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

Abstract

We have identified a detoxifying efflux carrier from Arabidopsis 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 Arabidopsis 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 Arabidopsis protein AtDTX1 (for Arabidopsis thaliana Detoxification 1). A large gene family of at least 56 members encoding related proteins was identified from the Arabidopsis 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 Cd(2+), 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.

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7

“…This strain is very useful for the analysis of various multidrug efflux pumps from micro-organisms (Morita et al, 1998;Mine et al, 1999;Masaoka et al, 2000) and plants (Li et al, 2002). E. coli KAM3 containing the mexXY or mexXY-oprA genes of PA7 showed increased resistance to a number of antibiotics, including erythromycin, ciprofloxacin, cefpirome, tetracycline and gentamicin (Table 2) to the same extent as E. coli KAM3 containing the mexXY genes of P. aeruginosa PAO1 (data not shown).…”

A molecular pathway for CO2 response in Arabidopsis guard cells

“…The mutant harboured a single T-DNA insertion in the exon of the At4g22790 gene, encoding a member of the MATE family ( Supplementary Fig. 1c) 41,42 . The RHC1 gene encodes a predicted protein of 491 amino acids with 12 predicted transmembrane helices and hydrophilic amino-and carboxy-terminal tails (36 and 34 amino acids, respectively) ( Supplementary Fig.…”

“…Taken together, we propose that RHC1 functions as a HCO 3 À -sensing component in the CO 2 signalling pathway in Arabidopsis guard cells. Because RHC1 is a member of the MATEtype transporter family 41,42 , it will be important to determine whether RHC1 has transporter activity and whether this activity is related to CO 2 sensing. We noted that stomatal movements in rhc1 mutant plants are not completely insensitive to changes in CO 2 concentration (Fig.…”

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

“…The nature of the substances transported by ALF5 and EDS5 remains unknown. Functional expression of an Arabidopsis MATE protein AtDTX1 in a bacterial mutant deficient in multidrug resistance indicates that AtDTX1 drives the efflux of positively charged plant alkaloids, antibiotics, Cd2 + , and other toxic compounds from the cytoplasm, possibly as a protondependent antiporter (Li et al, 2002).…”