Aluminum-resistant Arabidopsis mutants that exhibit altered patterns of aluminum accumulation and organic acid release from roots

Abstract

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L. V. Kochian [1998] Plant Physiol 117: 19-27).

Figure 1

Growth of Arabidopsis roots in hydroponic solution culture containing Al. Al-dependent root growth inhibition was compared for wild type (wt), alr-104, and alr-128. Seedlings were grown for 4 d in nutrient solution with no added AlCl₃, pH 4.2, and then transferred to nutrient solution containing varying concentrations of AlCl₃ and grown for an additional 2 d. Relative root growth increment in the presence of Al was expressed as (RL d 6 − RL d 4)/(RL d 4) × 100, where RL = root length. Error bars represent the se (n = 50).

Figure 2

Accumulation of Al and callose in roots after exposure to AlCl₃. A, Patterns of Al-dependent callose accumulation were examined for roots of wild type (wt), alr-104, and alr-128. Roots of 4-d-old seedlings were exposed to nutrient solution containing 75 μm AlCl₃ for 24 h, except for the first panel, in which no Al was added (−Al). Seedlings were then fixed, stained with 0.1% aniline blue, pH 9.0, and observed using epifluorescence microscopy. The top row represents bright-field images of the treated roots, and the bottom row are fluorescence images indicating callose accumulation. B, Patterns of Al accumulation by roots of wild type, alr-104, and alr-128 were observed using the Al-indicator dye morin. Five-day-old seedlings grown in nutrient solution without Al were exposed to nutrient solution containing 25 μm AlCl₃ for 1 h, except for the first panel, in which no Al was added (−Al). Roots were then stained with 100 μm morin, a stain that fluoresces when complexed with Al.

Figure 3

Organic acid exudation by roots of wild-type (wt) seedlings and alr mutants in the presence or absence of Al. One-hundred seedlings of wild type (Col-0), alr-104, alr-108, alr-128, and alr-131 were grown for 5 d in nutrient solution and then transferred to a solution consisting of 100 μm CaCl₂, pH 4.2, containing either 0 or 2.7 μm Al³⁺ for 24 h. Root exudates were collected, free Cl⁻ was removed, and samples were analyzed using an ion-chromatography system. Each panel represents the total picomoles of organic acid released by 100 roots during the 24-h period in the absence (−Al) or presence (+Al) of added Al. Error bars represent the se (n = 6).