Acid soils are prevalent in many regions of the world and present a range of stresses to plants. One of the major stresses caused by these soils is aluminum (Al), which is solubilized by the acidity into the soil solution. Soluble Al exists in its most toxic form as Al3+, which can inhibit root growth in many plant species at micromolar concentrations. The resulting reduced and damaged root system limits the capacity of plants to uptake water and nutrients, and increases their susceptibility to other stresses.
The mechanisms by which Al inhibits root growth are not well understood, despite extensive physiological investigations. The root apex is the most sensitive part of the root to Al because it is the site of cell division and cell elongation [1, 2]. Since Al is so reactive, it can interact with multiple structures in the apoplasm and symplasm of root cells. In the cell wall, Al primarily binds to the pectin matrix and thereby alters the physical properties of the cell wall [3, 4]. In the symplasm, sites of Al interaction include membrane constituents, ion channels, metabolic enzymes, components of signaling pathways, members of the cytoskeleton, and the DNA [3, 5]. Although some of the resulting cellular alterations have been proposed to affect cell division or cell elongation, a recent study conducted in Arabidopsis thaliana indicates that it is not Al toxicity that is directly responsible for the inhibition of root growth. Genetic and biochemical evidence suggest that the cells of the root apex have an ATR-controlled mechanism to monitor Al-induced DNA damage . In plants exposed to Al, this mechanism activates blockage of cell cycle progression and thus root growth. This active response of roots to Al may not protect individual plants, but it may help to safeguard plant populations by preventing the passage of damaged DNA to subsequent plant generations .
Plant species vary considerably in their degree of Al tolerance, and even genotypes within the same plant species vary in their ability to cope with Al. The mechanisms providing enhanced Al tolerance can be classified into external and internal mechanisms [5, 7]. The best-documented external mechanism is the efflux of organic acid anions, such as malate, citrate, and oxalate, from the roots in response to Al exposure . These organic acid anions effectively chelate Al and thereby detoxify Al in the rhizosphere. Other proposed external mechanisms involve secretion of proteins , root-mediated increase of the rhizosphere pH , and masking Al binding sites at the cell wall [11, 12]. Proposed internal tolerance mechanisms include chelation of Al by organic acid anions or phenolic compounds and sequestration of Al in the vacuole .
The genes responsible for the Al-induced efflux of malate and citrate have been recently isolated and demonstrated to represent major genes for Al tolerance in several plant species . The genes responsible for the efflux of malate belong to the ALMT (for Al-activated malate transporter) gene family [13–15] and those involved in the efflux of citrate to the MATE (for multidrug and toxin efflux protein) gene family [16–20]. All these genes encode membrane proteins, consistent with their role in facilitating the efflux of organic acid anions. Additional genes with putative roles in Al tolerance have been identified in Al-sensitive mutants of rice (Oryza sativa) and Arabidopsis. The rice mutants star1 (for sensitive to Al rhizotoxicity 1) and star2 were found to be mutated in genes encoding two proteins that form together an ATP-binding cassette (ABC) transporter . This complex mediates the transport of UDP-glucose to the cell wall, where the molecule is believed to play a role in masking Al binding sites. Similarly to star1 and star2, the Al-sensitive mutants als1 (for Al sensitive 1) and als3 of Arabidopsis are mutated in genes encoding ABC transporter-like proteins [22, 23]. Although the substrate of these proteins is not known, the mutant phenotypes and patterns of gene expression have led to the proposal that the two proteins transport and sequester Al to confer Al tolerance. ALS1 is believed to be involved in the intracellular transport of Al to the vacuole , whereas ALS3 appears to be necessary for the long-distance transport of Al to the aerial parts of the plant .
Further insight into the molecular mechanisms involved in Al toxicity and tolerance come from gene expression analyses. Genome-wide transcriptome analyses in roots of Arabidopsis have revealed a number of cellular processes that are altered in response to Al exposure. Examples are cell wall modification, protein metabolism, transport processes, and oxidative stress [24, 25]. In maize (Zea mays), wheat (Triticum aestivum), and Medicago truncatula, gene expression was analyzed in plant lines with contrasting levels of Al tolerance [26–28]. These studies have led to the identification of several candidate genes for Al tolerance, including previously identified genes encoding organic acid efflux transporters, genes controlling levels of reactive oxygen species (ROS), as well as genes involved in pectin modification and immobilization of Al by phosphate.
Forest trees generally tolerate high concentrations of Al . For example, seedlings of Norway spruce (Picea abies) and birch (Betula pendula) did not show any reduction in root growth at Al concentrations below 0.3 and 3 mM, respectively [30, 31]. In contrast, Al concentrations as low as 50 μM tend to affect the root growth of Arabidopsis and several crop plants (e.g. [24, 28, 32]). Since many forest tree species grow naturally in acid soils, it appears likely that such species have developed adaptive mechanisms that enable them to tolerate high Al conditions. Analyses of the root responses to Al in forest trees may thus broaden our understanding of Al tolerance mechanisms in plants.
In a previous study, we used clonal aspen (Populus tremula, clone Birmensdorf) to investigate Al-induced efflux of organic acid anions from roots . The results showed that Al concentrations ≥ 200 μM induced the efflux of citrate, while Al concentrations ≥ 500 μM enhanced the efflux of oxalate. At these concentrations, Al did not cause any visible symptoms at the root tips, indicating that the aspen clone examined tolerates high concentrations of Al. Using the same aspen clone, we investigated temporal patterns of root gene expression under Al stress. Changes in gene expression were assessed by application of the Affymetrix poplar genome array. The expression of selected genes was further analyzed by reverse-transcription PCR.