Changing Gly311 to an acidic amino acid in the MATE family protein DTX6 enhances Arabidopsis resistance to the dihydropyridine herbicides

In modern agriculture,frequent application of herbicides may induce the evolution of resistance in plants,but the mechanisms underlying herbicide resistance remain largely unexplored.Here,we report the char-acterization of rtp 1(resistant to paraquat 1),an Arabidopsis mutant showing strong resistance to the widely used herbicides paraquat and diquat.The rtp1 mutant is semi-dominant and carries a point mutation in the gene encoding the multidrug and toxic compound extrusion family protein DTX6,leading to the change of glycine to glutamic acid at residue 311(G311E).The wild-type DTX6 with glycine 311 conferred weak para-quat and diquat resistance when overexpressed,while mutation of glycine 311 to a negatively charged amino acid(G311E or G311D)markedly increased the paraquat and diquat resistance of plants,whereas mutation to a positively charged amino acid(G311R or G311K)compromised the resistance,suggesting that the charge property of residue 311 of DTX6 is critical for the paraquat and diquat resistance of Arabi-dopsis plants.DTX6 is localized in the endomembrane trafficking system and may undergo the endosomal sorting to localize to the vacuole and plasma membrane.Treatment with the V-ATPase inhibitor ConA reduced the paraquat resistance of the rtp1 mutant.Paraquat release and uptake assays demonstrated that DTX6 is involved in both exocytosis and vacuolar sequestration of paraquat.DTX6 and DTX5 show functional redundancy as the dtx5 dtx6 double mutant but not the dtx6 single mutant plants were more sen-sitive to paraquat and diquat than the wild-type plants.Collectively,our work reveals a potential mecha-nism for the evolution of herbicide resistance in weeds and provides a promising gene for the manipulation of plant herbicide resistance.