46 Plant Roots Under Aluminum Stress: Toxicity and Tolerance

Hideaki Matsumoto
Okayama University, Okayama, Japan

I.

DISTRIBUTION OF ACID SOILS AND THEIR NATURE

II. A.

ALUMINUM TOXICITY IN ACID SOILS Occurrence and Chemistry of Al

Acid soils occupy 3.95 billion ha (30%) of the worlds ice-free land area (Baligar et al., 1998), comprising both the tropical and temperate belts. The distribution of acid soil in selected regions in the world is shown in Table 1. Depending on the degree of weathering and soil acidity, acid soil can be classied into 10 groups. Oxisols and Ultisols are the major acid soils in the tropical region and occupy 22% (846 m ha) and 18% (727 m ha), respectively, of the acid soil area in the world. Inceptisols including the sulfate soils of many tropical river deltas are very acidic owing to acid formed upon oxidation of suldes. The level of acidication of acid soil generally reects the degree of weathering and leaching it has experienced (Baligar et al., 1998). In addition to the natural factors that aect weathering, agricultural farming processes such as the excessive supply of inorganic fertilizers or removal of cations by harvest lower the pH. Furthermore, the acidity of soils is gradually increased owing to environmental pollution and acid rain. Acid soils are infertile because they lack the basic nutrients, such as Ca2 , Mg2 , and K . Acid soils are characterized by high content of toxic elements such as Al, Mn, and Fe or deciency of Ca2 , Mg2 , K , N, and P. Most acid soils have low cation exchange capacity, leading to loss of essential minerals and to poor crop production.

Exchangeable Al and Mn are the major toxic elements in most acid soils. In most Oxisols and Ultisols, Al occupies 494% of the cation exchange sites (Baligar et al., 1998). Al is the most abundant metal in the earths crust. Aluminum exists in the soil in insoluble aluminosilicates or oxides. It has complicated chemical form and biological function. At pH < 5, Al3 exists as the octahedral hexahydrate, AlH2 O3 , often abbreviated as 6 Al3 . As the solution becomes less acidic, AlH2 O3 6 undergoes successive deprotonations to yield AlOH2 and AlOH . In neutral solution AlOH3 2 precipitates as gibbsite which redissolves in basic solutions owing to formation of tetrahedral AlOH as 4 aluminate anion. Time-dependent formation of polynuclear species may also take place (Martin, 1986). Since Al toxicity diers with the chemical form of Al, many studies have been done regarding this interaction, especially between Al3 and mononuclear hydroxy-Al. Generally Al3 is more phytotoxic than AlOH2 or AlOH , but Alva et al. (1986) and 2 Kinraide and Parker (1987) reported that dicotyledonous plants may be more sensitive to AlOH2 and AlOH than to Al3 . The dierence in behavior of Al 2 species between monocots and dicots may be related to the fact that dicots have a much higher CEC in their

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822 Table 1 Extent of Acid Soils in the World and Selected Regionsa Region Distribution class Acid land area (10 ha) Acid land area (%)c
Von Uexkull and Mutert (1995). Excluding South and East Asia. c Ice-free land area of the globe. Source: Baligar et al. (1998).
b a

Matsumoto

Global
6

Central America 37 35

South America 917 14

Africa 659 22

Asiab 532 76

Australia/ New Zealand 239 30

North America 662 30

Europe 391 37

3,950 30

cell wall than monocots, but further studies are needed to elucidate the cause. In the soil solution Al3 reacts not only with OH but also with phosphate, F , SO2 , silicate, and a large 4 number of organic ligands. Under specic conditions of OH/Al ratio, total Al, and stirring rate, AlO4 Al12 OH24 H2 O7 (Al13 polymer), which is 12 highly toxic, can be formed (Parker et al., 1989). Nevertheless, Al13 was not observed in soil solutions (Funakawa et al., 1993). Rhizotoxicity of the aluminate ion, AlOH , which is formed at an alkaline 4 pH was tested with wheat and red clover. Root elongation was inhibited to < 4% by 25 M AlOH at pH 8 4 but not at pH 8.9, where elongation was unaected. Thus, AlOH is nontoxic, and the inhibition at a 4 lower pH is attributable to the Al13 formed (Kinraide, 1990).

B.

Inhibition of Root Elongation by Al

Indeed, only the apical 23 mm of maize and pea roots need to be exposed to Al for the inhibition of root elongation to take place (Delhaize and Ryan, 1995; Matsumoto et al., 1996). In near-isogenic wheat (Triticum aestivum) lines diering in Al tolerance, root apices of Al-sensitive genotypes were stained with hematoxylin after a short exposure to Al (10 min h). Apices of Al-tolerant seedlings showed less intensive staining (Delhaize et al., 1993a). This indicates that inhibition of root elongation by Al varies among plant species or cultivars. Similar results were obtained with Al-tolerant wheat (Atlas 66) and Al-sensitive wheat (Scout 66) exposed to Al for 1 day (Sasaki et al., 1997b) (Fig. 2). These results suggest that (1) the dierences in Al accumulation in the root apex are related to dierences in Al sensitivity, (2) inhibition of root growth is related to the Al content in the root apex, and (3) tolerant cultivars possess a mechan ism that excludes Al from root apices (Rincon and Gonzales, 1992; Samuels et al., 1997). 1. Morphological Changes of Intact Roots and Root Cells Under Al Stress

Inhibition of root elongation is the rst visible symptom of Al stress. In most plant species, root elongation is markedly inhibited by Al at the mol level in a simple solution containing Ca2 alone. Inhibition of root elongation of Al-sensitive maize occurred within 30 min of Al treatment (Llungany et al., 1995). However, inhibition of root elongation is reduced in the presence of other ions, because the interaction with other coexisting ions reduces the toxicity of Al3 . Changes in the electric charge of the root surface by other ions, especially cations, aect the accessibility of Al3 . Root elongation in Al-sensitive wheat cultivar, Scout 66, was apparently inhibited by a 3-h treatment with 5 M Al, but that of Atlas 66 was inhibited to the same degree only by a 10-fold higher concentration of Al (Fig. 1). The root apex (root cap, meristem, and elongation zone) accumulated more Al and plays a major role in the Al perception mechanism.

Accumulation of coating materials on the epidermis of the apex and around the cap is commonly observed upon Al stress. Root meristem cells of canola (Brassica napus var. napus L. cv. barassa) plants, grown under control conditions, responded dierently from the much larger control cap cells (Clune and Copeland, 1999). Under mild Al stress (20 M, 24 h), these cells expanded and increased in number, but under more severe treatment (80 M Al, 24 h) they diminished in size and number. Furthermore, the distinct boundary between cells in the root cap meristem and the elongation zone was no longer apparent, and the outer layer of cells in the root cap appeared to be only loosely attached. After only 4 h in 80 M Al, many ultrastructural changes were evident in periph-

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Figure 1 Time course for wheat root elongation of Atlas 66 and Scout 66. Seedlings were in the presence and absence of 5 (Scout 66) or 50 (Atlas 66) M Al. Data are means (SE) of results from 10 roots. (From Sasaki, 1996.)

Figure 2 Hematoxylin-stained wheat roots of Atlas 66 and Scout 66. Seedlings were grown in the presence and absence of Al for 48 h. (From Sasaki et al., 1997b.)

eral root cells, including the appearance of numerous small vacuoles that occupied most of the cytoplasm. After 24 h, disorganization of the cellular contents of the peripheral root cap cells became obvious, and the plasma membrane was clearly separated from the cell wall. The cytoplasm was markedly reduced in volume and extensively vacuolated. Similarly, Al-induced vacuolations have been observed in several plant species (Ikeda and Tadano, 1993; Marienfeld et al., 1995). In Lemna minor, the vacuolation and numerous myelinlike whorls of membrane increased in the apical meristem of the root (Severi, 1991). Moreover, the vesicles produced by the Golgi apparatus were larger. However, whether vacuolation is related to the storage of Al remains to be determined. Al has a localized eect on auxin transport and unilateral application of Al inhibited root curvature. Inhibition of cell elongation in the elongation zone is the major outcome of the inhibition of root elongation. Shortening of the root elongation zone by Al is accompanied by an increase in the diameter and a decrease of the length of the cells in the second and third layers of the cortex of the elongation zone of Atlas 66 plants (Matsumoto, 2000; Sasaki et al., 1996) (Figs. 3, 4). The ratio of length to diameter of the cells in the control root was three to four times larger than that in the Al-treated roots, and cells in the second and third layers of the cortex were swollen laterally. The Al-induced inhibition of longitudinal cell expansion and cell swelling in the elongation zone might be related to the disorder of the cytoskeletal network. The orientation of the microtubules (MTs) is closely related to cell expansion. Longitudinally elongating cells have transversely oriented MTs. MTdisrupting agents promote lateral expansion but inhibit longitudinal expansion. Cortical MTs are known to be involved in the orientation of cellulose microbrils. Indeed, the disappearance of the cortical MTs in elongating cells of wheat roots that was observed under Al stress (Sasaki et al., 1997a) might be responsible for these changes in cell growth. Moreover, the timedependent eect of Al on MTs stability was correlated with that on the reduction of root growth. The eect of Al on the behavior of structural proteins has been investigated intensively in recent years. The actin network plays an important role in the plant cell. Al induced a signicant increase in the tension within the transvacuolar actin network in soybean cells (Grabski and Schindler, 1995). Al resulted in a reorganization of MTs in the inner cortex, but not outer cortex, and in the epidermis of the elongation zone of Zea mays (Blancaor et al., 1998). They also

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Figure 3 Photomicrographs of longitudinal sections of wheat roots of Atlas 66. Roots were treated with or without 20 M Al for 24 h. Bar indicates 0.2 mm. (From Sasaki et al., 1996.)

Figure 4 Effects of Al on the lengths and diameters of wheat root cells in the second layer from surface in Atlas 66. Roots were treated with or without 20 M Al for 24 h (a) or 48 h (b). Data are means (SE) of results from ve or six samples. (From Sasaki et al., 1996.)

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found that the auxin-induced reorientation and coldinduced depolymerization of MTs in the outer cortex were blocked by Al, suggesting that Al increased the stability of MTs in these cells. The changes of behavior of MTs against Al stress may depend on the growth phase of the cells (Sivaguru et al., 1999). This stability eect of Al in the outer cortex coincided with growth inhibition. Aluminate [AlOH ] at pH 10.0 induced 4 the bending of roots of salt-tolerant grass (Thinopyrum bessarabium A. Love). The roots under aluminate treatment displayed a number of morphological and structural malformations (Eleftheriou et al., 1993). The root cap decreased in size, and both the calyptrogen and the meristemic region occupied a smaller area. Amyloplasts in the root cap cells were hardly distinguishable and showed less evidence of sedimentation. The swollen cells of wheat roots were characterized by the drastic accumulation of lignin on their cell walls under Al stress (Sasaki et al., 1996). The decrease of cell viability of the elongation zone of wheat roots were also observed after 3 h of exposure to Al which coincided with the time required for the inhibition of the root elongation as well as for lignin deposition (Sasaki et al., 1997b). It is still unknown whether lignin deposition is involved in the mechanism of Al toxicity. The morphological changes in roots were characterized by the cracks on the root surface (Sasaki, 1996) (Fig. 5). Cracking might be caused by the outward pressure of the cells in the second and third layers of the cortex of wheat roots. The distal part of the elongation zone of maize roots, where cells are undergoing a preparatory phase for rapid elongation, is the primary target of Al inuence (Sivaguru and Horst, 1998). To understand the primary event of Al toxicity, we must know how the cells in the specic zones, i.e., elongation and/or transition zone of the root, accumulate Al and how their elongation is inhibited by ultrastructural alterations. Why do such events cause death of the cell? In other words, are the matured root cells resistant to Al toxicity after their elongation has ended? 2. Inhibition of Cell Division

Figure 5 Photographs with scanning electron microscope of wheat root surface of Atlas 66. Roots were treated without and with 50 M Al for 4 h in the presence of 0.1 mM CaCl2 . (From Sasaki, 1996.)

Cell division in root meristems of several plants is inhibited by Al (Clarkson, 1965; Morimura et al., 1978). However, cell division accounts for only 12% of the overall root elongation. Furthermore, cell cycle in plants takes about 1 day. However, the primal phenomenon of Al toxicity is the inhibition of root elongation that occurs within hour(s) of Al treatment.

Thus, attention has been largely paid to the inhibition of root cell elongation as the primary site of Al toxicity. On the other hand, the lethal consequence of Al toxicity might be inhibition of cell division (Matsumoto, 2000). Large amounts of Al accumulate in the developing lateral roots of pea roots where cells are actively dividing (Matsumoto et al., 1976a). Al was detected in nuclei of root hair cells by staining and by chemical determination of Al in puried nuclei prepared from Al-treated pea roots. Furthermore, Al accumulation in the nuclei of soybean root tips was detected with Al-sensitive stain lumogallion and confocal laser scanning microscopy (Silva et al., 2000). The nuclei isolated from pea roots treated with 1 mM AlCl3 at pH 5.5 for 1 day were fractionated; 73% of the total Al in nuclei was recovered in the chromatin fraction, and 94% of Al in chromatin was recovered in DNA (Matsumoto et al., 1977b). Expression of genetic information of DNA is regulated by structural changes of DNA and chromatin. Unwinding of double strands of DNA is a prerequisite

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for expression of genetic information, but separation of double strands of DNA was interrupted by Al (Fig. 6) (Matsumoto, 1991). Furthermore, the structural change of chromatin in pea roots treated with Al in vivo implied that Al induced the condensation and/or aggregation of chromatin (Matsumoto, 1988). These results suggested that the template activity of DNA and/or chromatin of pea roots for RNA synthesis is repressed by Al. This is caused by Al-induced structural alteration of DNA and chromatin through the association of the negative charge of phosphates of DNA and positively charged Al3 (Morimura and Matsumoto, 1978). Plant cells require dynamic cytoskeleton-based networks for various cell activities (e.g., dierentiation and division). With suspension of tobacco cells, Sivaguru et al. (1999) found that the

actively dividing log-phase cells were characterized with faint and larger phragmoplasts and unusually enlarged daughter nuclei after 6 h of Al treatment. After a 24-h treatment, no phragmoplasts and spindle MTs (SMT) from cells having metaphase plate chromosomes were observed. The disintegration of SMT and disorganization of phragmoplasts caused by Al might block cell division directly at the metaphase. As mentioned before, inhibition of cell division will be the cause for the complete inhibition of root elongation by Al and subsequent death. Therefore, elucidation of the detailed mechanism of cell division inhibition by Al will be required in order to understand the mechanism of Al toxicity (Matsumoto, 2000). C. 1. Site of Al Toxicity Apoplast

Figure 6 Proposed mechanism of inhibition of the transcription of RNA by Al. (A) Normal transcription of RNA on a sense strand of DNA template in the absence of Al. A short section of the double strands must open and, thus, only the sense strand can act as the template. (B) Transcription is inhibited in the presence of Al. Sections of the double strands (indicated by two arrows) are captured by Al polymers with various structures shown as Al Al Al, through the strong electrostatic interaction between phosphate groups with a negative charge and the large positive charge of the Al polymer. Thus, separation of the strands is blocked and limited synthesis of RNA results. In addition, the Al polymer with its large positive charge on one helix, shown as nAlm where n and m are highly variable and depend on coexisting factors, can associate with the other helix and cause aggregation of chromatin bers. (From Matsumoto, 1991.)

Although there is disagreement with regard to the site of Al toxicity, namely, symplastic or apoplastic, many investigators have stated that 3090% of the absorbed Al is localized in the apoplast (cf. Tice et al., 1992; Rengel, 1996). Two possible interpretations has been given to the role of CEC in connection with Al accumulation in the apoplast. On the one hand, high CEC will be associated with large quantities of Al accumulated in the apoplast. On the other hand, high CEC may prevent Al from entering the symplast, where it exerts its lethal eect. However, a clear relationship between root CEC and Al sensitivity and/or tolerance was not found across a wide range of plant genotypes (Grauer, 1992). As to the binding site of Al in the apoplast, pectin carboxyl was suggested as a plausible candidate although almost no evidence has been found to show the binding of Al to pectin in vivo (Matsumoto et al., 1977a; Horst, 1995). Although Al is bound by the negative charge of pectin, the binding capacity of pectin varies with the plant species, and the pectin content is extremely dierent between monocots and dicots. Even in the same species, the pectin content of the roots diers with the position on the root or with the chemical modication of pectin, such as methylation or demethylation changes with the physiological activity of the cell. A Ca-pectate membrane was used as a model system. There was a rapid reaction between Al and Ca pectate, but there was no dierence in Al remaining in solution even after 16 min. Only a slight decrease was observed after 24 h. The solution containing 29 M Al and 1 mM Ca reduced the ow through the Ca pectate membrane by > 80% compared to the

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solution containing 1 mM Ca only. These results suggest that an important eect of toxic Al is a reduction in water movement into roots (Blamey et al., 1993). Interactions of Al with other cell-wall components, such as enzymes, extensin and xyloglucan may aect the functional integrity of cell walls. In the roots of cotton seedlings, Al impaired the sucrose utilization for cell wall formation (Huck, 1972). Al also induced the production of cell-wall components of squash seedlings, especially of hemicellulose, (Van et al., 1994). Al stress increased the level of covalently bound cell wall proteins in pea roots (Pisum sativum cv. Alaska). In vitro and in vivo Al binding experiments have suggested that extensin has the highest capacity to bind Al among cell wall proteins (Kenjebaeva et al., 2001). 2. Plasma Membrane

The plasma membrane is one of the rst targets of Al (Haug, 1984; Matsumoto, 2000). Al binds readily to the plasma membrane because of its high content of phosphate such as phospholipids and the negative charge of the membrane surface. Al3 has a 560-fold higher anity for the phosphatidyl choline surface than Ca2 (Akeson et al., 1989). Structural and functional changes of the plasma membranes are induced by Al binding, although the evidence of Al binding to the plasma membrane in vivo is limited (Matsumoto et al., 1992). Al-induced changes in membrane behavior of intact root cortex cells of Quercus rubra root showed that Al altered the activation energy required to transport water (32%), urea (9%), and monoethyl urea (7%) across cell membranes as measured by the plasmometric method (Zhao et al., 1987). Al increased the lipid partiality of the plasma membrane at > 9 C but decreased it at temperatures < 7 C. These changes in membrane behavior are explainable if Al reduces membrane lipid uidity and kink frequency and increases packing density and the occurrence of straight lipid chains (Chen et al., 1991). It can be concluded that Al3 (1) increased membrane permeability to the nonelectrolytes, (2) decreased the membrane partiality for lipid permeators, and (3) decreased membrane permeability to water caused by increased activation energy. It is thus implied that Al3 alters the architecture of membrane lipids (Vierstra and Haug, 1978). Furthermore, Al3 inhibited the inux of cations and enhanced the inux of anions. This was caused by the Al-induced formation of positively charged layer at membrane surface inuencing ion movement to the binding sites of the transport proteins. A posi-

tively charged layer would retard the movement of cations to the plasma membrane. This is explained by the charge of the membrane surface potential, Zeta potential, through the binding of Al. It was also argued from the relationship between Al tolerance and surface negativity of plasma membranes (Wagatsuma et al., 1995). Al reduced the negative charge associated with phospholipids, i.e., depolarization of Zeta potential, and proteins by binding to these charged groups or shielding the surface charges. Alteration of K eux and H inux by Al also aect the Zeta potential as well as the potential dierence (PD) across plasma membrane (Sasaki et al., 1994a). A more direct eect of Al3 is its binding to transport proteins and impairs their function. For instance, Al3 blocks inward-rectifying K channels in root hairs of wheat (Triticum aestivum) and VDAC, channel-forming protein located in the outer mitochondrial membranes (Dill et al., 1987). Recently, a special interaction was found between depolarization of Zeta potential and decrease of H -ATPase of plasma membrane of squash roots treated with Al. The interaction was typically observed at root tips, 05 mm portion of the root (Ahn et al., 2001). However, the varietal sensitivity to Al3 is not based on the dierence in cell surface electrical potential (Kinraide et al., 1992), and inhibition of root growth by Al is not caused by the reduction in current or H+ inux at the root apex (Ryan et al., 1992). On the other hand, a dierent membrane potential depolarization of root cap cells preceded Al tolerance in snapbean (Phaseolus vulgaris L.) (Olivetti et al., 1995). The Al-tolerant cultivar Dade depolarized rapidly upon exposure to Al, but the Alsensitive cultivar Romano was only slightly depolarized. This might be related to the fact that Al reduces the K eux channel conductance in the tolerant Dade root cap cells, but does not aect it in the sensitive cultivar Romano. Further research is needed to understand the interrelationship between Al toxicity and/or tolerance and electrophysiology. One of the biochemical changes of the plasma membrane is the Al-dependent lipid peroxidation in the root tip of soybean (Glycine max). A close relationship existed between lipid peroxidation and inhibition of root elongation induced by Al and/or Fe toxicity and/or Ca deciency (Cakmak and Horst, 1991). Enhanced lipid peroxidation by oxygen free radicals can be a consequence of primary eects of Al on membrane structure. The tolerance mechanism against Al toxicity in terms of lipid peroxidation was proposed for tobacco suspension cells (Yamamoto et al., 1998). Lipid composition can be a determinant of the varietal

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dierence for Al sensitivity. The largest change of the lipid composition of the microsomal fraction in the root is sterylglucosides upon Al treatment between Al-resistant (PT741) and Al-sensitive (Katepwa) cultivar of Triticum aestivum (Zhang et al., 1997). 3. Calcium Ca2 is an essential element for root growth and much work was devoted to Al toxicity in terms of Ca2 . Alinduced changes in cell physiology, occurring in the cytoplasm and at the plasma membrane, might be caused by the disorder of Ca2 homeostasis. Al inhibited Ca2 inux into the root apex of Al-sensitive wheat cultivar Scout 66, but had a much smaller eect on the Al-tolerant cultivar Atlas 66. In the absence of Al3 both cultivars maintained similar rates of net Ca2 uptake (Huang et al., 1992). A similar inhibitory eect of Al was observed not only on Ca2 uptake but also on its translocation from the apical region. Furthermore, Ca2 transport activity due to the membrane potential was inhibited by externally added Al to isolated plasma membrane vesicles. But there was no dierence in the inhibition eect between tolerant and sensitive wheat cultivars (Jacob and Northcote, 1985; Sasaki et al., 1994b; Huang et al., 1996). Such results imply that Al blocks the Ca2 channels on the plasma membrane. The observed dierence in Al uptake between intact roots and membrane vesicles might be due to dierent chelation of Al with the dierent eux level of organic acids from the roots triggered by Al. The antagonistic eect between Ca2 and Al toxicity is well known. The question is how Al inhibits the physiological functions of Ca2 . Replacement of functional Ca2 from the membranes and from the cell walls in the root might be one reason although the evidence for such displacement in vivo is limited (Kinraide et al., 1992). Competition between Ca2 and Al3 is thought to either weaken the cell walls, by reducing the number of Ca crosslinks or by replacing Ca2 crosslinks with stronger Al3 ones, making them too rigid for growth. Displacement of apoplasmic Ca2 by Al is in part due to the competition for ligands, such as pectin carboxyl radical. It can also be caused by reduction of the negative potential dierence, on the surface of plasma membrane. In isolated cell walls equilibrated with 50 M Ca2 at pH 4.4, 100 M Al displaced > 80% of the bound Ca2 with half-time of 25 min (Reid et al., 1995). Ca2 was not displaced by Al in wheat (Ryan et al., 1997a), but the signal initiated or disrupted by excess Al inhibited the growth in the meris-

tem of Allium cepa (Schoeld et al., 1998). Al also impaired Ca-mediated plant defense responses to low pH conditions (Plieth et al., 1999). The disruption of Ca2 metabolism by Al was debated. In wheat seedlings, root growth can be severely inhibited by Al3 concentrations that do not aect Ca2 uptake, while the addition of other ameliorating cationse.g., 30 mM Na , 3 mM Mg2 , or 50 M Tris (ethylenediamine) cobalt (III), depressed Ca2 uptake (Ryan et al., 1994). Phytotoxic action of Al in the root hairs of Arabidopsis is not due to blockage of Ca2 -permeable channels required for Ca2 inux into the cytoplasm (Jones et al., 1998). On the other hand, Zhang and Rengel (1999) found that the increase of cytoplasmic free Ca2 ions ([Ca2 c ) in root apical cells was higher in Al-sensitive wheat (ES8) than in Al-tolerant wheat (ET8). The Al-related increase in [Ca2 c was correlated with inhibition of root growth and was reversible upon removing the ambient AlCl3 . At present, we are far from elucidating the mechanism of Al/Ca interaction. The question whether Al increases or decreases cytoplasmic Ca2 and how these alterations in Ca2 level are related to Al toxicity syndrome in plant roots should be solved. Technical advancement for the detection of free Ca2 in the cytosol of plant cells is also needed. 4. Callose

Callose (-1,3-D-glucane) synthesis is very sensitive to Al stress and has been considered to be a reliable marker for Al toxicity. Callose formation was induced by Al as low as 5 M and as early as 10 min after Al addition to a suspension of cultured cells of soybean (Horst, 1995). Callose formation is most intensive in the root apex and conned only to the outer cortical cell layers of soybean seedlings (Wissemeier et al., 1992). The callose concentration in the 10- to 30-mm root tip of cowpea was inversely related to the root elongation rate when the roots were subjected to Al concentration > 10 M. Furthermore, a negative correlation was found between the relative callose concentration and relative root elongation rates of three soybean genotypes diering in Al sensitivity. Therefore, Al-induced callose formation in root tips might be used as a selection criterion for Al sensitivity. Callose is synthesized by -1,3-glucane synthetase on the plasma membrane and activated by Ca2 . It is still unknown where the Ca2 required for callose synthesis comes from. Ca2 is released from the exchange sites of the cell wall and plasma membranes. Thus, an increase

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in the free Ca2 concentration and triggering callose synthesis cannot be excluded. Ca2 may not be the only signal for callose formation, and alteration in the plasma membrane architecture might also be important for callose synthesis (Jacob and Northcote, 1985). As callose is released into the apoplast after its synthesis on the plasma membrane, cell walls of root cells of Al-treated plants most likely contain callose depositions. What is the inhibitory function of callose under Al stress? Callose can be considered as a sealing system in plants. Callose is localized in the wall around the plasmodesmata, which appear to be structurally subdivided. Thus, the constricting force upon callose synthesis would be transmitted to the plasmodesmal core (Turner et al., 1994). This will inhibit the transport of cellular compounds through plasmodesmata under Al stress. A large increase of callose accumulation at the plasma membrane under Al stress was found in Al-sensitive wheat roots. Such an increase inhibited the cell-to-cell tracking of molecules through the plasmodesmata, resulting in the inhibition of root elongation. Furthermore, these events are markedly repressed in the presence of 2-deoxyD-glucose, which is a callose synthase inhibitor (Sivaguru et al., 2000). However, in an Al-tolerant Arabidopsis mutant, no direct relationship between Al uptake and callose formation was established (Larsen et al., 1996). D. Signal Transduction and Al Signal

tion of Al signal in barley roots is related to an increase of ABA. ABA induces both the ATP- and PPi-dependent H pump activity of the tonoplast (Matsumoto et al., 1996). Contrary to ABA, transport of exogenously applied [3H]indole-3-acetic acid to the meristemic zone was signicantly inhibited by Al in maize roots. The signaling pathway in the root apex mediating the Al signal may be responsible for the genotypic dierence in Al resistance (Kollmeier et al., 2000). The biochemical mechanism in terms of the transduction of Al signal is poorly understood. Protein phosphorylation may be involved because of the strong association of Al with phosphate.

E.

General Metabolism Affected by Al

Plants respond to Al stress quickly. Inhibition of root elongation is observed within less than an hour, and homeostasis of cytoplasmic free Ca2 is broken instantly after Al addition. Special attention has been paid to phosphoinositide-associated signal transduction. AlCl3 and Al-citrate inhibited phospholipase C (PLC) of the microsomal membrane in a dose-dependent manner in wheat roots. I50 was observed at 1520 M Al (Jones and Kochian, 1995). Binding of Al to microsomes and liposomes was found to be lipid dependent, with the signal transduction element PIP2 having the highest anity for Al with an Al:lipid stoichiometry of 1:1. These results suggest an Al eect on the signal transduction pathway that is associated with the mechanism of Al toxicity. How is the Al signal recognized by receptor and how is it transported into the cytoplasm at the root apices? Bennet and Breen (1991) proposed that the Al signal is perceived in the root cap of Zea mays. Matsumoto et al. (1996) speculated that the transduc-

The eect of Al on excised root apices and isolated mitochondria of wheat was investigated. O2 uptake by excised roots was reduced by 23% and 35% after 12- and 24-h treatment with 75 M Al. Mitochondria isolated from Al-treated roots had reduced oxidative capacity with supply of electrons to complexes I and II. It was found that initially Al aected electron ow through complexes I and II, and after longer exposure interacted with other sites in the mitochondria (de Lima and Copeland, 1994). Al tolerance of Phaseolus vulgaris (cv. Dade) was an inducible trait. In this cultivar, the resumption of root elongation during recovery from Al treatment was accompanied by increased rates of respiration. Respiration rates slowly declined over the 72-h treatment of Al-sensitive Romano. When partitioned into growth and maintenance expenditures, a larger proportion of root respiration of Al-treated Dade plants was allocated to maintenance processes, potentially reecting diversion of energy to metabolic pathways that oset the adverse eects of Al toxicity (Cumming et al., 1992). Carbon metabolism is also aected by Al. Al stress increased alcohol dehydrogenase activity in wheat (Triticum aestivum cv. Vulcan) roots. Sucrose synthase and lactate dehydrogenase were also increased in Altreated roots, suggesting that the early eect of Al on wheat roots may be a shift from aerobic to anaerobic metabolism. The rst two enzymes in the pentose phosphate pathway (G-6-PDH and 6-PGDH) decreased in Al-sensitive wheat cv. Grana. However, these two enzymes rst increased, but then decreased in Al-toler ant rye (Slaski et al., 1996). These results suggest that the mechanism of Al resistance involves the regulation of the pentose pathway.

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III. A.

ALUMINUM TOLERANCE MECHANISM Al Tolerance on Genetic Basis

based on identical Al tolerance in heterozygotes and Alt 1 homozygotes. This gene controls the excretion of malate upon Al stress. B. Genetic Basis of Al Tolerance

The bimodal distribution of phenotypes corresponding to 3:1 segregation ratio for Al tolerance/sensitivity in populations derived from crosses between tolerant and sensitive cultivars revealed the presence of single major genes for Al tolerance (Gain, 1998). In addition to major genes conferring major dierences in Al tolerance, there is also some evidence that minor genes or modier genes may play a role in modulating the eect of major Al tolerance genes. Other studies have suggested that genetic factors located on the long arm of chromosome 2D prevent accumulation of Al in root apical meristems of the BH1146 euploid wheat (Aniol, 1995). However, other genetic factors are also located on these chromosome segments that control Al detoxication in the root tips of Al-tolerant lines. The D genome of wheat may determine the tolerance to acid soil and consequently contribute to the increased adaptation of hexaploid wheats during their evolution. Atlas 66 is a well-known Al-tolerant cultivar of wheat. However, not all the genes for tolerance to Al in Atlas 66 are located on the D genome chromosome (Berzonsky, 1992). Furthermore, Al tolerance in wheat is a dominant trait and the majority of observed variability could be explained by hypotheses of two or three gene pairs, each gene aecting the same character with complete dominance of each gene pair. Al tolerance in the ditelosomic line of Chinese Spring wheat cultivar revealed that genes controlling this character are located on the short arm of chromosome 5A and the long arm of chromosomes 2D and 4D (Delhaize et al., 1993a). Conservation of the Al tolerance gene by various species was investigated. RFLP markers for a major wheat Al tolerance gene AltBH were found on the long arm of chromosome 4D, while in rye, the Al tolerance gene was located on chromosome 4, which harbors chromosome segments homologous to regions of wheat chromosome 4D. In barley, the Al tolerance gene Alp is almost certainly orthologous to the wheat AltBH gene due to the fact that the relative positions of Alp and AltHB with respect to a common set of molecular markers are virtually identical in both genomes (Berzonsky, 1992). In spite of eorts made so far, the genetics of Al tolerance is little known for any single species. As to the physiological functions of Al tolerance genes, Delhaize et al. (1993b) found that Al tolerance, controlled by the Alt gene in wheat, appeared to be dominant across a range of Al concentrations

Application of lime to acid soils to increase the soil pH is one strategy for alleviating Al toxicity. However, this technique is problematic from the economical and environmental points of view. Another strategy is to use Al-tolerant crops. Breeding may depend on classical techniques and/or transgenic plants to which Al tolerance genes are being introduced. Snowden et al. (1995) isolated several cDNA (wali 17) whose transcript accumulates in wheat under Al treatment. A cDNA library constructed from the mRNA of Al-treated roots of Al-sensitive wheat (cv. Victory). It was screened with a degenerate oligonucleotide probe derived from a partial amino acid sequence of the Al-induced protein TAl-18. Out of seven clones that initially hybridized with the probe, one encoding a novel 1,3--glucanase mRNA was upregulated in Altreated roots, with highest expression after 12 h. A second cDNA showed similarity to genes encoding cytoskeletal mbinlike protein. Unfortunately, a transgenic protein enriched with those genes was not constructed (Cruz-Ortega et al., 1997). Al ions bind to phospholipids, and the plasma membrane is a primary barrier to the entry of Al into the cells (Matsumoto, 1988; Kochian, 1995). Thus, a change in lipid composition of the plasma membrane could improve the resistance of the cell by excluding Al (Delhaize et al., 1999). Cloned wheat cDNA (TaPSS1) that codes for phosphatidyl serine synthase (PSS) was tested. Overexpression of PSS increased Al resistance in yeast. However, a high level of TaPSSI expression in Arabidopsis and tobacco led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS (Delhaize et al., 1999). An Arabidopsis blue-copper-binding protein gene, a tobacco glutathione S-transferase gene, a tobacco peroxidase gene, and a tobacco GDP dissociation inhibitor gene conferred a certain degree of resistance to Al (Ezaki et al., 2000). These lines also showed increased resistance to oxidative stress, suggesting a link between Al stress and oxidative stress in plants. One successful approach was the generation of transgenic tobacco and papaya with the citrate synthase (CS) gene from Pseudomonas aeruginosa with the 35S promoter of cauliower mosaic virus introduced using a Ti plasmid derived from a transfor-

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mation system. The idea was that organic acids serve as chelating agents and may prevent Al toxicity (Delhaize and Ryan, 1995; Fuente et al., 1997). Hematoxylin staining was employed to examine whether the increased Al tolerance of the CSb lines is due to the inhibition of Al uptake by the root tip. Following exposure to Al CSb lines showed a considerably lighter staining than the control. Apparently, the expression of a citrate synthase in the cytoplasm increases the concentration of citrate, which led to a higher rate of its eux; the higher synthesis and excretion of citrate confers Al tolerance.

C.

Organic Acids as Al-Chelating Substance

Since higher plants cannot move away from the acid soil, they have developed ways to reduce this edaphic stress. An eective strategy to reduce the stress is to chelate the Al3 in the rhizosphere and by that reduce its toxicity. Exclusion of malate from wheat (Delhaize et al., 1993b); citrate from maize, Cassia tora, and rye (Miyasaka et al., 1991; Zheng et al., 1998; Li et al., 2000b); and oxalic acid from taro and buckwheat (Ma and Miyasaka, 1998; Ma et al., 1997b) have been reported. Some plants exclude both malate and citrate under Al stress. Malate and oxalate are excreted instantly under Al stress, but citrate is excreted only after a lag phase. These results suggest that stored malate and oxalate are excreted while citrate is synthesized by a gene-regulated system. The organic acids were classied into three groups of Al detoxiers: (1) strong (citrate, oxalic, tartaric); (2) moderate (malic, malonic, salicylic); and (3) weak (succinic, lactic, formic, acetic phthalic) (Hue et al., 1986). The following facts support the role of excreted organic acids as detoxiers; During the rst 20 h of Al exposure, the root growth rate of both tolerant and sensitive maize varieties was severely inhibited. However, after this period, root growth was resumed in the tolerant plants, but remained severely inhibited in the Al-sensitive one. A dose-dependent citrate and malate exudation was observed from tolerant but not from sensitive roots (Jorge and Arruda, 1997; Yang et al., 2000). The root of the Al-resistant snapbean released 70 times more citrate in the presence of Al as in its absence, and the amount of citrate excreted was 10 times as much as that of Al-sensitive cultivars (Miyasaka et al., 1991). Similar results regarding malate exclusion were obtained with Al-tolerant and Al-sensitive isogenic wheat lines.

In > 36 lines of wheat cultivars diering in Al resistance that were screened, Al-stimulated malate release was correlated with Al resistance (Ryan et al., 1995). It was shown that addition of organic acids to the solution ameliorated Al toxicity in the root of Al-sensitive varieties and reduced dramatically the loss of viability of root cells (Li, 2000). This may be explained by the fact that complexes of Al with di- and tricarboxylic acids were not transported through root cell membranes (Ma et al., 1998). Excretion of organic acids is Al3 specic and is not induced by other trivalent cations (Ma et al., 1997b). The loss of a certain chromosome arm resulted in a decrease in Al resistance in ditelosomic wheat lines and decreased rates of root apical malate release concomitant with decreased Al exclusion (Kochian, 1998). Exposure to Al induced depolarization of the membrane potential (Em) in Al-tolerant wheat cultivars but not in an Al-sensitive cultivars. Depolarization was specic to Al. Al-induced depolarization of root cap cell membrane potentials is probably linked to malate release (Papernick and Kochian, 1997). Al3 triggers the opening of the putative malatepermeable channel. Several antagonists of anion channels inhibited the Al-stimulated eux of malate. The anion channel antagonist niumate inhibited the current in whole-cell measurements by 83% at 100 M Al. Patch clamp recordings revealed a multistate channel with single-channel conductance of between 27 and 66 ps. This is a good candidate to be the transport system facilitating Al-induced malate release (Ryan et al., 1997b). K-252a, a potent inhibitor of protein phosphorylation, reduced dramatically the excretion of malate from Al-treated wheat, suggesting the involvement of protein phosphorylation for the regulation of malate excretion under Al stress (Osawa and Matsumoto, 2001). Transgenic introduction of the bacterial cytosolic citrate synthetase gene into tobacco and papaya resulted in Al tolerance (Fuente et al., 1997). Excretion of organic acids from the root apex where Al injury is located seems to be a reasonable strategy for the eective use of carbon and energy by the plant (Delhaize and Ryan, 1995). Such experimental evidence indicates that the mechanism of Al exclusion that depends on the chelation of Al3 with excreted organic acids is an eective strategy. However, it is unclear whether the quantities of organic acids released are adequate to explain the insensitivity to Al of the more tolerant genotypes. Consumption of the excreted organic acids by soil bacteria should also be considered. The question why dierent plant species excrete

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Matsumoto

dierent organic acids in dierent ways upon Al stress also remains to be solved. The eux of organic acids is probably switched on or o by the Al3 stress, because continuous excretion of organic acids from roots would consume carbon and energy in extraordinary amounts. The precise mechanism remains to be elucidated, and a more integrative, multifaceted model of tolerance is needed (Parker and Pedler, 1998). The role of intracellular organic acids on Al tolerance was investigated using the Al-tolerant plant Hydrangea macrophylla. Leaves of hydrangea may contain up to 15.66 mmol Al kg1 fresh weight. About 77% of the total Al exists in the cell sap (Ma et al., 1997a). The ligand in the Al complex of hydrangea leaves was citric acid with a molecular ratio of 1 Al to 1 citrate. The puried Al:citrate complex from hydrangea leaves did not inhibit the root elongation of maize and did not decrease the cell viability although both parameters were strongly inhibited by the same concentration of AlCl3 . This means that Al in the form of Al:citrate is not toxic. Also, buckwheat contains large amounts of Al compared to other crop plants but can grow normally under Al stress. This suggests that buckwheat can detoxify Al. The measurement of 27 Al-NMR revealed that the Al in buckwheat leaves and roots existed as an Al:oxalate (1:3) complex. The Al:oxalate (1:3) complex is the least toxic complex compared to other complexes. Buckwheat contains a large amount of oxalate regardless of Al stress and can excrete oxalate immediately after the plant is exposed to Al. Even if some Al is incorporated into the plant tissues, it is immediately chelated there. D. Protein Expression in Roots Under Al Stress

band in PT741 suggested a potential role of these proteins in mediating resistance to Al, associated with the tonoplast. Antibodies raised against tonoplast H+ATPase and H+-PPiase did not crossreact with 51kDa protein, although Al stress induced these enzymes in the barley roots (Matsumoto et al., 1996; Taylor et al., 1997). E. Mucilage

Wheat genotypes diering in Al tolerance were compared for qualitative and quantitative dierences of their proteins. However, no conclusive evidence for upregulation of a certain protein that confer Al tolerance was reported (Delhaize et al., 1991; Ownby and Hruschka, 1991). Most of the changes in protein expression associated with Al stress probably result from the eects of Al on cell metabolism. Another approach was to look for proteins that are characterized by their binding capacity to Al. Appearance of the 23-kDa peptide in root exudates cosegregated with the Al-resistant phenotype in F2 populations and had a signicant Al-binding capacity (Basu et al., 1999). Similarly, a 51-kDa membrane-bound protein accumulated in the root tip of Al-tolerant wheat (PT741) under Al stress. The specic induction of the 51-kDa

The root apices of most plant species are covered by mucilaginous substances that are excreted from root cap cells (see also Chapter 3 by Sievers et al. in this volume). The meristem and cap region where Al toxicity is dominant are coated with mucilage that ranges in thickness from 50 m to 1 mm. Mucilage consists mainly of polysaccharides > 2 106 daltons. Abundant sugars are glucose, galactose, and arabinose. Uronic acids are smaller in amount but characteristic of the mucilage. Mucilage has various protective functions against toxic metals in the soil and has a high Al-binding capacity. Fifty percent of the total Al of root apices of cowpea was associated with mucilage (Horst et al., 1982). Al bound to mucilage of wheat roots accounted for $ 2535% of the Al remaining after desorption by citric acid. The Al in rhizosphere is bound to mucilage that blocks the entry of Al into the root. When the mucilage was periodically removed from the root tips of cowpea with a brush, inhibition of root elongation was increased. Apparently, binding of Al to mucilage is a mechanism of Al tolerance. A good correlation exists between mucilage volume and Al tolerance. Organic acids released into a mucilage droplet would diuse slowly; thus, the mucilage droplet would form a region of high concentration of organic acids where Al is captured before reaching the root surface. However, a protective role of the mucilage against Al injury is dicult to reconcile with the lack of evidence that mucilage excretion aected by Al. On the contrary, disappearance of mucilage is one of the rst visible symptoms of Al toxicity (Puthota et al., 1991). The role of mucilage in the protection of roots against Al toxicity depends on the amount of mucilage excreted and how strongly Al bounds to mucilage. Mucilage from maize roots is strongly bound to Al but failed to prevent Al-induced inhibition of root elongation (Li et al., 2000a). Approximately 50% of the total Al of the root apices was located in the mucilage of cowpea, while only 922% of Al in maize root was bound to mucilage. The binding is decreased by the lower content of uronic acids (3%) in maize muci-

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lage as compared to 11.5% in cow pea mucilage (Li et al., 2000a). It will be necessary to determine the Albinding sugar component in mucilage as well as total amount of mucilage and kinetic data of synthesis and excretion of mucilage in order to understand the role of mucilage in Al resistance. F. pH in the Rhizosphere

Solubility of Al depends strongly on pH. Thus, maintenance of a high solution pH may reduce the solubility and toxicity of Al. An increase in the pH of dilute nutrient solution from 4.5 to 4.6 caused a 26% decline in soluble Al concentration (Blamey et al., 1983). This suggests that even a slight pH change can aect the toxicity of Al as well as tolerance. However, measurement of pH should be done carefully because the change of pH near the root surface is important. For this purpose, a vibrating microelectrode was used to measure pH at a radial distance of 20 and 50 m from the surface of the root tip of a wild-type and of an Al-tolerant Arabidopsis (Degenhardt et al., 1998). The Al-tolerant Arabidopsis mutant alr-104 showed a clear increase of pH at the rhizosphere in the presence of Al, but the wild type did not. Al exposure of alr-104 induced a twofold increase in net H inux localized at the root tip. The increased ux raised the root surface pH of alr-104 by 0.15 unit, suggesting that Al resistance in alr-104 is mediated by pH change in the rhizosphere. Dierence in Al resistance between wild type and alr-104 disappeared when roots were grown in pHbuered medium. It is interesting that no dierence in root H uxes between wild type and alr-104 was detected in the absence of Al.

old leaves is thicker and Al may have accumulated in the cell wall during thickening. The formation of new roots was greatly accelerated after 1 month of Al treatment and thereafter the growth of tops was positively aected (Matsumoto et al., 1976b). Tea is a sensitive plant for phosphate nutrition and markedly inhibited by the excess of phosphate. Konishi et al. (1985) showed that maximum growth of tea occurred with coexistence of Al with phosphate. The reduced growth of tea at 0.8 mM phosphate was dramatically stimulated by the presence of 1.6 mM Al with new root formation. Al plays a regulatory role in the eective absorption and utilization of phosphate. Another strategy of tea plants against Al toxicity is binding of most of Al to catechin, phenolics, and organic acids (Nagata et al., 1992). The mechanism of Al tolerance is generally carried out by internal detoxication or excretion of chelators, but the mechanism of benecial eect of Al on the growth has not been clearly demonstrated.

V.

CONCLUDING REMARKS

IV.

BENEFICIAL EFFECT OF ALUMINUM ON PLANT GROWTH

Plants that contain > 1000 ppm Al are called Al accumulators. Among the 259 plant families, 37 Al accumulator species were found and most of them are arborescent cryptogams (Chenery and Sporne, 1976). Al accumulators can grow in acid soils, and growth of some of them is even promoted. Tea (Camellia sinensis) may contain as much as 30,000 ppm Al in old leaves but only 600 ppm in young leaves. The specic locations of Al in epidermis of old leaves and in the cell lumen of bean and barley root was demonstrated by the staining of Al with aluminon and by x-ray (EMX) microanalysis (Waisel et al., 1970; Matsumoto et al., 1976b). The secondary cell wall of epidermal cells of

Inhibition of root elongation caused by Al toxicity is one of the most deleterious factors for plant growth in acid soils. Al3 concentrations as low as 1 mol at pH 4.55.0 inhibit root elongation within 1 h. Absorbed Al is localized at the root apex, where it inhibits cell functions. It is therefore important to know the eect of Al on the processes of cell elongation and cell division at the root apex. There are several unsolved problems underlying the mechanism of Al toxicity. The receptor of the Al signal on root cell membrane and how the signal is transmitted remain unknown. Does the signal work only in the apoplast? If so, there must be a signal transduction system through the plasma membrane into the symplast. Structural proteins like tubulin and actin are candidates for participation in that system. Another possibility is that Al itself is active in the symplast. In this case, we must know the mechanism of Al transport through the plasma membrane. The role of organic acids, both intracellular and extracellular, in the mechanism of Al tolerance has been claried markedly during the last decade. Al-tolerant plants accumulate less Al than sensitive ones, and formation of chelaters reduces Al toxicity. The major organic acids are citric, malic, and oxalic acids. Why do dierent plant species excrete dierent organic acids by the same Al signal? Is there any other Al-chelating compounds of plant origin other than organic acids? Is

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834

Matsumoto Berzonsky WA. 1992. The genomic inheritance of aluminum tolerance in Atlas 66 wheat. Genome 35:689693. Blamey FPC, Edwards DG, Asher CJ. 1983. Eects of aluminium, OH:Al and P:Al molar ratios, and ionic strength on soybean root elongation in solution culture. Soil Sci 136:197207. Blamey FPC, Asher CJ, Kerven GL, Edwards DG. 1993. Factors aecting aluminium sorption by calcium pectate. Plant Soil 149:8794. Blancaor EB, Jones DL, Gilroy S. 1998. Alterations in the cytoskeleton accompany aluminum-induced growth inhibition and morphological changes in primary roots of maize. Plant Physiol 118:159172. Cakmak I, Horst WJ. 1991. Eect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiol Plant 83:463468. Chen J, Suco E, Stadelmann EJ. 1991. Aluminium and temperature alteration of cell membrane permeability of Quercus rubra. Plant Physiol 96:644649. Chenery EM, Sporne KR. 1976. A note on the evolutionary status of aluminium-accumulators among dicotyledons. New Phytol 76:551554. Clarkson DT. 1965. The eect of aluminium and some other trivalent metal cations on cell division in the root apices of Allium cepa. Ann Bot N S 29:309315. Clune TS, Copeland L. 1999. Eect of aluminium on canola roots. Plant Soil 216:2733. Cruz-Ortega R, Cushman JC, Ownby JP. 1997. cDNA clones encoding 1,3-B-glucanase and a mbrin-like cytoskeletal protein are induced by Al toxicity in wheat roots. Plant Physiol 114:14531460. Cumming JR, Cumming AB, Taylor GJ. 1992. Patterns of root respiration associated with the induction of aluminium tolerance in Phaseolus vulgaris L. J Exp Bot 43:10751081. Degenhardt J, Larsen PB, Howell SH, Kochian LV. 1998. Aluminum resistance in the Arabidopsis mutant alr-104 is caused by an aluminum-induced increase in rhizosphere pH. Plant Physiol 117:1927. Delhaize E, Ryan PR. 1995. Aluminum toxicity and tolerance in plants. Plant Physiol 107:315321. Delhaize E, Higgins TJV, Randall PJ. 1991. Aluminium tolerance in wheat: analysis of polypeptides in the root apices of tolerant and sensitive genotypes. In: Wright PJ, Baligar VC, Murrmann RP, eds. PlantSoil Interactions at Low pH. Dordrecht, Netherlands: Kluwer Academic Publishers, pp 1071 1078. Delhaize E, Craig S, Beaton CD, Bennet RJ, Jagadish VC, Randall P. 1993a. Aluminum tolerance in wheat (Triticum aestivum L.). Plant Physiol 103:685693. Delhaize E, Ryan PR, Randall PJ. 1993b. Aluminum tolerance in wheat. II. Aluminum-stimulated excretion of malic acid from root apices. Plant Physiol 103:695 702.

the amount of excreted organic acids sucient for Al chelation in the rhizosphere in acid soil? (See also Chapter 36 by Neumann and Romheld in this volume.) Much progress has been made in the molecular aspects of Al binding to oxalic acid and excretion mechanism of malic acid. However, an important problem still to be solved is the regulatory mechanism of the synthesis and excretion of organic acids upon the Al signal. The knowledge of the cell responses to the short-term eects of Al is expected to help us to understand the whole-plant responses and would lead to the improvement of crop production under long-term eects of Al. ACKNOWLEDGMENT The author wishes to thank Mrs. S. Rikiishi for her careful preparation of the manuscript. REFERENCES
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