Functional Cloning and Characterization of a Plant Efflux Carrier for Multidrug and Heavy Metal Detoxification

TLDR

Multidrug resistance is impaired in the bacterial KAM3 mutant, which cannot grow on norfloxacin, and the Arabidopsis genome encodes a large family of related efflux proteins. The authors employed functional cloning by transforming KAM3 cells with an Arabidopsis cDNA library and selecting for colonies that restored growth on norfloxacin‑containing medium. They identified AtDTX1, a novel 12‑transmembrane protein that localizes to the plasma membrane and acts as an efflux carrier for plant alkaloids, antibiotics, other toxic compounds, and cadmium, thereby conferring multidrug resistance.

Abstract

We have identified a detoxifying efflux carrier from <i>Arabidopsis</i> using a functional cloning strategy. A bacterial mutant, KAM3, is deficient in multidrug resistance and does not survive on medium containing norfloxacin. After transformation of KAM3 cells with an <i>Arabidopsis</i> cDNA library, transformants were selected for restored growth on the toxic medium. One cDNA clone that complemented KAM3 encodes a novel protein with twelve putative transmembrane domains and contains limited sequence homology to a multidrug and toxin efflux carrier from bacteria. We named this <i>Arabidopsis</i> protein AtDTX1 (for<i>Arabidopsis thaliana</i> Detoxification 1). A large gene family of at least 56 members encoding related proteins was identified from the <i>Arabidopsis</i> genome. Further functional analysis of AtDTX1 protein in KAM3 mutant demonstrated that AtDTX1 serves as an efflux carrier for plant-derived alkaloids, antibiotics, and other toxic compounds. Interestingly, AtDTX1 was also capable of detoxifying Cd²⁺, a heavy metal. Further experiments suggest that AtDTX1 is localized in the plasma membrane in plant cells thereby mediating the efflux of plant-derived or exogenous toxic compounds from the cytoplasm.