Plant Physiol. (1998) 117: 19-27

Aluminum Resistance in the Arabidopsis Mutant alr-104 Is Caused by an Aluminum-Induced Increase in Rhizosphere pH¹

Jörg Degenhardt, Paul B. Larsen, Stephen H. Howell, and Leon V. Kochian^*

United States Plant, Soil, and Nutrition Laboratory, United States Department of Agriculture-Agriculture Research Station, Tower Road, Cornell University, Ithaca, New York 14853 (J.D., P.B.L., L.V.K.); and Boyce Thompson Institute, Tower Road, Cornell University, Ithaca, New York 14853 (S.H.H.)

	ABSTRACT

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A mechanism that confers increased Al resistance in the Arabidopsis thaliana mutant alr-104 was investigated. A modified vibrating microelectrode system was used to measure H⁺ fluxes generated along the surface of small Arabidopsis roots. In the absence of Al, no differences in root H⁺ fluxes between wild type and alr-104 were detected. However, Al exposure induced a 2-fold increase in net H⁺ influx in alr-104 localized to the root tip. The increased flux raised the root surface pH of alr-104 by 0.15 unit. A root growth assay was used to assess the Al resistance of alr-104 and wild type in a strongly pH-buffered nutrient solution. Increasing the nutrient solution pH from 4.4 to 4.5 significantly increased Al resistance in wild type, which is consistent with the idea that the increased net H⁺ influx can account for greater Al resistance in alr-104. Differences in Al resistance between wild type and alr-104 disappeared when roots were grown in pH-buffered medium, suggesting that Al resistance in alr-104 is mediated only by pH changes in the rhizosphere. This mutant provides the first evidence, to our knowledge, for an Al-resistance mechanism based on an Al-induced increase in root surface pH.

	INTRODUCTION

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Al is the most abundant metal in the earth's crust and occurs in a number of different forms in the soil. In neutral and basic soils, Al is mostly found as oxide or silicate precipitates that are not toxic to plants. However, in very acidic soils (pH < 5.0), Al speciates to a soluble octahedral hexahydrate form, commonly called Al³⁺, which is believed to be the primary phytotoxic Al species (Kochian, 1995). The initial and most dramatic symptom of Al toxicity is inhibition of root growth, which results in a reduced and damaged root system and can lead to mineral deficiencies and water stress. Al toxicity is a primary factor limiting agronomic production in acidic soils, which constitute more than 30% of the world's arable land (Von Uexkull and Mutert, 1995). The root apex is the primary target of Al toxicity, and the reduction in root growth is detectable within minutes after Al addition (Ryan et al., 1993; Jones and Kochian, 1995).

Cultivar or variety differences in Al resistance have been reported in a number of crop plants (for review, see Carver and Ownby, 1995). Two categories of Al-resistance mechanisms have been proposed: tolerance to higher concentrations of Al in the root symplast, and the ability to exclude Al from the root apex (Taylor, 1991; Delhaize and Ryan, 1995; Kochian, 1995). Whereas little is known about mechanisms of symplastic tolerance (Aniol, 1984), an Al-exclusion mechanism has recently been described. Delhaize et al. (1993a, 1993b) demonstrated in isogenic lines of wheat that the presence of Al induced the release of more malate from the root apex in the Al-tolerant genotypes. Like several other organic acids, malate chelates Al³⁺ in the rhizosphere and prevents Al uptake into the root. In these studies, it was shown that resistance segregated as a single dominant locus termed alt1. Malate release was subsequently shown to correlate with Al resistance in a number of other wheat cultivars (Ryan et al., 1995). A similar exclusion mechanism has been observed in maize, in which an Al-induced release of citrate at the root apex was reported (Pellet et al., 1994).

As early as the mid 1960s, Foy et al. (1965) proposed an Al-exclusion mechanism that involves increases in rhizosphere pH. Alkalinization of the rhizosphere would reduce the concentration of Al³⁺ in favor of less-toxic Al species such as Al hydroxides and Al phosphates (Martell and Motekaitis, 1989). There have been many reports of a general correlation between Al resistance and transient increases in growth solution pH for several species, including wheat (Foy et al., 1967, 1974; Mugwira et al., 1976, 1978; Mugwira and Elgawahry, 1979; Foy and Fleming, 1982; Fleming, 1983; Dodge and Hiatt, 1992), barley (Foy et al., 1967), pea (Klimashevsky and Bernadskaya, 1973), rye (Mugwira et al., 1976, 1978), and triticale (Mugwira et al., 1976, 1978; Mugwira and Patel, 1977), but to date, there have been no direct demonstrations of this Al-resistance mechanism. In most of these reports, it was not clear whether the pH differences were the cause of Al resistance or if they were the result of Al-induced inhibition of root function in the sensitive cultivars.

All of these studies were based on pH measurements of the bulk solution, which were shown to be problematic in two respects. First, the N source of the growth medium can have a significant impact on rhizosphere pH, since uptake of NO₃ leads to an alkalinization of the medium, whereas NH₄⁺ uptake can cause rhizosphere acidification (for review, see Taylor, 1988). Therefore, the ratio of NO₃ to NH₃ in the growth medium of each experiment can have a substantial effect on the pH of the growth solution (Taylor, 1991). Second, bulk-solution measurements reflect pH changes associated with the whole root and not specifically the root tip, which is the primary site of Al toxicity. For this reason, Miyasaka et al. (1989) used pH microelectrodes to map the surface pH along wheat roots and showed that the Al-resistant wheat cv Atlas maintained a slightly higher pH (approximately 0.15 pH unit) at the root tip, but not the mature parts of the root, compared with the Al-sensitive cv Scout. In these experiments, it was not determined whether the differences in rhizosphere pH at the apex led to Al resistance, or whether they merely reflected differences in root function after the onset of Al toxicity in cv Scout.

Recently, we have taken a molecular-genetic approach to gain a better understanding of Al toxicity and resistance by isolating Al-sensitive and Al-tolerant mutants in Arabidopsis thaliana. We previously reported on Al-sensitive Arabidopsis mutants. This trait was described by eight different complementation groups (Larsen et al., 1996). In the companion paper (Larsen et al., 1998), we describe a family of five Al-resistant mutants that map to two different loci on the Arabidopsis genome. All of these alr mutants exclude Al from the root apex to a greater degree than wild type. Four of the mutants mapped to the same location on chromosome 1 and used an Al-exclusion mechanism associated with the increased release of malate and citrate, whereas the other mutant mapped to chromosome 4.

We describe the Al-resistant chromosome 4 mutant alr-104, which does not exhibit enhanced root organic acid release. In this study, we investigated whether increased Al resistance in alr-104 was caused by an increased rhizosphere pH around the root apex. For these studies, we used a vibrating H⁺ microelectrode system that allowed us to measure root ion fluxes with a very high degree of spatial and temporal resolution (Kochian et al., 1992; Smith et al., 1994). We modified this technique to measure very small roots such as those found on Arabidopsis seedlings, and devised a method to quantify root H⁺ fluxes in a gel film containing a complex nutrient solution. alr-104 showed an Al-inducible increased H⁺ influx at the root tip, which resulted in a higher rhizosphere pH in this region (compared with wild type). The increase in apical rhizosphere pH in alr-104 accounts for greater Al tolerance in this mutant. This report provides the first direct evidence to our knowledge for an Al-tolerance mechanism based on a modification of apical rhizosphere pH.

	MATERIALS AND METHODS

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The genetic and general physiologic characteristics of Al resistance in wild type and alr-104 and alr-128 mutants of Arabidopsis thaliana Heyn. ecotype Columbia (Col-0) used in this study are described in the companion paper (Larsen et al., 1998). Arabidopsis seedlings were grown on a 2-mm layer of a gel (0.15% gellan gum; Gell-Gro, ICN) covering a microscope slide; the gel contained a nutrient solution as described by Larsen et al. (1996) (2 mm KNO₃, 0.1 mm KH₂PO₄, 2 mm MgSO₄, 0.25 mm [NH₄]₂SO₄, 1 mm Ca[NO₃]₂, 1 mm CaSO₄, 1 mm K₂SO₄, 1 µm MnSO₄, 5 µm H₃BO₃, 0.05 µm CuSO₄, 0.2 µm ZnSO₄, 0.02 µm NaMoO₄, 0.1 µm CaCl₂, 0.001 µm CoCl₂, and 1% Suc, pH adjusted to 4.2). The gel was bounded by a plastic support glued to the slide. Four to six seeds of wild type and alr mutants (alr-104 and alr-128) were sown onto the gel layer, and the slide was placed at a 30^o angle in a growth chamber with a 16-h day/8-h night cycle for 4 to 5 d.

During incubation the lower end of the slides was submerged in nutrient solution of the same composition. The liquid medium was changed daily to prevent depletion of nutrients in the gel. Twelve hours before an experiment, the slides were oriented horizontally and the medium in the gel layer was equilibrated with 200 mL of nutrient solution. For the Al treatments the nutrient solution also contained 300 µm AlCl₃. The pH of Al-containing solutions was adjusted before the addition of AlCl₃, as described in detail by Larsen et al. (1996). When buffered nutrient solution was used, 10 mm Homo-Pipes (Research Organics, Cleveland, OH) was added before adjustment of pH and addition of AlCl₃.

Root-Growth Measurements

Measurement of Root H⁺ Fluxes with a Vibrating H⁺ Microelectrode

View larger version (39K): [in this window] [in a new window]   Figure 1. Experimental setup for measurement of H+ fluxes around Arabidopsis roots. The roots were growing within a thin layer of gel equilibrated with nutrient solution. The microelectrode was mounted vertically and perpendicular to the root and vibrated along its long axis between 5 and 35 µm from the root surface. The gel was held on a microscope slide within a chamber containing the appropriate nutrient solution. The solution was constantly exchanged using a peristaltic pump.

The efficiency of the vibrating H⁺ microelectrode to detect a H⁺ gradient within the gel was determined by following a procedure modified from Smith et al. (1994). An artificial H⁺ gradient was set up and measured with the vibrating H⁺ microelectrode, and the measured gradient was compared with the theoretical gradient values determined from diffusion equations. The following describes the modifications that were made to account for the buffering effect of the gel and the nutrient solution. A gel consisting of 0.15% gellan gum in nutrient solution was adjusted to pH 4.0 and introduced into a micropipette (tip diameter, approximately 5 µm). The pipette was mounted onto a microscope slide and embedded in a 2-mm layer of the same gel at pH 6.0. The ionic strength of the gel was adjusted by addition of 99 µm KOH to provide a cation for counterdiffusion and to minimize osmotic water flow between the two phases. After 4 h a stable H⁺-diffusion gradient developed around the micropipette tip, which served as an ion source (data not shown). The H⁺ gradient was measured with a pH microelectrode and compared with the expected diffusion gradient derived from the appropriate diffusion equation (Jaffe and Levy, 1987; Kühtreiber and Jaffe, 1990; Smith et al., 1994).

Measurement of Root Surface pH with a H⁺ Microelectrode

5

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	RESULTS

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Efficiency of the H⁺-Selective Vibrating Microelectrode

View larger version (17K): [in this window] [in a new window]   Figure 2. Efficiency of the vibrating H+-selective microelectrode. The electrode was vibrated at different positions from an H+ source (a micropipette containing gelled nutrient solution at pH 4.0) placed in a pH 6.0 nutrient solution. At each point, the potential difference (representing the H+ gradient between the end points of the vibration) was determined (). Comparison of these data with the theoretical values for the H+ gradient calculated according to Fick's law () allowed for determination of the efficiency of the system.

Al Effects on H⁺ Fluxes at the Arabidopsis Root Tip

View larger version (47K): [in this window] [in a new window]   Figure 3. Vector diagram of net H+ fluxes along an Arabidopsis root tip. Orthogonal flux measurements (shown as thin vectors for one point 600 µm from the root tip) were taken at several positions at radial distances of 20 and 50 µm from the root surface. The length of the vector represents the flux magnitude. Addition of the orthogonal vector components determines the magnitude and direction of the net H+ current (bold vectors). The root thickness is not drawn to scale with the flux measurements.

To compare root H⁺ fluxes between wild type and alr-104, we focused our flux measurements on the region of maximal influx (between 0 and 500 µm from the root tip). In this region the H⁺ current is primarily perpendicular to the root surface, which allowed us to measure the fluxes with a microelectrode vibrating at a 90^o angle with respect to the root. Data from 8 to 12 roots were averaged to account for the differences between individual roots. In nutrient solution without Al, there was no difference in maximal H⁺ influx between alr-104 and wild type (approximately 120 pmol cm² s¹) (Fig. 4A). Also, at regions adjacent to the point of maximum H⁺ influx, no substantial flux differences were found between mutant and wild type.

View larger version (17K): [in this window] [in a new window]   Figure 4. Influence of Al exposure on net H+ influx along Arabidopsis root tips. H+ influx was measured along wild-type () and alr-104 () roots in the absence (A) and presence (B) of 300 µm AlCl3. C, Root H+ influx along wild-type () and alr-128 () roots in the presence of 300 µm AlCl3. Average net influx and se for 8 to 12 roots are shown.

H⁺-flux measurements were repeated in the presence of 300 µm AlCl₃. Because the gel matrix binds Al³⁺, the Al³⁺ activity at 300 µm AlCl₃ in the gel-layer system is comparable to the Al³⁺ activity in liquid nutrient solution containing 30 µm AlCl₃, based on a bioassay of Al³⁺ activity (inhibition of root growth in wild-type seedlings; data not shown). After 12 h of incubation in Al-containing nutrient solution, the net H⁺ influx in alr-104 roots had increased to 180 pmol cm² s¹ at the point of maximal H⁺ flux, whereas the influx in wild-type roots remained at approximately 100 pmol cm² s¹ (Fig. 4B). This approximately 80% increase in H⁺ influx can be seen consistently along the first 500 µm of the root. Seedlings of the Al-resistant mutant alr-128, which is one of the four alr mutants mapping to the same locus on chromosome 1 (alr-104 maps to chromosome 4), were compared with wild-type seedlings in a separate experiment. These seedlings, which released increased amounts of Al-binding organic acids, did not have a detectable increase in H⁺ influx in the presence of Al (Fig. 4C).

Al Effects on Rhizosphere pH at the Arabidopsis Root Tip

View larger version (20K): [in this window] [in a new window]   Figure 5. Influence of Al exposure on rhizosphere pH along the surface of Arabidopsis root tips. The root surface pH was measured along roots of wild-type () and alr-104 () roots in the absence (A) and presence (B) of 300 µm AlCl3. Average root surface pH and se for 8 to 12 roots are shown.

Al Resistance in alr-104 Is Dependent on Rhizosphere pH Alteration

The root growth rate of the seedlings was determined in Al-containing medium that was either unbuffered or buffered at pH 4.4 or 4.5, respectively (Fig. 6). As previously demonstrated, the growth rate of alr-104 in unbuffered medium surpassed that of wild type in the presence of Al, which was consistent with an increased resistance to Al. When the pH of the medium was raised by 0.1 pH unit (from 4.4 to 4.5), the growth rate of both wild-type and alr-104 roots in the presence of Al was nearly doubled. Therefore, an increase in rhizosphere pH of 0.1 unit conferred a significant increase in Al resistance.

View larger version (25K): [in this window] [in a new window]   Figure 6. Influence of Al and pH on the root growth rate of wild-type (WT), alr-104, and alr-128 seedlings grown in unbuffered and buffered medium. Seedlings were grown in a thin gel layer equilibrated with nutrient solution containing 300 µm AlCl3 with or without 10 mm Homo-Pipes adjusted to either pH 4.4 or 4.5. The average root growth rate and se of 12 seedlings after 12 h of incubation in the appropriate medium are shown.

Included in this experiment was another Al-resistant mutant, alr-128. This mutant displays a degree of Al resistance similar to alr-104 and was shown to release increased amounts of organic acids into the rhizosphere (Larsen et al., 1998). In buffered conditions that abolished Al resistance in alr-104, the Al resistance of alr-128 was maintained (Fig. 6). This suggests that Al resistance in alr-128 is independent of rhizosphere pH alteration, whereas Al resistance in alr-104 appears to involve a pH-mediated mechanism in which the roots of the mutant alkalinize the rhizosphere. The increased rhizosphere pH around the root apex of alr-104 should drive the speciation of Al toward less-toxic Al species, which would reduce Al toxicity. The similar growth rate of alr-104 and wild type in buffered conditions also demonstrates that the increased Al resistance of alr-104 is attributable solely to a mechanism based on rhizosphere pH alteration.

The root-growth studies were repeated without the addition of Al to determine whether the difference in acidic stress between pH 4.4 and 4.5 had an effect on the root growth rate. The results shown in Figure 7 suggest that this small pH difference in the nutrient solution does not have a significant influence on the root growth rate by itself.

View larger version (22K): [in this window] [in a new window]   Figure 7. Influence of rhizosphere pH on Arabidopsis root growth in pH-buffered medium. Wild-type (WT) and alr-104 seedlings were grown in a thin gel layer equilibrated with medium containing 10 mm Homo-Pipes at either pH 4.4 or 4.5. The average root growth rate and se of 12 seedlings after 12 h of incubation in the appropriate medium are shown.

	DISCUSSION

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H⁺-Flux Measurements along the Arabidopsis Root Tip

It was necessary to modify the extracellular vibrating microelectrode technique to study ion fluxes in very small Arabidopsis roots embedded in a low-density gel matrix. To minimize disturbance of the gel, the electrode was vibrated perpendicular to the root and along the long axis of the microelectrode. H⁺ influx was measured along the root tip, which reached approximately 120 pmol cm² s¹ at the point of maximum influx. The two-dimensional mapping of the H⁺ influx indicated that most influx is localized within a rather small region in the root elongation zone, approximately four to eight epidermal root cells in length. The spatial pattern of net H⁺ current at the Arabidopsis root tip was similar to that of total ionic current (deduced from measurements of extracellular electric fields) measured in other species, including barley (Weisenseel et al., 1979), Lepidum sativum (Behrens et al., 1982), Trifolium repens (Miller et al., 1986), and tobacco (Miller et al., 1988).

In the Al-resistant Arabidopsis mutant alr-104, the increased H⁺ influx was induced by Al. Despite the change in flux intensity, no alteration in the spatial pattern of H⁺ influx was observed. Therefore, it is likely that the ion-transport processes in alr-104 are the same as those in the wild-type root tip, but are stronger in the presence of Al. However, the nature of the increased net H⁺ influx in alr-104 has yet to be determined. Because we are measuring a net H⁺ uptake into the root apex, a stimulation of this flux could be caused by a stimulation of unidirectional H⁺ influx or a decrease in H⁺ efflux. A decreased H⁺ efflux presumably would be caused by Al interaction with the plasma membrane H⁺-ATPase. Mutations in the H⁺-ATPase regulatory mechanism in alr-104 might allow a direct or indirect effect by Al.

The apparent increased H⁺ influx in alr-104 could also be attributable to the efflux of solutes that are protonated when they are released into the acidic rhizosphere. In terms of organic and inorganic acids, only pyruvate was found to be released at a higher rate in alr-104 than in wild type (see Larsen et al., 1998). However, increased release of pyruvate was constitutive and not induced by Al in alr-104. Furthermore, pyruvate is not protonated significantly when it is transported from a neutral cytoplasm to a rhizosphere with a pH around 4.0, and is therefore unlikely to have an effect on rhizosphere pH.

The increased net H⁺ influx in alr-104 could also be caused by an alteration in the H⁺-coupled transport system (H⁺ symport or antiport). One possible change is the uptake of N (as NO₃ or NH₄⁺), which is closely coupled to H⁺ transport. It has been shown from bulk-solution pH measurements that the uptake of NO₃ and NH₄⁺ is often associated with pH changes (for review, see Miller and Smith, 1996). In many of these studies, interpretation is often complicated because NO₃ and/or NH₄⁺ were eventually depleted in the hydroponic medium, causing dramatic changes in the pH of the bulk solution. In the experiments described here, roots of Arabidopsis plants were equilibrated with a large volume of nutrient solution, providing a constant NH₄⁺/NO₃ ratio. It is possible, however, that an Al-induced difference in NH₄⁺ or NO₃ uptake in alr-104 is the cause of the altered rhizosphere pH. That is, it is possible that in alr-104, Al exposure stimulates NO₃ influx or inhibits NH₄⁺ uptake, which in turn could increase rhizosphere pH.

Because of the complex nutrient requirements for maintaining Arabidopsis root growth, it will be difficult to identify an ion-transport process associated with Al resistance in alr-104. The mutation in alr-104 is a single mutation on chromosome 4 that was isolated from a population of ethyl methylsulfonate-mutagenized seeds. It is therefore likely to be a point mutation that confers a loss of function, although the inducibility of the H⁺ influx with Al suggests a gain of function. Another example of Al inducibility of resistance comes from the work of Delhaize and colleagues (1993a, 1993b) with wheat, in which the Al-resistance locus Alt1 was shown to confer an Al-induced Al resistance based on organic acid release. The cloning of the alr-104 gene in Arabidopsis is currently being pursued in our laboratories and, when successful, could shed some light on the mechanism of Al-induced rhizosphere pH increase.

pH Differences at the Root Surface Are Responsible for Al Resistance in alr-104

Differences of 0.1 to 0.2 pH unit were also reported in bulk-solution measurements with several other wheat cultivars (Taylor and Foy, 1985a, 1985b). In all of these studies, it was not shown whether the small pH differences conferred increased Al resistance. It is important to note that the magnitude of the pH differences measured at the root surface might be even greater at the plasma membrane surface within the cell wall. Because the pH gradient is generated at the plasma membrane of root cells, the electrically charged cell wall might act as a barrier for ion release. Thus, it is possible that the pH difference between the plasma membrane surface and the external solution is greater than that measured in the unstirred layer adjacent to the root.

To determine whether Al resistance in alr-104 is indeed caused by a small (0.1-0.2 pH unit) rhizosphere alkalinization, we performed root-growth studies in nutrient solution in which the unstirred layer adjacent to the root was pH clamped with high concentrations of Homo-Pipes. The extent of buffering was sufficient to avoid the formation of pH gradients at the root surface, but we do not know how far the buffering extends into the root apoplast. Homo-Pipes is a biological buffer with a pK of 4.29 and we have found that it does not bind Al³⁺ (data not shown).

From these root-growth studies, two important pieces of information were obtained. First, when we abolished root-surface pH gradients with Homo-Pipes, Al resistance in alr-104 disappeared, suggesting that the increased Al resistance of alr-104 is solely the result of a mechanism based on rhizosphere pH alteration. Second, when rhizosphere pH was buffered at pH 4.5, a significant increase in Al resistance was observed compared with findings from Al-toxicity studies conducted in a solution buffered at pH 4.4. These results indicate that in alr-104, the small Al-induced increases in rhizosphere pH (0.1-0.2 unit) are sufficient to account for the observed Al resistance. In alr-128, in which we have demonstrated a correlation between Al resistance, Al exclusion from the root tip, and increased release of malate and citrate (Larsen et al., 1998), we were able to show that Al resistance does not involve changes in rhizosphere pH.

The increased Al resistance of the Arabidopsis mutant alr-104 appears to be caused by an Al-induced alkalinization of the rhizosphere. This increased alkalinization is localized to the root tip, which is the site of Al toxicity. Although this mechanism has often been proposed in the literature, these findings are the first strong evidence to our knowledge for an Al-resistance mechanism involving a rhizosphere pH barrier in higher plants. This mechanism of Al resistance in alr-104 is different from previously described Al-resistance mechanisms, which were based on the exudation of Al-chelating organic acids. In future studies we will investigate the role of ion-transport processes in the Al-induced alkalinization of the rhizosphere. We are also focusing on the isolation of the alr-104 gene by map-based cloning to better understand this mechanism of Al tolerance on a molecular level.

	FOOTNOTES

   Received May 27, 1997; accepted November 20, 1997.

	ACKNOWLEDGMENTS

The authors thank Jon E. Shaff for generous help with technical aspects of the vibrating probe. Dr. Peter J.S. Smith is acknowledged for advice regarding the efficiency measurements.

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