Development of a Novel Aluminum Tolerance Phenotyping Platform Used for Comparisons of Cereal Aluminum Tolerance and Investigations into Rice Aluminum Tolerance Mechanisms

The genetic and physiological mechanisms of aluminum (Al) tolerance have been well studied in certain cereal crops, and Al tolerance genes have been identified in sorghum (Sorghum bicolor) and wheat (Triticum aestivum). Rice (Oryza sativd) has been reported to be highly Al tolerant; however, a direct comparison of rice and other cereals has not been reported, and the mechanisms of rice Al tolerance are poorly understood. To facilitate Al tolerance phenotyping in rice, a high-throughput imaging system and root quantification computer program was developed, permitting quantification of the entire root system, rather than just the longest root. Additionally, a novel hydroponic solution was developed and optimized for Al tolerance screening in rice and compared with the Yoshida's rice solution commonly used for rice Al tolerance studies. To gain a better understanding of Al tolerance in cereals, comparisons of Al tolerance across cereal species were conducted at four Al concentrations using seven to nine genetically diverse genotypes of wheat, maize (Zea mays), sorghum, and rice. Rice was significantly more tolerant than maize, wheat, and sorghum at all Al concentrations, with the mean Al tolerance level for rice found to be 2- to 6-fold greater than that in maize, wheat, and sorghum. Physiological experiments were conducted on a genetically diverse panel of more than 20 rice genotypes spanning the range of rice Al tolerance and compared with two maize genotypes to determine if rice utilizes the well-described Al tolerance mechanism of root tip Al exclusion mediated by organic acid exudation. These results clearly demonstrate that the extremely high levels of rice Al tolerance are mediated by a novel mechanism, which is independent of root tip Al exclusion.

genetically diverse panel of more than 20 rice genotypes spanning the range of rice Al tolerance and compared with two maize genotypes to determine if rice utilizes the well-described Al tolerance mechanism of root tip Al exclusion mediated by organic acid exudation. These results clearly demonstrate that the extremely high levels of rice Al tolerance are mediated by a novel mechanism, which is independent of root tip Al exclusion.
Aluminum (AI) is the most abundant metal in the earth's crust, constituting approximately 7% of the soil (Wolt, 1994). Al is predominately found as a key component of soil clays; however, under highly acidic soil conditions (pH < 5.0), Al3+ is solubilized into the soil solution and is highly phytotoxic. Al3+ causes a rapid inhibition of root growth that leads to a reduced and stunted root system, thus having a direct effect on the ability of a plant to acquire both water and nutri ents. Approximately 30% of the world's total land area and over 50% of potentially arable lands are acidic, with the majority (60%) found in the tropics and subtropics (von Uexkull and Mutert, 1995 acidic soils are a major limitation to crop production, particularly in the developing world. As a whole, cereal crops (Poaceae) provide an ex cellent model for studying Al tolerance because of their abundant genetic resources, large, active research communities, and importance to agriculture. In addi tion, work in one cereal species can rapidly translate into impact throughout the family. Previous research has focused on understanding the genetic and phys iological mechanisms of Al tolerance in maize (Zea mays), sorghum (Sorghum bicolor), and wheat (Triticum aestivum). The most recognized physiological mecha nism conferring Al tolerance in plants involves exclu sion of Al from the root tip (Miyasaka et al., 1991;Delhaize and Ryan, 1995;Kochian, 1995;Kochian et al., 2004aKochian et al., , 2004b. The exclusion mechanism is primarily mediated by Al-activated exudation of organic acids such as malate, citrate, or Oxalate from the root apex, the site of Al toxicity (Ryan et al, 1993(Ryan et al, , 2001Ma et al., 2001). These organic acids chelate Al in the rhizo sphere, reducing the concentration and toxicity of Al at the growing root tip (Ma et al., 2001). Phosphate has also been identified as a class of root exudates in volved in cation chelation and therefore can be con sidered a potential exudate involved in Al exclusion from the root tip (Pellet et al., 1996).
Al-activated malate and citrate anion efflux trans porters have been cloned from wheat (ALMT1; Sasaki et al., 2004) and sorghum (SbMATE;Magalhaes et al., 2007), and root citrate efflux transporters have been implicated in Al tolerance in maize (Pineros and Kochian, 2001;Zhang et al, 2001). Recently, a maize homolog of sorghum SbMATE was shown to be the root citrate efflux transporter that plays a role in maize Al tolerance (Maron et al., 2010). Although organic acids have been shown to play a major role in Al tolerance in these species, another exclusion mecha nism has been identified in an Arabidopsis (Arabidop sis thaliana) mutant, where a root-mediated increase in rhizosphere pH lowers the Al3+ activity and thus participates in Al exclusion from the root apex (Degenhardt et al., 1998). Furthermore, there is clear evidence that tolerance in maize cannot be fully ex plained by organic acid release (Pineros et al., 2005). These types of findings strongly suggest that multiple Al tolerance mechanisms exist in plants. Rice (Oryza sativa) has been reported to be the most Al-tolerant cereal crop under field conditions, capable of withstanding significantly higher concentrations of Al than other major cereals (Foy, 1988). Despite this fact, very little is known about the physiological mechanisms of Al tolerance in rice. Two independent studies have identified increased Al accumulation in the root apex in susceptible compared with Al-tolerant rice varieties, but no differences were observed in organic acid exudation or rhizosphere pH (Ma et al., 2002;Yang et al., 2008). These studies suggest that rice may contain novel physiological and/or genetic mech anisms that confer significantly higher levels of Al tolerance than those found in other cereals. A more thorough analysis is required to clarify the mechanism of Al tolerance in rice. Cultivated rice is characterized by deep genetic divergence between the two major varietal groups: Indica and Japonica (Dally and Second, 1990;Garris et al., 2005;Hu et al., 2006;Londo et al., 2006). Exten sive selection pressure over the last 10,000 years has resulted in the formation of five genetically distinct subpopulations: indica and aus within the Indica vari etal group, and temperate japonica, tropical japonica, and aromatic/groupV within the Japonica varietal group (Garris et al., 2005;Caicedo et al., 2007;K. Zhao and S. McCouch, personal communication).
(Note: When referring to varietal groups, the first letter will be capitalized, while lowercase letters will be used to refer to the subpopulation groups.) Subpopulation differences in trait performance are often significant, particularly with respect to biotic and abiotic stress (Champoux et al., 1995;Lilley et al., 1996;Garris et al. 2003;Xu et al., 2009). This can lead to confusion because trait or performance differences may be con founded with subpopulation structure, leading to false positives (type 1 error; Devlin and Roeder, 1999;Pritchard and Donnelly, 2001;Yu et al., 2006;Zhao et al., 2007). Therefore, it is important to consider the subpopulation origin of genotypes being compared when studying the genetics and physiology of Al tolerance in rice.
Al tolerance screening is typically conducted by comparing root growth of seedlings grown in hydro ponic solutions, with and without Al (Pineros and Kochian, 2001;Magalhaes et al., 2004;Sasaki et al., 2004). Sorghum and maize are often screened for Al tolerance in Magnavaca's nutrient solution (Pineros and Kochian, 2001;Magalhaes et al., 2004;Pineros et al., 2005), while rice seedlings are typically grown in Yoshida's solution (Yoshida et al., 1976). Furthermore, Al concentrations used to screen for Al tolerance in maize (222 um), sorghum (148 um), and wheat (100 um) are significantly lower than those used for screening Al tolerance in rice (1,112-1,482 um;Wu et al, 2000;Nguyen et al., 2001Nguyen et al., , 2002Nguyen et al., , 2003. These differences in chemical composition of the nutrient solutions make it difficult to directly compare plant response to Al across these cereals. In rice, the high Al concentrations required to observe significant differences in root growth between susceptible and resistant varieties also complicate Al tolerance screening due to the precipitation of Al along with other elements. The result is that control (-A1) and treatment (+A1) solu tions may differ with regard to essential mineral nutrients that react with Al, leading to differences in growth not directly attributable to Al. Additionally, because the active form of Al that is toxic to root growth is Al3+, any Al that precipitates out of solution has no effect on root growth (Kochian et al., 2004a). In a hydroponic solution, Al may be found in one of four forms: (1) as free Al3+, where it actively inhibits root growth; (2) precipitated with other elements and es sentially unavailable to inhibit plant growth; (3) dif ferent hydroxyl monomers of Al, which are not believed to be toxic to roots (Parker et al., 1988); or (4) complexed with other elements in an equilibrium between its active and inactive states. The degree to which Al inhibits root growth is primarily dependent upon the activity of free Al3+ in solution (Kochian et al., 2004a). The objectives of this study were to (1) develop and optimize a suitable nutrient solution and high throughput Al tolerance screening method for rice; (2) quantify and compare differences in Al tolerance between maize, sorghum, wheat, and rice; and (3) use the developed screening methods to determine if rice utilizes the organic acid-mediated Al exclusion mech anism that is observed in maize, sorghum, and wheat.  Table I).

Optimization of Nutrient Solution Composition for Al
Furthermore, 40% of the total Al added to the solution was lost as a precipitate.
To address these problems, we developed an opti mized nutrient solution, hereafter referred to as mod ified Magnavaca's solution, which minimizes the concentration of Al necessary to elicit significant levels of root growth inhibition in rice seedlings (Table II) Table I).
Plant growth experiments were conducted using seven diverse rice genotypes to investigate whether the two nutrient solutions had different effects on seedling root growth under control conditions (-A1) and whether there were differential responses to the total Al added to each solution. The average total root growth (TRG) of the seven genotypes after 3 d of growth in the two control solutions was virtually identical, 60.58 cm in modified Magnavaca solution and 59.47 cm in Yoshida's solution (Fig. 1A). However, when the same A1C13 concentrations were used in the two treatment solutions, the average TRG of the seven genotypes was 40% to 50% less in the modified  Fig. 1A). These results demonstrate that inhibition of TRG is not determined by the total amount of Al added to a solution but rather by the activity of available Al3+ in the solution. Figure 1 displays the correlation coeffi cients of TRG as a function of total Al (r2 = 0.40) added and available soluble Al (not precipitated; r2 = 0.86), demonstrating that available soluble Al is a much better predictor of root growth inhibition than total Al added.
Al Tolerance Phenotyping Platform RRG of the longest root is the most commonly used parameter for estimating Al tolerance in cereals. We compared estimates of Al tolerance based on RRG of the longest root and RRG of the total growth of the root system to determine whether the longest root mea surement would serve as a useful proxy for estimating , the correlation coefficient for the relationship between RRG of the longest root and RRG of the total root system was r2 = 0.172 (Supplemental Fig. SI). Based on this analysis, it was determined that the RRG of the longest root was not a good proxy for RRG of the total root system because a genotype may appear tolerant based on longest root measurements when, in fact, total root growth is inhibited (Fig. 2). To obtain accurate estima tions of total root growth, we used a custom root digital imaging system developed in our laboratories to quantify root length parameters for the thin, fibrous root systems of rice. The system was based on digital photography and semiautomatic measurement of in dividual primary, secondary, and tertiary roots using RootReader2D software (for details, see "Materials and Methods")- In this system, the length of the total root system can be reliably measured, and we are able to capture high-quality digital images of each root system.

Comparison of Al Tolerance between Cereal Species
When Al tolerance was directly compared between diverse genotypes of maize, sorghum, wheat, and rice, at three Al3+ activity levels, 8.75, 27, and 160 llm, rice was consistently more tolerant than the other cereals, maize was intermediate, and sorghum and wheat were the most sensitive (Fig. 3). The genotypes used in this analysis were selected to represent the range of known Al tolerance within each species; that is, we selected varieties classified as Al sensitive, intermedi ate, and tolerant for each species to ensure adequate representation of variation within as well as between the species. At all Al3+ activities, the order of Al tolerance among the four cereal species was consistent: rice > maize > sorghum > wheat.
To ensure that we were able to observe the full distribution of Al tolerance in each of the four cereal Thus, at 27 llm Al3+, rice was significantly more toler ant than all the other species examined (P < 0.001). The differences in tolerance between rice and the other species became even more apparent at 160 llm Al3+. Growth was essentially abolished in all sorghum and wheat genotypes screened and severely inhibited in all maize genotypes. Of the nine maize genotypes screened, root growth in all but two genotypes was inhibited over 90% (RRG < 0.1).   compared with 4,062 ug Al g"1 (se = 140) in B73. The mean root apex Al concentration of rice was greater than 900 u-g Al g-1 higher than that of Cateto, which was much more susceptible at 160 um Al3+ than any rice genotype. Three rice genotypes accumulated higher Al concentrations than the B73 genotype yet were between three and seven times more Al tolerant (Supplemental Table S2).

Investigation into the Role of Root Exudates in Rice
Al Tolerance Root exudation of citrate, malate, and phosphate, the three root excreted Al-binding ligands implicated in cereal Al tolerance, was quantified in 21 rice geno types (10 Japonica and 11 Indica) evaluated under control (?Al) and treatment (+A1) conditions (Supple mental  Fig. S2). When the relationship between root exudates and Al tolerance was analyzed independently in each rice varietal group, it revealed that within the Indica group, there was a small correlation between Al tolerance and malate (r2 = 0.24) and phosphate exudation (r2 = 0.13; Fig. 6, A-C).
In the more Al tolerant Japonica group, there was a negative correlation between Al tolerance and phosphate exudation (r2 = 0.18) and no correlation between citrate and malate exudation (Supplemental Fig. S2).
In the tolerant maize variety Cateto, which has previously been reported to utilize an Al-activated citrate exudation Al tolerance mechanism (Pineros et al., 2005), we observed Al-activated citrate exuda tion, and exudation rates were significantly higher in Cateto than in any rice variety.  Table S2).

Investigation into the Role of Root Exudates in Al Exclusion
When levels of organic acid exudation were com pared with Al accumulation in root apices across all rice genotypes, we observed a slightly negative corre lation between citrate exudation and root tip Al con centration (r2 = 0.06) and no relationship between malate or phosphate exudation and root tip Al accu mulation (Supplemental Fig. S2). When exudation levels were compared within each varietal group independently, there was a significantly negative correlation in the Indica group between citrate exudation (r2 = 0.47) and Al accumulation and a slightly negative correlation between malate (r2 = 0.07) and phosphate (r2 = 0.075) exudation and root tip Al accumulation (Fig. 6, D-F). Therefore, it appears that in the Indica varietal group, citrate exudation is asso ciated with Al exclusion from the root apex, but this Al exclusion does not confer Al tolerance. In the Japonica varietal group, we observed no relationship between  The precipitation issues confound the ability to quantify rice Al tolerance, as it is difficult to design a nutrient solution with reproducible levels of Al as well as the essential elements P, sulfur, and Fe, which can also impact root growth. Al3+ is highly reactive and readily precipitates with other essential elements; in the Yoshida's +A1 solution, both P and Fe were re duced to such low levels that it was difficult to distinguish between root inhibition due to Al and that due to lack of P and Fe. P and Fe are typically present in nutrient solutions as P04~ and Fe3+, and it has been well documented that different concentra tions of P and/or Fe can lead to alterations in root growth and architecture (Lynch and Brown, 2001;Williamson et al. 2001;Lopez-Bucio et al, 2003;Ward et al., 2008) The differential root growth responses observed in +A1 treatments between the two nutrient solutions were consistent with Geochem-EZ predictions.
It is generally accepted that the primary rhizotoxic form of Al is Al3+; thus, when a large proportion of Al is precipitated in the Yoshida's solution, it becomes unavailable to affect root growth (Kochian et al., 2004a). The increased root growth inhibition in the modified Magnavaca's Al solution can be attributed to one or a combination of three factors: (1) less of the added Al is precipitated with sulfur and P compared with Yoshida's, leaving more Al in the active (rhizo toxic) form; (2) the citrate in Yoshida's solution added as an Fe chelate preferentially complexes with Al, whereas the modified Magnavaca's uses an HEDTA chelate, which chelates Fe preferentially over Al; and (3) the modified Magnavaca's solution has a lower overall ionic strength than the Yoshida's solution, which increases the activity coefficient (and hence the concentration of thermodynamically relevant ion in solution) of a trivalent ion. Also, as the nutrient solution ionic strength decreases, it prevents the roots from being protected from Al3+, as the Al ions have less competition for negatively charged sites within the root cell wall and root plasma membrane by decreasing the concentrations of other cations that can shield Al3+ from these negative sites.

Importance of Quantifying the Whole Root System in Al
Tolerance Studies Rice seedling root systems are fibrous and can have multiple primary, secondary, and tertiary roots within a few days after germination. There is also significant genetic variation in rice root architecture among vari eties, ecotypes, and/or subpopulations. The pheno typic variation in root growth habit per se among varieties and ecotypes must be taken into consider ation when determining Al tolerance. To date, pub lished results on Al tolerance in maize, sorghum, and rice have all used the growth of the longest root(s) as the assay for Al tolerance (Wu et al., 2000;Nguyen et al., 2001Nguyen et al., , 2002Nguyen et al., , 2003Pineros and Kochian, 2001;Magalhaes et al., 2004;Xue et al., 2006Xue et al., ,2007. Although this approach has proven useful in assessing Al toler ance in other cereals, as demonstrated by the cloning of Al tolerance genes in wheat and sorghum, our results suggest that Al tolerance based on RRG of the longest root is not the best predictor of Al tolerance in rice. Using a set of 225 diverse rice genotypes and the RootReader2D software, we determined that the cor relation between the RRG of the longest root and the RRG of the total root system was weak (r2 = 0.17; Supplemental Fig. SI). Furthermore, in two quanti tative trait locus (QTL) mapping studies where Al tolerance was evaluated based on both assays, we identified some of the same, but also some novel, major effect Al tolerance QTLs that were only detected by TRG-RRG (A. Famoso, K. Zhao, L. Kochian, and S. McCouch, personal communication).
Our observa tions in this study are consistent with studies in maize, wheat, sorghum, soybean (Glycine max), sugarcane (Saccharum officinarum), and tobacco (Nicotiana taba cum), where all have reported severe inhibition of lateral roots in sensitive genotypes (Hetherington et al., 1988;Bushamuka and Zobel, 1998;Silva et al., 2001;Brichkova et al., 2007). We thus conclude that the RRG of the total root system is clearly a much better quantitative indicator of rice Al tolerance than RRG of the longest root, and our newly developed automated image capture and computational deter mination of growth of the total root system makes it feasible to use this parameter in large-scale genetic and physiological studies.

Comparison of Al Tolerance between Cereal Species
In this study, we demonstrated that young rice seedlings (3 d old) tolerate significantly higher con centrations of Al3+ than maize, sorghum, or wheat, consistent with the superior Al tolerance of rice ob served in previous hydroponic Al3+ concentrations and field studies (Foy, 1988;Wu et al., 2000;Nguyen et al., 2001Nguyen et al., , 2002Nguyen et al., , 2003Magalhaes et al., 2004;Sasaki et al., 2004). Yet, we know little about the genes and physiological mechanisms responsible for the high levels of Al tolerance in rice. Other cereals, such as rye (Secale cereale), have been reported to exhibit high levels of Al tolerance (Gallego and Benito, 1997).
However, the Al concentrations in which rye has been screened are four times lower than those at which rice is screened (Gallego and Benito, 1997;Gallego et al., 1998;Li et al, 2000;Collins et al, 2008). This suggests that rice is a very useful model for characterizing the mechanisms conferring high levels of Al tolerance in cereals. Rice also has an abundance of genetic and genomic resources, including several sequenced genomes, high density of genotyping arrays, the avail ability of numerous immortal mapping populations, and extensive germplasm collections (www.gramene.org and www.irri.cgiar.org).
Despite the fact that sorghum has been previously demonstrated to exhibit higher Al tolerance than wheat (Sasaki et al., 2006;Caniato et al., 2007), in this study sorghum and wheat seedlings exhibited similar levels of Al tolerance after 3 d in Al solutions. A likely explanation for this discrepancy is the extended time in Al required to observe the Al tolerance response in sorghum (5-6 d; Magalhaes et al., 2007). Thus, the degree of Al tolerance observed for sorghum in our study is less than would be predicted if the plants were grown in Al solution for up to 6 d (Caniato et al., 2007;Magalhaes et al., 2007).

Rice Must Employ a Novel Al Tolerance Mechanism
Organic acid-mediated root tip Al exclusion has been reported in numerous plant species, explaining most of the phenotypic variation in wheat (Sasaki et al., 2004, 2006), sorghum (Magalhaes et al., 2007, and Arabidopsis (Hoekenga et al., 2003) and a portion of the variation in Al tolerance in maize (Pineros et al., 2005). As a species, rice is two to five times more Al tolerant than wheat, sorghum, and maize, yet this study demonstrated that there is no significant corre lation between Al exclusion from the root apex and root growth in Al. This indicates that the roots of tolerant rice varieties can continue to grow even with significant Al accumulation into the root tip. Thus, rice must employ unique mechanisms of Al tolerance not found in other cereal species.
Unlike Japonica, the more susceptible Indica varieties do exhibit a significant negative correlation between rates of citrate exudation and Al concentrations in the root tip (r2 = 0.47), although this response is not corre lated with Al tolerance (RRG of the total root system).
However, rates of root exudation of malate (r2 = 0.24) and phosphate (r2 = 0.13) showed a weak positive correlation with Al tolerance in Indica varieties, but not Al exclusion. These findings suggest that malate and/ or phosphate exudation may function at least in part to chelate Al3+ within the apoplast of the root tip, rather than exclude Al3+ from entering the root tip. The primary function of root exudates in Al tolerance is believed to be the exclusion of Al from the root apex, but this alone is not responsible for the high levels of Al tolerance in rice. The clearest evidence for this comes from experiments where the wheat Al tolerance gene (ALMT1) was transformed into rice, resulting in Al induced gene expression and enhanced malate exuda tion but no effect on Al tolerance (Sasaki et al., 2004).
However, when the ALMT1 gene was transformed into barley (Hordeum vulgare), an Al-susceptible species, Al tolerance was increased by more than 100%. Multiple rice Al tolerance QTL studies have identified a region on chromosome 1 that is in close proximity to the rice MATE family member that is a homolog of the sorghum Al tolerance gene (SbMATE), leading to the hypothesis that this gene may be underlying these QTLs. SbMATE functions in sorghum Al tolerance as an Al-activated root citrate efflux transporter that excludes Al from the root tip, with differences in Al tolerance across sorghum genotypes directly related to gene expression (r2 = 0.98; et al., 2007).

Magalhaes
Quantitative RT-PCR was  Based on these observations, it appears that rice, as a species, is capable of withstanding significantly higher Al concentrations both in the soil solution and the root tip than other cereals. All but one rice line was more tolerant than the most tolerant maize line (Cateto), yet all but one rice genotype accumulated more Al in the root apex. Some rice genotypes that accumulated 50% to 100% more Al in their root tips were two to five times more Al tolerant than other rice genotypes that accumulated less root tip Al (Supplemental Table   S2). Based on the lack of correlation between rice Al exclusion and Al tolerance, and the relatively high levels of Al accumulation in rice compared with maize, we conclude that rice utilizes one or more novel Al tolerance mechanisms. At this time, we have little information regarding the nature of this new Al tolerance mechanism. Because the majority of the Al in the root tip resides in the apoplast (Kochian, 1995), it is logical to speculate that the root cell wall may play a role in the high level of Al tolerance observed in rice. Recent work from Jian Feng Ma's laboratory supports this speculation based on the map-based cloning of an Al-sensitive knockout mutant locus in rice (Huang et al., 2009). This resulted in the identification of two mutant genes, STAR1 and STAR2, which encode two interacting proteins that form an ATP-binding cassette transporter complex. Transport studies via the STAR1/ STAR2 transporter complex in oocytes showed that the transporter mediates the efflux of UDP-Glc, presum ably into the root apoplast, leading the authors to speculate that cell wall modification may play a role in rice Al tolerance. Furthermore, a study conducted by Yang et al. (2008) provided evidence that cell wall Polysaccharides may be involved in rice Al tolerance. It is known that the growing root tip is the site of Al toxicity (Ryan et al., 1993); however, the mechanism by which Al inhibits root growth in plants is still unclear. Based on observations that Al tolerance in wheat, sorghum, and maize is related to the plant's ability to exclude Al from the growing tip, but not from the mature root regions, researchers have inferred that Al in these species poisons proteins and/or structural components of the root tip that are critical to cell growth, elongation, and/or division. In rice, where there is no significant correlation between Al accumu lation in the root tip and Al tolerance, it appears that the mechanism of toxicity must be categorically dif ferent than in other species. We hypothesize that at some point in evolution, the lineage leading to modern species of Oryza experienced a dramatic shift in its position within the landscape of plant response to Al, demonstrating greatly enhanced ability to grow under high concentrations of Al. If this hypothesis is true, identifying the genes/alleles underlying Al tolerance or susceptibility among rice varieties will provide limited insight into novel plant Al tolerance mecha nisms. To fully understand the novelty of the mecha nism^) of Al toxicity and tolerance found in rice, it will be necessary to undertake very specific physio logical, biochemical, and molecular experiments in a phylogenetic context. Thus, rice appears to hold the key to understanding how and when, over the course of evolution, a lineage of plants experienced a dra matic genetic change that led to enhanced levels of Al tolerance and will provide critical insights that are likely to help move this capability into other species that are critical to human survival.

MATERIALS
AND METHODS

Plant Material
A set of seven to nine genotypes of rice (Oryza sativa), maize (Zea mays),

Plant Growth Conditions
Seeds were germinated in rolled germination paper at 26?C to 30?C for 3 to 5 d under dark conditions. Wheat and sorghum seeds were surface sterilized with 10% bleach and rice with 20% bleach for 15 to 20 min. Maize seeds were treated with a fungicide treatment of Captan400, Trilex, and Allegiance. Upon germination, seedlings were transferred to control (-A1) solutions for 24 h, then 20 uniform seedlings were photographed and root length was quantified using RootReader2D. Subsequently, 10 seedlings were transferred to fresh control solution and 10 seedlings to Al treatment solution. After 3 d in the respective treatments, roots were photographed and measured, mean root growth in control and +A1 treatment was calculated for each genotype, and RRG was determined: RRG = treatment root growth/control root growth. Plants were grown in 9-L tubs with 48 plants per tub, and the plants were supported with eight foam strips (six plants per strip) with a slit cut into the foam to anchor the stem. Aeration was provided in all experiments, except for experiments comparing the nutrient solutions, in which only rice lines were compared. Plant growth chamber conditions for the maize diversity screen and species comparison experiments were 26?C (day)/23?C (night), while the rice diversity screening conditions were 30?C (day)/26?C (night). All exper iments were conducted with 12-h days and a light intensity of 450 mmol photons m~2 s-1.

Nutrient Solutions
The control (-A1) Yoshida's nutrient solution was prepared as described previously (Yoshida et al., 1976), and the pH was adjusted to 4.0 with 1 n NaOH. The Yoshida treatment (+A1) solution was identical to the control but contained 35 pL L_1 (1,297 ^m) A1C13. The control (-Al) modified Magnavaca's nutrient solution was modified from Magnavaca et al. (1987). The treatment (+A1) modified Magnavaca's solution contained 540 /xm A1C13, added after pH adjustment to 7.8 with KOH to prevent Al precipitation, and the final pH was adjusted to 4.0 with 1 n HCl.

ICP-ES Analysis of Nutrient Solutions
ICP-ES elemental profiling was conducted on all elements, except nitrogen, in both nutrient solutions. To determine the available concentration of each element, 1 L of each nutrient solution per treatment was made and analyzed to confirm elemental composition. Four 50-mL samples of each nutrient solution