Mechanism Of Epinodosin-Mediated Polar Auxin Transport And Auxin Signal Transduction To Inhibit The Root Growth Of Arabidopsis Thaliana

Auxin affects all aspects of plant growth and development,including the growth,division and differentiation of plant cells,as well as the process of plant development from embryo to reproductive development,what?s more,auxin affects plant growth and development through complex regulatory networks such as synthesis,polar transport and signal response.Epinodosin is a kind of diterpene compounds extracted from Isdon japonica var.galaucocalyx.It has been proved that diterpenes have obvious allelopathic potential to lettuce and other pla nts,participating in regulating the growth and development of plants: low concentration promotes plant growth while high concentration inhibits growth.We speculate that epinodosin may affect plant growth and development by mediating auxin pathway including auxin polar transport and auxin signal transduction.This study,on the basis of Arabidopsis thaliana,a model plant,uses 16 auxin pathway-related functional deficiency mutants,9 transgenic reporter lines and 7 fusion protein marker lines to study the mechanism of epinodosin inhibiting the growth and development of Arabidopsis thaliana roots,which aims to provide a scientific basis for revealing the chemical ecological effects of this class of compounds.The main results of this study are as follows:(1)60,80 and 100 ?M epinodosin inhibited the root growth of WT obviously.After the treatment of epinodosin,the length of primary roots and the number of lateral root and lateral root primordia decreased,and the development of lateral roots at every stage is inhibited.(2)60 and 80 ?M epinodosin increased the activity of CYCB2 in the root tip meristem of the transgenic line CYCB2;2::GUS,which indicated that the number of S/G2-phase cells increased and the cell cycle arrest was formed.At the same time,it was found that the number of cortical cells between meristem and elongation was inhibited by epinodosin,which indicates the decrease of cell production in root meristem;60 and 80 ?M epinodosin also decreased the length of cells in elongation zone.These results suggest that epinodosin can inhibit the primary roots and lateral roots by reducing mitosis and inhibiting the elongation of the cels in the meristem.(3)Treatment of transgenic Lines DR5::GUS and DR5::GFP with 60 and 80 ?M epinodosin,GUS staining and GFP fluorescence intensity of lateral root and primary root tip of Arabidopsis thaliana seedlings were significantly increased,which indicates that DR5 activity increased,auxin level of root tip increased,and the auxin in the apical part of the root tip accumulated.(4)The effects of 60 and 80 ?M epinodosin on the growth of primary and lateral roots of WT as well as pin1,pin2,pin3-4,pin4-3,pin5-2,pin7-2,aux1 mutants were compared.The results showed that epinodosin significantly inhibited the primary length and the number of lateral root and lateral root primordium of WT,but did not show the inhibitory effect on lateral root and lateral root primordium of pin3-4,pin4-3,pin5-2 and aux1 mutants,the lower concentration of epinodosin(60 ?M)also showed no inhibitory effect on the lateral root pin1,pin7-2 mutants.Compared with WT,the lower concentration of epinodosin(60 ?M)showed a lower length inhibitory effect on pin3-4,pin4-3 and aux1 mutants,that is,it shows the inhibition and recovery effect.These results suggest that pin1,pin3,pin4,pin5,pin7 and aux1 mutants mediates the inhibition of lateral root formation by epinodosin(60 and 80 ?M),while pin3,pin4,aux1 mutants mediates the inhibitory effect of epinodosin(60 ?M)on the growth of the primary roots.(5)The fusion protein lines(PIN1::PIN1:GFP,PIN2::PIN2:GFP,PIN3::PIN3:GFP,PIN4::PIN4:GFP,PIN7::PIN7:GFP,AUX1::AUX1:YFP)of Arabidopsis thaliana and transgenic lines(PIN1::GUS,PIN2::GUS,PIN3::GUS,PIN4::GUS,PIN7::GUS and AUX1::GUS)were treated with 60 and 80 ?M epinodosin.The results showed that except for the increase of PIN1 protein abundance,the abundance of PIN2,PIN3,PIN4,PIN7 and AUX1 protein decreased significantly.Except for the increase of PIN1 transcriptional activity,the transcriptional activity of PIN2,PIN3,PIN4,PIN7 was significantly inhibited.These results suggest that the increase of PIN1 expression and the decrease of PIN2,PIN3,PIN4,PIN7 and AUX1 expression may result in the accumulation of auxin at root tip.(6)The effects of 60 and 80 ?M epinodosin on the growth of primary and lateral roots of WT and auxin signal pathway mutants(tri1-1,iaa17/AXR3,iaa19/msg2,iaa28-myc,iaa31-1,arf7-1,arf10-1,arf16-1,arf19-1)were compared.The results showed that epinodosin significantly inhibited the primary root length and lateral root primordia of WT,but did not show the inhibitory effect on the primary root and lateral root primordia of the mutants iaa17/AXR3,iaa19/msg2,iaa28-myc,and there was no inhibitory effect on the number of lateral root and the lateral root primordium of the mutants tri1-1,iaa31-1,arf19-1.The response of other mutants to epinodosin was consistent with that of WT.60 and 80 ?M decreased the activity of HS::AXR3-3NT-GUS,which indicated that epinodosin could induce the degradation of IAA/AXR3.These results suggest that iaa17/AXR3,iaa19/msg2,iaa28-myc mutants mediate the inhibitory effect of epinodosin(60 and 80 ?M)on the formation of primary and lateral roots,while tri1-1,iaa31-1,arf19-1 mutants only mediate the inhibition of lateral root formation.In conclusion,60,80 and 100 ?M epinodosin regulate the expression of auxin export carriers PIN1,PIN2,PIN3,PIN4,PIN7 and auxin import carrier AUX1 at molecular level,promote auxin transport to the top and inhibit auxin transport to the base,which interferes with the balance of auxin concentration in the root tip of Arabidopsis thaliana,activates the signal transduction pathway of growth factor,and finally inhibits the growth of root at the tissue level.