, probably as a result of Al³⁺ amelioration of H⁺ toxicity (24).

Iron Uptake

Iron acquisition in Strategy II plants (gramineous plants) involves secretion of mugineic acids (MA) and uptake of MA–Fe³⁺ complexes (67). Chang et al. (134) demonstrated that exposure to 100mM Al for 21 h depressed biosynthesis and secretion of 2′-deoxymugineic acid in wheat.

Water Channels

Aluminum is reported to reduce permeability of the plasma membrane to water, perhaps through reduced aquaporin (water channel) activity. Milla et al. (135) found that expression of a rye (Secale cereale L.) gene encoding for aquaporin (water channel) was decreased by aluminum.

Signal Transduction

Interference with Phosphoinositide Signal Transduction

Under in vitro conditions, aluminum interacted strongly with the phosphoinositide signal transduction element, the plasma-membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP₂) (136). In animals, cleavage of the plasma membrane lipid, PIP₂, by phospholipase C (PLC) releases inositol 1,4,5-triphosphate (IP₃) into the cytoplasm. Then, IP₃ could produce a signaling cascade by binding to a Ca²⁺ channel and releasing Ca²⁺ into the cytosol. In microsomal membranes of wheat roots, aluminum≥20 µM dramatically inhibited PLC activity (136). Under in vitro conditions, aluminum was shown to block the PLC-activated cleavage of PIP2 to IP3 (136).

Transduction of Aluminum Signal

Cell wall-associated kinases could serve as a connecting molecule between the cell wall and the cytoplasmic cytoskeleton. These kinases span the plasma membrane, with the extracellular portion covalently bound to pectin in the cell wall and the cytoplasmic portion containing kinase activity. Recently, expression of a cell wall associated kinase (WAK1) in arabidopsis was induced within 3 h of exposure to aluminum (89). Sivaguru et al. (89) hypothesized that WAK1 could be involved in the aluminum signal transduction pathway.