Aluminum resistance in the Arabidopsis mutant alr-104 is caused by an aluminum-induced increase in rhizosphere pH

Abstract

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.

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.

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.

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.

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.

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.

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.

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.