Loss of AtPDR11, a plasma membrane-localized ABC transporter, confers paraquat tolerance in Arabidopsis thaliana.

SUMMARYParaquat is one of the most widely used herbicides in the world. However, no paraquat transporter has beenisolated in plants. Here, we describe paraquat-tolerant mutant pqt24-1, isolated from an activation-tagginglibrary on the basis of its tolerance to 2 l M paraquat in the seedling stage. Molecular analysis revealed that theT-DNA was inserted in the 13th exon of At1g66950, which encodes AtPDR11, a member of the ATP-bindingcassette transporter superfamily. As a result, AtPDR11 was knocked out in the mutant. Loss-of-functionmutations of AtPDR11 led to reduced paraquat accumulation in plant cells. In addition, the AtPDR11 proteinwasspecificallylocalizedintheplasmalemma,suggestingAtPDR11asapotentialtransporterofparaquat.Thisconclusion was supported by kinetic analysis of paraquat import. Further studies showed that the transcriptlevel of AtPDR11 could be strongly induced by paraquat and other abiotic stresses including H 2 O 2 , indicatingpossible up-regulation of AtPDR11 expression by oxidative stress signaling. Thus, our data suggest thatparaquat is an opportunistic substrate of AtPDR11 and the enhanced paraquat tolerance of pqt24-1 is due toreduced uptake of paraquat into plant cells.Keywords: ABC transporter, oxidative stress, AtPDR11, At1g66950, Paraquat, herbicide.INTRODUCTIONParaquat (N,N¢-dimethyl-4,4¢-bipyridinium dichloride) is oneof the most widely used herbicides in the world. It is quick-acting and non-selective, and kills green plant tissue oncontact by diverting electrons from photosystem I tomolecular oxygen, thus generating toxic oxygen forms(Summers, 1980).The molecular mechanism of paraquat resistance is stillonly partially understood. Several hypotheses have beenproposed to explain paraquat resistance in higher plants,including sequestration of paraquat to the vacuoles andenhanced activity of antioxidative enzymes (Hart and DiTomaso, 1994; Szigeti, 2005). It has been demonstrated thatparaquat transport across the plasma membrane isperformedbyanenergy-dependentandsaturabletransporterthatiscompetitivelyinhibitedbyputrescine(Hartet al.,1992,1993;DiTomasoet al.,1993).Thesestudiessuggestthatplantresistance to paraquat could also be improved by changingthe activity of its transporter(s). However, no paraquat trans-portershavebeenisolatedinhigherplants.ATP-binding cassette (ABC) proteins constitute a largeand ubiquitous superfamily found in all phyla (Verrieret al., 2008). ABC proteins have a characteristic modularstructure consisting of a single or double set of two basicstructural elements: a hydrophobic transmembranedomain usually made up of six membrane-spanninga-helices and a cytosolic nucleotide-binding domain thatis involved in ATP binding (Rea, 2007). Plant ABC proteinscan be assigned to 13 sub-families on the basis of proteinsize (full, half or quarter molecule), orientation (forward orreverse), the presence or absence of idiotypic transmem-brane and/or linker domains, and overall sequence simi-larity (Rea, 2007).Most ABC proteins are integral membrane proteins andact as ATP-driven transporters (ABC transporters) for a verybroad range of substances, including ions (Larsen et al.,2005; Kim et al., 2007; Huang et al., 2009), carbohydrates(Shiet al.,2007;Panzeriet al.,2011),lipids(Awaiet al.,2006;Bird et al., 2007; McFarlane et al., 2010; Song et al., 2010),xenobiotics (van den Brule et al., 2002), auxin (Cho et al.,2007; Wu et al., 2010a) and abscisic acid (Kang et al., 2010;Kuromori et al., 2010, 2011).More than 130 ABC transporter-encoding genes havebeen identified in the Arabidopsis genome, 40% of whichare full-molecule ABC transporters (Schulz and Kolukisaoglu,