Supplementary notes

In a previous study in which we examined the genome-wide response of Arabidopsis to the xenobiotic compound trinitrotoluene (TNT) via cDNA microarrays, we found that Atwbc19 (At3g55130) was among upregulated genes. Given the role of ABC transporters in cellular detoxification we sought to further characterize this gene. Initial characterization of mutants revealed that root growth of Atwbc19 knockout mutants on media containing kanamycin was much slower than that of other ABC mutants. We specifically compared the kanamycin resistance level of mutants of Atwbc19 (SALK_107731) and two close homologues Atwbc18 (SALK_100187) and Atwbc16 (SALK_119868) that share over 76% similarity in amino acid sequence with Atwbc19 (see Supplementary Fig. 5). The mutants were T-DNA insertional mutants transformed with plasmid pROK2 containing an nptII gene, under the control of the nopaline synthase (nos) promoter. Therefore, we also compared their NPTII protein synthesis levels by ELISA assays. On media without kanamycin, there was no significant difference in root growth between the 3 lines. On media with kanamycin, root growth of line SALK_107731 was not different than that of SALK_119868 (0.31+0.04 and 0.30+0.03 cm, respectively) and clearly less than that of SALK_100187 (1.82+0.25 cm) as illustrated in Supplementary Figure 1. However, the amount of NPTII protein synthesized was more than 1000-fold higher in the SALK_107731 mutant compared to the other two mutants. Thus, these results strongly suggested that Atwbc19 had a role in kanamycin resistance while its two close homologues did not. The estimated level of resistance conferred by Atwbc19, under the control of its native promoter is equivalent to 1 μg NPTII per mg fresh weight, an