Cloning And Function Analysis Of Paraquat Resistant Genein Arabidopsis Thaliana

Paraquat(PQ) is one of the most widely used herbicides in the world for controlling weeds in crops. It acts in the presence of light to induce generation of reactive oxygen species(ROS) and desiccate the green parts of plants through a quick penetration from the leaf surface. Paraquat has benefited no-tillage agriculture for nearly 50 years since it applied. However, enhanced tolerance to paraquat was evolved in many weeds due to excessive use. Studies on paraquat resistance in weeds and model plants, Arabidopsis and tobacco illustrate two mechanisms of paraquat resistance:(1) sequestrating paraquat away from target sites in chloroplasts;(2) increasing capability to scavenge ROS. In this study, we identified the novel mutants resistant to paraquat in Arabidoosis, and analyzed one mutant function.1. Identification of paraqaut resatant mutantsPQ was used for screening the XVE inducible activation lines provided by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. 8 paraquat resistant mutants were isolated, designated as pqr1~8. Under normal conditions, no significant difference in growth and development between mutants and wild-type plants was observed. T-DNA insertion was detected in pqr1, pqr3, pqr5, pqr6, pqr7, but not in pqr2, pqr4, pqr8. The vector carries a kanamycin resistant marker, while pqr2 showed kanamycin sensitivity. Therefore, T-DNA insertion was not responsible for the paraquat resistance phenotype in pqr2.Compared with wild-type, pqr2 showed a reduced accumulation of superoxide, while treated with paraquat, as revealed in leaves stained with NBT. Stained with Evans blue, the reduced cell death was observed in pqr2 leaves. These results indicate that the paraquat-induced generation of ROS and resulting cell death are inhibited by the pqr2 mutation.2. Map-basde cloning of paraquat resistance gene in pqr2Paraquat resistance gene PQR2 was identified in pqr2 by map-based cloning. DNA sequencing analysis of PQR2 revealed a mutation, characterized as a T-to-A transition at nucleotide 453 in the PQR2 CDS, which converts a tyrosine into a terminator at residue 151. This tyrosine residue is highly conserved in related proteins across different kingdoms. Full length CDS of PQR2 was amplified to construct complemention vector under the control of 35 S promoter. Transgenes fully restored the paraquat sensitive phenotype, similar to that of wild-type plants, thereby confirming that the pqr2 mutant phenotype is caused by a mutation in PQR2.3. Function alylasis of PQR2According to annotations on TAIR webisite, PQR2 encodes a polyamine uptake transporter. It was reported that paraquat can be transported through polyamine transpoter in plants. In this study, pqr2 exhibited reduced root length compared with wild-type when when treated with 600 μM and 700 μM putresine. It indicated that PQR2 was involved in polyamine transport.4. ABA inhibited paraquat resistanceABA is a very important stress hormone in plants. In this study, ABA and ABA synthesis inhibitor Na2WO4 were used to treat wild-type and pqr2. The results showed that ABA decreased paraquat tolerance in pqr2, while Na2WO4 enhanced paraquat tolerance in wild-type. Drawing information from RT-PCR, expression level of PQR2 in wild-type and pqr2 was remarkably enhanced by exogenous ABA and significantly decreased by Na2WO4. Moreover, compared with wild-type, expression level of PQR2 in pqr2 was significantly lower. Taken together, ABA induced expression of polyamine transpoter, followed by incresde efficiency of paraquat tansport. As a result, paraquat resistance was decreased in Arabidopsis.5. Gene expression level was althered in pqr2Paraquat altered gene expression by generateing oxidative stress. We screened 21 genes which can be regulated by paraquat. RT-PCR analysis revealed that expression level of 9 genes was down-regulated in pqr2 under PQ、ABA and Na2WO4 treatment, including protein kinase genes: AT5G07180 and AT3G46370; ROS synthesis genes: AT3G11180 and AT1G31690; phoprotection and stess resopse related genes: AT1G77120, AT4G37930, AT3G52960 and AT5G01600; transcription factor: AT1G75390. The results indicated that PQR2 could inhibit expression of these genes directly or through ABA signal pathway.