Aluminum Toxicity Tolerance
By Dr. E. Delhaize,
CSIRO Division of Plant Industry,
Acid soils are prevalent on the earth’s surface and have been estimated to occur on about 30% of the land area. A description of the types of soils that are acid and their distribution is provided by von Uexküll and Mutert (1995). Many of the soils used for agriculture, particularly those in developing countries where forests have been cleared, are considered sufficiently acidic that they restrict the growth of many susceptible plant species. Soils may be acid naturally or may become acidic due to the activities of humans. These activities can include farming practices that result in acidification or acid rain as a consequence of industrial processes (see Kennedy 1992 for a comprehensive account of acid soils and acid rain).
While low pH can restrict plant growth in its own right, in most cases it is the dissolution of toxic metals, particularly aluminum (Al), which restricts plant growth. Aluminum is the most abundant metal in the earth’s crust and comprises some 7% of its mass. Dissolution of just a small fraction of the aluminum compounds in soils can result in serious Al toxicity to susceptible species.
Fortunately not all forms of Al are toxic and as indicated above, it is the soluble forms that are implicated in the toxicity of acid soils. In general, trivalent cations are toxic to plants and Al3+ is considered to be the major phytotoxic form although some studies have implicated the di- and monovalent forms in toxicity. Aluminum hydrolyzes in solution and Al3+ dominates under acidic conditions while Al(OH)2+ and Al(OH)2+ species are prevalent at pHs between 5 and 7. As the pH increases, the solid phase Al(OH)3 can form and under alkaline conditions, Al(OH)4- is the dominant species. Many ligands bind avidly to Al3+ making the chemistry of Al in soils difficult to understand and predict (Ritchie 1995). Even in solutions of known composition, the effects of various forms of Al on roots can be difficult to analyze.
Although Al is considered the major limitation to plant growth on acid soils, other metals such as manganese can also be present at toxic concentrations. In addition many acid soils have poor fertility due to deficiencies of calcium, magnesium, phosphorus and molybdenum. This Chapter will focus primarily on the toxicity of Al and mechanisms of Al tolerance in plants.
There are several recent reviews that discuss mechanisms of Al tolerance and toxicity in plants. These include reviews by Kochian (1995), Matsumoto (2000), Ma et al. (2001), and Ryan et al. (2001) and the reader is directed to these articles as well the Update link under this section for more detailed discussions on Al tolerance and toxicity.
Fig.1. Effect of Al on root growth of wheat genotypes that differ in Al tolerance at the Alt1 locus. Seedlings were grown in a solution consisting of 0.2mM CaCl2 and 10 mM Al for 5 days. The Al tolerant genotype (on the right) shows little or no effect of Al on root growth while the sensitive (on the left) is severely inhibited.
Fig.2. Hematoxylin stain to identify Al tolerant genotypes of wheat. Seedlings of Al tolerant and sensitive genotypes were grown in 0.2 mM CaCl2 without (top two roots) or with 50 mM Al (bottom two roots; Al-sensitive is the lowest) and then stained with hematoxylin. Intense staining of the sensitive root apices indicates accumulation of Al in these root tips.
There is considerable
variation within and between plant species in their ability to tolerate Al and
this variation within some species has allowed breeders to develop genotypes
able to grow on acid soils. The use of Al tolerant germplasm complements liming
practices that are aimed at neutralizing the acidity. However, in many
agricultural systems, the application of lime as the sole way of managing acid
soils is either too costly or takes many years for the lime to be effective
particularly where the acidity occurs at depth. On acid soils, Al tolerant
species can be used in place of Al sensitive species to maintain production.
For example, in pastures alfalfa can be replaced with more Al-tolerant pasture
species but the drawback with this approach is that the nutritional quality of
the alternative pastures might not match that of alfalfa. Where there is variation within a species,
this can be used by breeders to enhance the Al tolerance of elite genotypes.
For example, Al tolerance in wheat (Triticum
aestivum) is controlled by a small number of major dominant genes and these
have been exploited in breeding programs to enhance the Al tolerance of
cultivars (for example see Johnson et al. 1997; Berzonsky 1992; Kerridge and
Kronstad 1968). In the case of wheat, much of the germplasm that confers Al
tolerance can be traced back to
Fig.3. Organic acids able to form 5- or 6-membered ring structures with Al3+ protect plants from Al toxicity
There is now considerable evidence implicating a role for organic acids in the Al tolerance mechanisms of a range of plant species. Some organic acids are able to complex Al3+ into forms that are not toxic to plants. Hue et al. (1986) assessed the ability of a range of different organic acids to
protect plant roots from Al toxicity in hydroponic culture. They found that organic acids with hydroxyl and carboxyl groups able to form stable ring structures with Al3+ that consisted of 5- or 6- bonds conferred the greatest protection from Al toxicity (Figure 3). Organic acids commonly found in plants that fit this criterion are citric, oxalic and malic acids. Aluminium tolerance mechanisms postulated to involve organic acids can be divided into external and internal detoxification with some plant species apparently using both types of mechanisms.
Some plant genotypes can tolerate Al because they exclude it from the root apices. For example, in wheat a range of techniques have shown that Al-tolerant genotypes accumulate less Al in root apices than Al sensitive genotypes. We now know that Al tolerant genotypes of many species exude organic acids in response to Al (see Ryan et al. 2001 and Ma et al. 2001). These organic acids chelate the Al and therefore protect the roots from Al toxicity. For example in response to Al exposure, wheat exudes malate, whereas snapbeans, maize, Cassia toru and soybean exude citrate, and buckwheat exudes oxalate. Triticale, rapeseed, oats, radish and rye exude both malate and citrate in response to Al. In several of these examples the efflux of organic acids occurs primarily from the root apices and this makes good sense since this is the part of the root system most susceptible to Al toxicity. Furthermore, the finding that Al-tolerant genotypes of several of these species exude considerably more organic acid than the corresponding Al-sensitive genotypes supports the idea that the efflux of organic acid is an Al tolerance mechanism.
Fig.4. A model showing how Al-activated malate efflux protects wheat root tips from Al toxicity.
Some of the more convincing evidence relating organic acid efflux to Al tolerance comes from a study of near-isogenic wheat lines that differ in Al tolerance at a single genetic locus (the Alt1 locus). These near-isogenic lines are very useful for studying the physiology of Al tolerance because the lines are essentially identical to one another except for their tolerance of Al. This avoids comparisons between genotypes that are genetically dissimilar, even if they are of the same species, and avoids the possibility of having more than one tolerance mechanism responsible for the phenotype. In this study, Al was found to stimulate 5 to 10 fold more malate from root apices of the Al-tolerant line than the near-isogenic sister line (Delhaize et al. 1993; see figure 4 for a proposed model for the role of malate in Al tolerance of wheat). Furthermore, high rates of Al-stimulated malate exudation co-segregated with the Al tolerance phenotype and this provided additional evidence for a role of organic acids in the Al tolerance mechanism. Work by Papernik et al. (2001) using deletion lines of the moderately Al-tolerant wheat, Chinese Spring, showed that three
genetic loci associated with the loss of Al tolerance all resulted in reduced Al-activated malate efflux. These observations provide strong genetic evidence in support of a role for malate in the Al tolerance mechanism of wheat.
The amount of organic acid exuded from root apices need not detoxify all the Al in the soil surrounding the root system but should be sufficient to detoxify the Al that immediately surrounds the root apices. However, efflux needs to continue to replace organic acids that diffuse away from the root apex as well as to replace organic acids that are broken down by microorganisms. We can envisage the organic acids acting as a protective sheath around the root apex as it moves through an acid soil. As the Al is chelated by the organic acid, the amount of free Al3+ is reduced which then reduces the amount of organic exuded since efflux, up to a point, is determined by the concentration of Al in solution. In this way, the loss of organic acid, with the associated metabolic cost to the plant, is reduced.
Two general patterns of Al-stimulated efflux of organic acids have been observed from roots. In response to Al, Pattern I is typified by a rapid efflux of organic acid which remains constant with continued exposure to Al. In Pattern II by contrast, there is a considerable lag phase before maximal efflux is observed. The kinetics of Pattern I suggests that Al activates a pre-existing transport mechanism for malate and recent evidence has implicated a role for anion-channels in the transport of the organic acid (see below). Little is known about the mechanism of Pattern II-type responses although the lag phase is consistent with the induction of genes and the synthesis of proteins. These proteins could be involved in transporting organic acids out of the root cells and/or in the synthesis of organic acids.
Fig.5. The electrochemical gradients in root cells favor the efflux of malate.
At the prevailing pH of the cytoplasm (approx 7), organic acids are dissociated from their protons and exist largely as anions. Both the electrochemical gradient across the plasma membrane (approx. negative 200mV) and the concentration gradient for the organic anions will serve to
drive the efflux of organic anions out of the cells (Figure 5). Since they are charged molecules, organic anions are unlikely to move through the hydrophobic lipid bilayer of the plasma membrane unassisted. Damage to the plasma membrane will lead to the release of organic ions but this is an uncontrolled process and is likely to result in cell death as a whole range of metabolites leak out of the cell. The Al-stimulated efflux of organic acids is a controlled process and either stops or is reduced when Al is removed from the medium. Clearly, rupture of the plasma membrane is not responsible for the efflux because, as noted above, in response to Al, only one or two organic anions are exuded from roots of any given species. Furthermore, efflux is observed from the Al tolerant genotypes and less so from the sensitive genotypes where damage to the plasma membrane is more likely as a result of the Al toxicity.
Ion channels are proteins that span membranes and allow the passive flow of ions down their electrochemical gradients (see the following reference or links for more detailed information about ion-channels:
Fig.6. A model to explain the mechanism of Al activated efflux in root tip cells of wheat. Three possibilities are shown where Al interacts with either (1) the channel protein directly or (2) a component of the plasma membrane or (3) enters the cell to trigger the opening of the channel and malate efflux. The malate external of the cell chelates Al3+ to render it non-toxic.
These transporters can be specific for particular ions and be gated or activated by particular compounds. The finding that antagonists of anion
channels inhibited the Al-activated efflux of organic anions from some species suggested a role for anion channels in the transport of the organic anions out of cells. Recent evidence from research groups using the patch clamp technique (see this link for a general introduction of the patch clamp technique has yielded direct evidence for the presence of Al-activated anion channels in root cells of plants known to secrete organic anions. Ryan et al (1997) first showed that Al activates an anion channel present on the plasma membrane of apical-root cells. The characteristics of this channel are consistent with the properties of malate efflux that is observed from intact root apices of Al-tolerant wheat. More recently, Zhang et al. (2001) have found that the Al-activated channel in wheat is permeable to malate and that the channels in a pair of near-isogenic wheat lines that differ in Al tolerance (See above) have different properties. Similar channels have been observed in the plasma membrane of apical root cells from Al-tolerant maize (Pineros and Kochian 2001 and Kollmeier et al. 2001). These channels are permeable to citrate and can be activated by Al in isolated patches of outside-out plasma membrane. Activation of the channel in patches of membrane indicates that soluble secondary messengers are not required for activation and that the channel is either activated directly by Al or that a different protein on the plasma membrane acts as a receptor for Al (see Figure 6 for a model of possible mechanisms involved in activation of malate efflux). Taken together, these findings provide evidence that the Al tolerance gene in wheat and maize either encodes the anion channels themselves or proteins involved in transducing the signal from Al to channel activation.
Some plant species have the remarkable ability of accumulating Al in shoots and roots. Clearly these Al-tolerant species have evolved mechanisms that maintain the Al in non-toxic forms within the plant as well as mechanisms that allow the Al to move through the plant and across a range of membranes. Evidence points to a role for organic acids in complexing internal Al in buckwheat, hydrangea and melastoma. Using 27Al-nuclear magnetic resonance, researchers have identified Al complexed to oxalate in buckwheat and melastoma and to citrate in hydrangea (Watanabe 1998; Ma et al. 1998; Ma et al. 1997). Both citrate and oxalate are strong chelators of Al thereby protecting cellular components from the phytotoxic effects of Al. In buckwheat the organic acid that complexes Al differs in various tissues of the plant. Al is likely to be taken up by buckwheat as Al3+ but how this occurs is not known. Inside roots the Al is chelated with oxalate to form a 1:3 Al:oxalate complex. In the xylem the predominant form is Al-citrate as a 1:1 complex and once translocated to shoot cells, the 1:3 Al:oxalate complex is reformed. Buckwheat is a species that also exudes oxalate in response to Al and its high level of Al tolerance may be a result of both external and internal Al detoxification mechanisms. Little is known of the way that Al:organic acid complexes are transported across membranes but is likely to involve specific transporters.
While there is considerable evidence associating organic acids in the Al tolerance mechanisms of many species, other species apparently use mechanisms that do not rely on organic acids. For instance, Brachiaria decumbans, an extremely Al-tolerant species, does not secrete organic acids in response to Al and so must possess different ways of dealing with toxic levels of Al in the soil solution (Wenzl et al. 2001). Since the phytotoxic form of Al is largely dependent on pH, a mechanism based on increasing the pH around root apices should provide a degree of protection from Al. Evidence in support of such a mechanism comes from a study of an Al-tolerant Arabidopsis mutant (alr1). This mutant was found to exhibit an Al-induced increase in pH in the solution immediately surrounding the root apex and this would have resulted in a decrease in Al3+ activity (Dengenhardt et al. 1998). Other evidence has implicated the efflux of phosphate as an Al tolerance mechanism. Phosphate complexes Al and it has been found to be released along with malate, from the root apices of Atlas, a very tolerant wheat cultivar (Pellet et al. 1996). Unlike the efflux of malate, phosphate efflux was found to be constitutive without a requirement for Al to activate the mechanism of efflux.
There is some evidence that Al toxicity may be due, at least in part, to oxidative stress and the observations that Al-stress induces the synthesis of proteins typical of oxidative stress responses supports this idea. For example Al induces the expression of genes that encode peroxidases, glutathione S-transferase, and blue-copper proteins. The observation that overexpression of some these induced proteins in Arabidopsis results in increased Al tolerance as well as increased tolerance to oxidative stress strengthens this link between Al toxicity and oxidative stress (Ezaki et al. 2000). These findings also establish that increased Al tolerance can be conferred to plants by processes that are independent of organic acids.
Work in yeast has also shown that Al tolerance can be conferred by overexpression of genes that are unrelated to organic acid efflux. MacDiarmid and Gardner (1998) screened a yeast genomic library to identify genes that when over-expressed in yeast conferred Al tolerance. They identified two related genes that they named ALR1 and ALR2. These genes encode membrane-bound proteins that have the characteristics of Mg transporters. Indeed, when the genes are inactivated, the yeast is unable to grow unless high concentrations of exogenous Mg2+ are supplied. It is not clear how overexpression of these genes confers Al tolerance but one hypothesis is that the proteins encoded by these genes are the primary sites of Al toxicity in yeast. Disruption of Mg2+ uptake by Al leads to cell death due to Mg deficiency whereas high level expression of the ALR genes allows Mg transport to be maintained at normally toxic Al concentrations.
Yeast has proved to be a powerful and effective tool for cloning plant genes through complementation of mutants or simply by overexpressing plant genes and screening for a particular phenotype. Taking this approach, a range of plant genes have been found that confer Al tolerance when expressed in yeast but none of the genes appear to encode proteins involved in either organic acid biosynthesis or efflux. One of these genes encodes a phosphatidyl serine synthase, an enzyme involved in the biosynthesis of phospholipids (Delhaize et al. 1999). Since phospholipids are key components of the lipid bilayer of membranes, it is conceivable that a change in the lipid composition of the plasma membrane is the basis for the enhanced Al tolerance.
In some species the available germplasm for Al tolerance is limited. For example, although there is some Al tolerant germplasm in alfalfa and barley, the range of Al tolerance is limited compared to some other species. Since organic acids have been strongly implicated in Al tolerance (see above), a logical approach is to manipulate the biosynthesis and efflux of organic acids. To date genes encoding transporters for organic anions have not been cloned. However many of the genes encoding enzymes involved in organic acid biosynthesis or catabolism have been cloned and this provides an opportunity to modify these pathways. The first report of such an approach described the expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco (de la Fuente et al. 1997). The production of citrate by the condensation of oxaloacetate with acetylCoA is the first committed step in the tricarboxylic acid cycle and is catalyzed by citrate synthase which is normally present in the mitochondrion. In the case of the Pseudomonas citrate synthase gene, the enzyme was found in the cytoplasm of the transgenic plants. The transgenic lines expressing the bacterial citrate synthase gene had up to 10-fold greater internal citrate in roots and had greater citrate efflux than the control line. This resulted in increase Al tolerance and more recently benefits in phosphorus nutrition were also apparent due to organic acids solubilizing poorly-soluble forms of phosphate (Lopez et al. 2000). However, this approach appears to be subject to environmental influences as another group, using these same plants as well as ones engineered to express the citrate synthase gene to a much higher level, were unable to reproduce these findings (Delhaize et al. 2001).
Overexpression in Arabidopsis of a plant gene encoding the mitochondrial form of citrate synthase resulted in enhanced citrate accumulation and efflux (Koyama et al. 2000). This demonstrates the potential to increase organic acid secretion by overexpressing plant genes involved in organic acid metabolism. Other genes encoding organic acid biosynthetic enzymes whose expression could be manipulated to enhance production of organic acids include malate dehydrogenase, phosphoenolpyruvate carboxylase and isocitrate dehydrogenase.
Other approaches to increase the Al tolerance of plants is to over-express genes involved in protecting plants from oxidative stress. As discussed above his has already been demonstrated to be effective in Arabidopsis a very Al sensitive species. The increase in Al tolerance using this strategy was relatively small and it is yet to be applied to species important in agriculture. A more effective level of Al tolerance may be achieved by combining the over-expression of several of the genes involved in protection from oxidative stress within the same plant. Additional specific information on the genetics of Al tolerance can be found under Biotech Issues on this site.
Although considerable progress has been made in understanding Al-tolerance mechanisms based on organic acid efflux, much is still to be learned of the molecular mechanisms underlying the activation of anion-channels by Al. For instance, we need to better understanding of the processes involved in how a cell initially senses Al that then leads to channel gating and organic acid efflux. In addition the genes encoding these anion channels need to be cloned. As indicated above, there are clearly Al tolerance mechanisms operating in plants that do not rely on organic acids but to date little is known about these mechanisms. Some progress has been made in genetically modifying plants to enhance their Al tolerance and future work is needed to ensure that sufficient levels of Al tolerance are obtained to be useful for agriculture.
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