Interaction Mechanism Between TIR1-IAA7 And Typical Chiral And Achiral Auxin Herbicides

Auxin herbicides are the most widely used for controling broad-leaved weed in the world,and some of them have chiral structures.Through in-depth research on auxin signal transduction pathways,it has been found that auxin herbicides can alternatively"mimic"the role played by IAA in the regulation of plant hormones,affecting plant growth and development,senescence and death,thus showing herbicidal activity.Previous studies have confirmed that phenoxycarboxylic acid 2,4-D,can interact with the receptor protein TIR1-IAA7 in the auxin signaling pathway(TIR1,Transport Inhibitor Response 1;IAA7,Auxin/Indole-3-Acetic Acid transcriptional repressors),while other species such as quinolinic acids,benzoic acids and pyridine pyrimidinic acids,are there similar interactions?For chiral auxin herbicides,are there enantioselective intereactions with TIR1-IAA7?However,there are currently few reports.We used MCPA,quinclorac,chloramben,and aminocyclopyrachlor as the representatives of the above-mentioned four types of auxin herbicides,molecular docking simulation,radioligand analysis,surface plasmon resonance technology and transcriptomics was used comprehensively to analysis of the interaction between the tested herbicides and TIR1-IAA7,the mutant of TIR1 was used for verification the resistance of those herbicides,which provide a scientific basis for further mechanism of action of auxin herbicides.At the same time,with chiral auxin herbicides DCPP and MCPP as representatives,the comprehensive application of isotope tracing technology and the above analysis methods was used to study and clarify the molecular mechanism of the enantioselective herbicidal activity of chiral phenoxycarboxylic acid herbicides.The main results are as follows:1)The expression and purification preparation methods of TIR1 and IAA7proteins have been established.The target genes TIR1 and IAA7 are highly expressed in the insect baculovirus system(Bac to Bac system)and E.coli system respectively.The purified target protein is active and provides the foundation for subsequent experiments.2)The comprehensive results of molecular docking,in vitro binding ability analysis and resistance verification confirmed that TIR1 is one of the targets of the phenoxycarboxylic acid herbicide MCPA and the benzoic acid herbicide chloramben.IAA7,as another important protein,may recognize MCPA and chloramben together with TIR1.The results of molecular docking showed that the 8 herbicides of MCPA,2,4-dbutyric acid,chloramben,dicamba,aminopyralid,aminocyclopyrachlor,quinclorac and quinmerac can be combined with TIR1-IAA7,and interact with important amino acid residues such as Ser438 and Arg436.The lowest binding energy presents the law as follow:MCPA=chloramben<quinclorac<aminocyclopyrachlor<the other four herbicides.When the aromatic ring,pyridine ring or pyrimidine ring has halogen substitution and carboxylic acid group,it is easier to combine with TIR1-IAA7.When the ring structure has an amino group,it is easy to cause electrostatic interaction with the leucine-rich region in the active pocket.The results of in vitro binding analysis showed that when the concentration of the herbicide was excessive,compared with aminocyclopyrachlor and quinclorac,MCPA and chloramben bound more TIR1-IAA7,the binding speed was faster,and needed more time to dissociate.The K_i of MCPA and chloramben was 138.3 n M and1132 n M,respectively,while aminocyclopyrachlor and quinclorac were incompatible with TIR1-IAA7(K_i was greater than 10000 n M).The results of resistance assays showed that mutant of TIR1 was resistant to MCPA and chloramben significantly,as the concentration increased,the resistance weakened to disappear;at low concentration,mutant of TIR1 was resistant to aminocyclopyrachlor slightly;mutant of TIR1 was not resistant to quinclorac.The results of transcriptome analysis further showed that quinclorac and aminocyclopyrachlor are not compatible with TIR1-IAA7,but they can cause differential expression of genes such as IAA16,IAA18 and IAA26,activate the auxin signaling pathway,and inhibit photosynthesis.That is,these two herbicides may affect plant growth by inhibiting photosynthesis and causing downstream reactions after interacting with other auxin receptor proteins.3)The comprehensive results of molecular docking,in vitro binding ability analysis and mutant reverse verification confirmed that TIR1 is one of the targets of phenoxycarboxylic acid herbicides DCPP and MCPP.IAA7,as another important protein,may recognize these two herbicides together with TIR1,and preferentially binds R-DCPP and R-MCPP.The results of molecular docking showed that DCPP and MCPP can bind to TIR1-IAA7,and interact with important amino acid residues such as Ser438 and Arg436.The lowest binding energy of R configurations was lower than the S configurations.The results of in vitro binding ability analysis showed that compared with S-MCPP and S-DCPP configurations(K_i is greater than 10000 n M),R-MCPP and R-DCPP bound more TIR1-IAA7,and the dissociated to equilibrium later.K_i was 61.14n M and 118.8 n M,respectively.q RT-PCR results further showed that the expression levels of IAA7 in Arabidopsis treated with R-DCPP and R-MCPP were up-regulated,while the expression levels of S-DCPP and S-MCPP groups did not change significantly.The expression levels of TIR1 did not show significant difference.The resistance assays results also showed that mutant of TIR1 had significant resistance to R-DCPP and R-MCPP,but not to S-DCPP and S-MCPP.4)Arabidopsis absorpted and transported chiral isomers of DCPP and MCPP differently.At 144 h after application,90.13%of R-DCPP and 90.24%of R-MCPP were absorbed,respectively,71.22%of S-DCPP and 85.75%of S-MCPP were absorbed.The R configurations content of each part was greater than the corresponding S configurations.Further experiments confirmed that in the nucleus where TIR1-IAA7is located,the content of R configurations of DCPP and MCPP were higher than the corresponding S configurations,and the content of R configurations in nucleoprotein was greater than the corresponding S configurations.The molecular enantioselective mechanism of the herbicidal activity of chiral phenoxycarboxylic acid herbicides DCPP and MCPP is:Compared with the S configurations,the R configurations of the two herbicides are easier to combine with TIR1-IAA7 and are easily absorbed and transported by Arabidopsis to the cell nucleus.The research provides theoretical guidance for the structural optimization and further development of lead herbicide compounds based on the TIR1-IAA7 target.