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 characterization of rtp1 (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 paraquat 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 Arabidopsis 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 sensitive to paraquat and diquat than the wild-type plants. Collectively, our work reveals a potential mechanism for the evolution of herbicide resistance in weeds and provides a promising gene for the manipulation of plant herbicide resistance.

在现代农业中，频繁使用除草剂可能会诱导植物产生抗性，但除草剂抗性的机制在很大程度上仍未被探索。在这里，我们报告了rtp1（对百草枯 1 具有抗性）的特性，这是一种拟南芥突变体，对广泛使用的除草剂百草枯和敌草快具有很强的抗性。rtp1 _突变体是半显性的并且在编码多药和有毒化合物挤出家族蛋白DTX6的基因中携带点突变，导致残基311（G311E）处的甘氨酸变为谷氨酸。具有甘氨酸 311 的野生型 DTX6 在过表达时赋予了较弱的百草枯和敌草快抗性，而将甘氨酸 311 突变为带负电荷的氨基酸（G311E 或 G311D）显着增加了植物对百草枯和敌草快的抗性，而突变为带正电荷的氨基酸酸（G311R 或 G311K）损害了抗性，表明 DTX6 残基 311 的电荷特性对拟南芥的百草枯和敌草快抗性至关重要植物。DTX6 定位于内膜运输系统，并可能经历内体分选以定位于液泡和质膜。用 V-ATPase 抑制剂 ConA 处理降低了rtp1突变体的百草枯抗性。百草枯释放和摄取分析表明，DTX6 参与了百草枯的胞吐作用和液泡隔离。DTX6和DTX5显示为dtx5 dtx6双突变体的功能冗余，但不是dtx6单突变植物对百草枯和敌草快的敏感性高于野生型植物。总的来说，我们的工作揭示了杂草抗除草剂抗性进化的潜在机制，并为控制植物抗除草剂抗性提供了一个有前景的基因。