Determination of Paraquat in Arabidopsis Tissues and Protoplasts by UHPLC-MS/MS

Background

Paraquat (PQ, 1,1-dimethyl-4,4-bipyridinium dichloride, or commonly called methyl viologen) is a nonselective herbicide that has been widely used in many countries for over half a century (Farrington et al., 1973; Fussell et al., 2011). It acts on chloroplasts and kills plants in several hours. PQ accepts electrons from photosystem I and transfers them to molecular oxygen, thus generating highly toxic reactive oxygen species (ROS) and causing severe oxidative stress in green tissues (Farrington et al., 1973; Fussell et al., 2011;Hawkes, 2014). In the past decades, a lot of PQ-resistant weeds have been found in the field. Studying plant resistance mechanisms to PQ not only helps to understand how weeds evolve herbicide resistance, but also promotes the understanding of plant antioxidant stress mechanisms. In Arabidopsis, a number of PQ-resistant mutants have been reported, including rmv1 (resistant of methyl viologen 1) (Fujita et al., 2012), par1 (paraquat-resistant 1) (Li et al., 2013), atpdr11 (pleiotropic drug resistance 11) (Xi et al., 2012), rcd1 (radical-induced cell death 1) (Fujibe et al., 2004), pst1 (photoautotrophic salt tolerance 1) (Tsugane et al., 1999), par2 (paraquat-resistant 2) (Chen et al., 2009), pqt3 (paraquat tolerance 3) (Luo et al., 2016), dtx6 (Lv et al., 2021; Xia et al., 2021), and atpqt11 (paraquat tolerance 11) (Huang et al., 2022), and the mutant genes responsible for this resistance have been identified. The resistance mechanisms revealed so far have been classified into three types: impaired uptake and transport of PQ, enhanced sequestration of PQ, and enhanced ROS scavenging ability (Nazish et al., 2022).

We identified a new PQ-resistant mutant rtp1 (resistant to paraquat 1), in which a MATE (multidrug and toxic compound extrusion) family protein DTX6 is disrupted (Lv et al., 2021). MATE family proteins are a type of transporters with distinct substrate specificity. To elucidate the resistance mechanism of DTX6, it is important to measure the PQ level in cells after treatment. The isotopic labeling method has been used to track exogenous molecules in plants; however, radiolabeled PQ is becoming increasingly difficult to obtain, and the manipulation of radioisotopes needs very complex approval and inspection procedures. Therefore, we established a nonradioactive method to determine PQ content in cells by combining ultra-high-performance liquid chromatography (UHPLC) and mass spectrometry (MS) systems. The UHPLC-MS/MS can determine PQ by using the multiple reaction monitoring mode of triple-quadrupole mass spectrometry. It has high selectivity and sensitivity and can achieve a resolution of 10^-15 mol/L on column. Compared with traditional radiolabeling, the UHPLC-MS/MS method is more environmentally compatible and easier to implement.