Study On The Auxin Transport Pathpawys Of Echinochloa Crus-galli Var.zelayensis Resistant To Quinclorac

Quininclorac is an auxinic herbicide that bring out the release of ethylene and the coproduction cyanide by interferes the plant auxin pathways,which leads to plant death finally.It is currently known that Echinochloa crus-galli var.zelayensis resistant to quinclorac produces only a small amount of ethylene and cyanide after treatment with quinclorac,which may be the mechanism of quinclorac resistance.However,there have been no studies on how quinclorac affects the growth hormone pathway of ceratin.Therefore,in this study,Echinochloa crus-galli var.zelayensis was studied on the auxin transport pathways of resistant and susceptible biotypes treated with quinclorac.The effect of quinclorac on root development and auxin distribution of the two biotypes was compared.The sequences of the auxin transporters EcABCB1,EcABCB4,and EcABCB19 were analyzed,and the effect of quinclorac on the expression of auxin transporter protein genes was compared.It suggests that the difference in the auxin transport pathways between resistant and susceptible Echinochloa crusgalli var.zelayensis biotype may be related to resistance to quinclorac.The study on effect of the auxin transport inhibitor NPA(1-Naphthylphthalamic acid)and quinclorac on the root development of susceptible and resistant Echinochloa crus-galli var.zelayensis seedlings was conducted by culture dish bioassay.The results showes that NPA had a significant inhibiting effects not only on the length of the primary root,but also on the number of lateral roots and the fresh weight of the plant.And the inhibiting effects on the susceptible population was higher than that of the resistant population.Quinclorac also strongly inhibited the root development of susceptible populations.The primary root length,number of lateral roots,lateral root density and fresh weight of plants were significantly lower than that of water control at different concentrations of quinclorac,and the inhibiting effects increased with increasing quinclorac concentration.within the resistant biotype,quinclorac treatment had less effect on the extension of the main root,lateral rooting,and fresh weight of the plant than on the susceptible biotype.The quinclorac and NPA have the same inhibition phenotype on the root system of the seedling of Echinochloa crus-galli var.zelayensis,which indicates that quinclorac may have the same mode of action as NPA,and that it can always inhibit the roots development by affecting the polar transport of auxin.The effect of quinclorac on the distribution of auxin in resistant and susceptible Echinochloa crus-galli var.zelayensis was studied.Using enzyme-linked immunosorbent assay kits,the changes in auxin levels in different parts of the resistant and susceptible biotype were determined after treatment with quinclorac.The results showed that after treatment with 50 ?M quinclorac,the content of auxin in the root tip and aboveground part increased significantly,reaching 4:12 and 3.29 times of the control,respectively,while the content of auxin in the upper root tissue did not change significantly.However,in the resistant population,the content of auxin in the apical tissue increased significantly,and there was no change in the aboveground and upper root tissues.Besides,under the treatment with auxin transport inhibitor NPA,the auxin content in the root tip and aboveground part of the susceptible Echinochloa crus-galli var.zelayensis also increased,but the increase of the resistant population was significantly smaller than that of the susceptible population.It can be inferred that quinclorac and NPA have similar function of inhibiting the polar auxin transport,which resulted in changes in the distribution of auxin in the plants.However,the effect of auxin distribution on the resistant biotype was significantly less than that of the susceptible type indicating that the differences in the auxin transport pathway may involved in its resistance to quinclorac.NPA can specifically inhibits the ABCB transporter involved in the polar auxin transport in plants.Therefore,we got the three most common gene fragments encoding different ABCB transporters(ABCB1,ABCB4,and ABCB 19)through clone by PCR and sequencing at NCBI for simultaneous comparison with ABCB transporter genes in rice,Arabidopsis,and other plants.Alignment and phylogenetic analysis were performed to determine the auxin transportor gene of the Echinochloa crus-galli var.zelayensis,which were named by EcABCBl,EcABCB4,and EcABCB19,respectively.In the analysis of the three carrier protein sequences mentioned above,were similar to rice OsABCB and Arabidopsis thaliana AtA BCB,and they all contained multiple ABC transmembrane transport functional domains.The subcellular localization of proteins was found to be similar to that of OsABCB and AtABCB and localized to the plasma membrane.Based on the OsABCB and AtABCB expression profiles,it was concluded that EcABCB1 may be the vector responsible for the export of auxin in the root tip cells,EcABCB4 may be located in the root tip cells responsible for the auxin input,and EcABCB19 may participate in the auxin to the base of the aerial parts transport.The effect of quinclorac on the expression of EcABCB1,EcABCB4 and EcABCB19 was studied by RT-PCR.The results showed that the expression level of ABCB4 gene increased significantly under 50 ?M quinclorac treatment and the expression level of ABCB1 gene was significantly decreased in the root apical auxin exporter.The amount dropped significantly.In resistant biotypes,the expression level of ABCB1 gene in the roots increased significantly to 2.85 times after treatment at 6 hours after treatment,and then decreased gradually,but it was still significantly higher than before treatment.The amount of ABCB4 gene expression in roots was treated with the quinclorac increased significantly after six hours,but the increase was significantly lower than that of susceptible biotypes,and then gradually decreased to similar levels without quinclorac treatment;ABCB19 gene expression levels increased significantly at 6 hours,then gradually decreased,but the expression level was still significantly higher than the sensitivity biotype.In summary,after treated with quinclorac,the expression of the apical auxin-stimulated auxin-input protein,ABCB4 in susceptible Echinochloa crus-galli var.zelayensis increased significantly,and the expression level of the apical auxin output protein,ABCB1,decreased significantly.The expression of ABCB19 in the root growth hormone to the basic transporter protein was significantly decreased,resulting in the accumulation of root growth hormone at the root tip,which destroyed the normal concentration gradient of root growth hormone,indicating that the growth of the root system was inhibited;The expression of basital auxin transporter ABCB19 was significantly decreased by quinclorac and the auxin increased significantly in shoots,indicating that quinclorac interfered with the basital auxin transport in the shoot of,which may cause the growth of leaves.Elevated hormone concentrations induce the release of ethylene,which ultimately leads to the death of the plant.In the resistant biotypes,the expression of ABCB1 gene in the root tip was significantly increased after treatment with quinclorac,and the amplitude of ABCB4 gene expression was significantly lower than that of the susceptible biotype,and the root auxin concentration significantly lower than susceptible biotypes has less influence on the concentration gradient of growth hormones in the roots,so that the roots can grow and develop normally,while the expression of ABCB19 gene in shoots was not affected by quinclorac treatment,and the content of auxin in resistant population shoots is obviously less than that in the susceptible population.which indicate that the basital auxin transport in the shoot is less affected,and the growth hormone concentration can be kept stable,resulting in less ethylene and the plant maintaining normal growth.