AtPQT11, a P450 enzyme, detoxifies paraquat via N-demethylation

Paraquat is one of the most widely used nonselective herbicides in agriculture. Due to its wide use, paraquat resistant weeds have emerged and is becoming a potential threat to agriculture. The molecular mechanisms of paraquat resistance in weeds remain largely unknown. Physiological studies indicated that the impaired translocation of paraquat and enhanced antioxidation could improve paraquat resistance in plants. However, the detoxification of paraquat via active metabolism by plants has not been reported to date. Here we report that an activated expression of At1g01600 encoding the P450 protein CYP86A4 confers paraquat resistance as revealed by the gain-of-function mutant paraquat tolerance 11D (pqt11D), in which a T-DNA with four 35S enhancers was inserted at 1646 bp upstream the ATG of At1g01600. The paraquat resistance can be recapitulated in Arabidopsis wild type by overexpressing AtPQT11 (At1g01600), while its knockout mutant is hypersensitive to paraquat. Moreover, AtPQT11 also confers paraquat resistance in E. coli when overexpressed. We further demonstrate that AtPQT11 has P450 enzyme activity that converts paraquat to N-demethyl paraquat nontoxic to Arabidopsis, therefore detoxifying paraquat in plants. Taken together, our results unequivocally demonstrate that AtPQT11/ CYP86A4 detoxifies paraquat via active metabolism, thus revealing a novel molecular mechanism of paraquat resistance in plants and providing a means potentially enabling crops to resist paraquat.

A Paraquat-Inducible Protein B-like (pqiB) Gene From Chromobacterium Violaceum Confers Tolerance to Paraquat in Transgenic Tobacco

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is a contact non-selective herbicide, widely used in agriculture in several countries. Proteins induced by paraquat have been the subject of great interest because of the possibility of conferring herbicide resistance when introduced into crops. In this work, we analyzed a paraquat-inducible protein B-like ( cvpqiB ) gene, isolated from Chromobacterium violaceum, in conferring tolerance to paraquat in transgenic tobacco. A DNA fragment containing the pqiB coding sequence was isolated from the C. violaceum ATCC12472 genome, inserted into the pCAMBIA1390 vector, under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and used in Agrobacterium -mediated transformation of Nicotiana tabacum cv. Havana. Analysis of the regenerants revealed the incorporation of cvpqiB into the tobacco genome and its transmission in a Mendelian fashion to the progeny of transgenic plants. Sensitivity assays using tobacco leaves demonstrated that the transgenic plants were tolerant to concentrations up to 50 µM paraquat, whereas the wild-type (WT) plants exhibited intolerance to concentrations higher than 1 μM of the herbicide. Paraquat-treated leaves of the transgenic plants also exhibited significantly reduced electrolyte leakage and their chlorophyll content was not impacted as observed in the WT plants. Besides, in contrast to the WT, negligible amounts of hydrogen peroxide (H 2 O 2 ) were detected in paraquat-treated seedlings of the transgenic plants, as revealed by 3,3’-diaminobenzidine (DAB) staining. Collectively, these results indicate that the cvpqiB gene is functional in plants and may be further used in the genetic engineering of crop plants aiming paraquat tolerance.

Overexpression of EiKCS confers paraquat-tolerance in rice (Oryza sativa L.) by promoting polyamine pathway

Paraquat is an important bipyridine herbicide by acting on the photosynthetic system of the plants and generating reactive oxygen species leading to cell death, whereas the mechanism of the paraquat resistance remains to be explored. In this study, a putative paraquat-resistant gene EiKCS from goosegrass (Eleusine indica L.) was isolated and overexpressed in a transgenic rice (Oryza sativa L.). This transgenic rice (KCSox) was treated by exogenous spermidine and paraquat and then was analyzed by qualitative and quantitative proteomics. Overexpressing of EiKCS enhanced paraquat tolerance in KCSox by the accumulation of endogenous polyamines whose dominant presences of polyamines benzoylation derivatizations in rice were C18H20N2O2, C28H31N3O3, and C38H42N4O4. The mechanism underlying the improving tolerance enhanced antioxidant capacity of ROS systems and light-harvesting in photosynthesis in KCSox rice leaves to reducing paraquat toxicity. The protein β-Ketoacyl-CoA Synthase (EiKCS) encoded by the EiKCS gene promoted the synthesis and metabolism of proteins of the polyamine pathway. Three cofactors CERs were identified and positively correlated with the function of EiKCS on very-long-chain fatty acids (VLCFAs) biosynthesis via promoting the polyamine pathway and inhibiting the links with the TCA pathway and fatty acid pathway to responding to the paraquat tolerance in the KCSox rice, which also caused the prolongation of the overproduction of spermine and a transient increase of intracellular malondialdehyde (MDA). These results expanded the polyamines pathway manipulated in cereals using genetic engineering to clarify the mechanism of paraquat-tolerance.

Loss of rice PARAQUAT TOLERANCE 3 confers enhanced resistance to abiotic stresses and increases grain yield in field

ABSTRACTPlants frequently suffer from environmental stresses in nature and have evolved sophisticated and efficient mechanisms to cope with the stresses. To balance between growth and stress response, plants are equipped with efficient means to switch off the activated stress responses when stresses diminish. We previously revealed such an off-switch mechanism conferred by Arabidopsis PQT3, knockout of which significantly enhances resistance to abiotic stresses. To explore whether the rice homolog OsPQT3 is functionally conserved, we generated three knockout mutants with CRISPR-Cas9 technology. The OsPQT3 knockout mutants (ospqt3) display enhanced resistance to oxidative and salt stress with elevated expression of OsGPX1, OsAPX1, and OsSOD1. More importantly, the ospqt3 mutants show significantly enhanced agronomic performance with higher yield compared with the wild type under salt stress in greenhouse as well as in field conditions. We further showed that OsPQT3 was rapidly down regulated in response to oxidative and other abiotic stresses as AtPQT3. Taken together, these results support our previous findings that AtPQT3 acts as an off-switch in stress response, which is well conserved in rice. Therefore, PQT3 locus provides a promising candidate for crop improvement with enhanced stress resistance by gene editing technology.

Role of the Ascorbate–Glutathione Cycle in Paraquat Tolerance of Rice

Out of 1,343 mutant lines of rice mutated by sodium azide from the parental Japonica-type variety ‘Tainung 67’ (TNG67), a paraquat-susceptible line 1192 and a paraquat-tolerant line 72 were selected using whole seedlings at the four-leaf stage and leaf segments at the tillering stage as test materials. Further selection from progenies of these two mutant lines yielded the susceptible 1192-11 (S) and tolerant 72-16 (T), which were studied herein. Chlorophyll fluorescence, electrolyte leakage, and lipid peroxidation were measured for leaf segments of rice following treatment with 0.1 mM paraquat. A comparison of these responses among the three rice lines (TNG67, 72-16, and 1192-11), revealed a higher tolerance to paraquat in the tolerant mutant line 72-16 and the parental variety TNG67 than in the susceptible mutant 1192-11. Analysis of the antioxidative system in paraquat-treated leaf segments showed that the reduced form of glutathione (GSH) and the ratio of GSH to total glutathione increased by 3.5-fold within 6 h after treatment (HAT) and up to 5-fold 9 HAT in the T line, as compared with the S line. In view of the high activities of both dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in paraquat-treated leaves of TNG67 and the T line, the antioxidative effect of the ascorbate–glutathione cycle is hereby proposed to play an essential role in paraquat tolerance of rice. Pretreatment of rice segments with spermine enhanced DHAR and GR activities as well as paraquat tolerance of the S line. These results suggest that the activity of ascorbate–glutathione cycle induced by spermine is involved in rice tolerance to this herbicide. Although kinetics studies showed no significant difference among the three rice lines in paraquat inhibition of GR, a lower affinity of enzyme to substrate (Km) in TNG67 and the T line and a higher maximal reaction rate (Vmax) in the T line for the oxidized glutathione substrate (GSSG) were detected. These observations further implicate the importance of glutathione reductase in paraquat tolerance of rice.