Study On The Relationship Between The Dynamic Balance Of Auxin And Resistance To Quinclorac In E.crus-galli Var.zelayensis

Echinochloa crusgalli var.zelayensis is one of execrable Echinochloa.spp.weeds in rice fields.In recent years,Echinochloa spp.has become one of the most serious weeds in rice fields,which has seriously threatened the growth of rice fields in China.Quinclorac is a quinolinic acid hormone herbicide,which is a special herbicide for controlling weeds in rice fields.However,with the long-term single use of same herbicides,the control effect of quinclorac to Echinochloa spp.is decreasing year by year,even led to the development of resistance to quinone in many parts of China.At present,domestic studies have shown that ethylene biosynth esis,cyanide degradation and photosynthesis protection mechanism have a certain relationship with quinclorac resistance,and the research on the relationship between auxin biological pathway and quinclorac resistance is basically blank,the auxin biological pathway was mainly divided into four processes:Auixn homeostasis,growth hormone transport,signal transduction and downstream hormone response gene expression.The auxin homeostasis process includes auxin biosynthesis,conjugation,metabolism and so on.In order to further clarify the relationship between the dynamic balance of auxin and the quinclorac resistance was studied.The resistant populations SSXB-R2,JCWJ-R1 and the sensitive population JNNX-S were selected as the acticle populations,study the mode of action of quinclorac and the relationship between the auxin homeostasis and quinclorac resistance.The main results and conclusions are as follows:The effects of quinclorac and IAA with different concentrations on seedling growth were studied.The results have showed that the treatment with quinclorac can cause symptoms of leaf bending,growth inhibition of seedlings,stem curvature,foliar chlorosis and tissue decay,which is consistent with the symptoms of IAA overdose.It could also be found that the percentage of plant height,root length,above-ground and underground fresh biomass weight under quinclorac and/or IAA treatments were all decreased significantly compared to CKO and exhibited a negative correlation with resistance level in general.The IAA content in the above-ground and underground parts after treated with quinclorac were evaluated in the three biotypes with A enzyme-linked immunosorbent assay(ELISA)kit,respectively.The results have showed that IAA contents in above-ground and/or underground extracts from the sensitive biotypes JNNX-S were induced significantly after treated by quinclorac,at 24HAT,the IAA content reached 2.1068,1.5988 times compare to the initial levels at 0 HAT in JNNX-S,while in the JCWJ-R1 biotypes,it was only 1.4202,1.2721 times respectively,the content of IAA at 24 HAT in the high resistant biotypes increase by 1.3079 and 1.1204 times.It was speculated that quinclorac may have a similar mode of action as IAA.The auixn homeostasis was studied to determine whethe it plays a role in the resistance to quinclorac.Sequences of homologous genes involved in auxin homeostasis were searched in transcriptome sequencing database of E.crus-galli var.zelayensis(SRP130228).Thirteen genes involved in auxin homeostasis were identified,in which six genes(EcYUCCA4,Ec YUCCA5,Ec YUCCA9,Ec YUCCA10,EcTDC,EcAMI1)are responsible for auxin biosynthesis,six genes(EcGH3.1,EcGH3.3,EcGH3.5,EcGH3.8,EcGH3.11 and EcUDP)are involved in amino acid conjugation for IAA,and the rest one(EcDAO)are involved in auxin oxidation.These sequences were aligned on the NCBI website before used for design primers for Q-PCR.Amplified sequences were blasted to confirm they were desired gene fragments.The expression profile of these 13 genes in both aboveground and underground parts of E.crusgali var.zelayensis were analyzed by real time PCR.The results showed that the expression of EcYUCCA5 and EcYUCCA9 in the above-ground part of the sensitive biotype JNNX-S declined significantly,the gene EcYUCCA10 was drastically induced to nearly 4 folds at 24 HAT.In the middle resistant biotype JCWJ-R2,the expression level of EcYUCCA9 was down-regulated.However,in the resistant biotype SSXB-R2,most of Auxin genes did not have a significantly changes after quinclorac treatment.In the underground parts of JNNX-S,the expression of EcYUCCA4 and EcYUCCA5 were down regulated significantly at 24HAT.The expression of EcYUCCA10 was increased by 12 folds,which was significantly higher than that in JCWJ-R1 and SSXB-R2.In the middle resistant biotype,EcYUCCA4 was up-regulated significantly.There was no significant difference in resistant biotype.The genes involved in auxin conjugation were explored.The results showed that in the above-ground parts,the expression of EcGH3.1,EcGH3.3 and EcGH3.11 decreased significantly and the gene EcGH3.8 were drastically induced in JNNX-S at 24 HAT,while in SSXB-R2 they all keep stable,as no significant differences could be observed.In the middle resistant biotype JCWJ-R2,the expression level of EcGH3.8 was up-regulated 16 times,the gene EcUDP was up-regulted 26 times.In the under-ground parts,the expression of EcGH3.1,EcGH3.5 and EcUDP were all significantly increased in SSXB-R2 at 24 HAT,the expression of EcGH3.5,EcGH3.8 and EcUDP were significantly increased in JCWJ-R1.In sensitive biotype,the gene EcGH3.8 and EcUDP were significantly increased.The expression of EcDAO was up-regulated significantly in all the three biotypes at 24 HAT,in which a negative correlation between the increasing extents and the resistance level was suggested.However,in the underground parts,there was no significant difference.The activities of two major oxidative enzymes against IAA,POD and IAAO,are determined.In the above-ground parts,the activity of POD reached 1.6520 and 1.4843 times of the initial activity after 24HAT in the resistant biotypes SSXB-R2 and JCWJ-R1.The sensitive biotype JNNX-S reached 1.6205 times at 12 HAT,then decreased to 1.1961 times at 24HAT.In the underground parts,the activity of POD was increased in all the three biotypes and a negative correlation between the increased activity and resistance level could be observed.The activity of POD reached 1.4009,1.5907 and 1.7995 times compare to the initial activity respectively.As for the IAAO,the activity of IAAO was decreased along the treated time and a negative correlation between the activity and resistance level in all the three biotypes.The activity of IAAO decreased to 0.6489,0.4599 and 0.2598 times compare to initial activity at 24HAT.In the under-ground parts,there was no significant difference.In conclusion,the expression levels of IAA auxin biosynthesis related genes EcYUCCA4,EcYUCCA5 and EcYUCCA9 were declined significantly in sensitive biotypes,the gene EcYUCCA10 was significantly up-regulated,the expression levels of EcYUCCA4 and EcAM11 were down-regulated;IAA conjugated genes EcGH3.1,EcGH3.3 and EcGH3.11 declined significantly,EcGH3.8 and EcUDP were significantly up-regulated,EcGH3.5 was down-regulated;As for roots of sensitive biotypes,the expression levels of IAA synthetic genes EcYUCCA4 and EcYUCCA5 were significantly down-regulated,the gene EcYUCCA10 was significantly up-regulated and the expression of EcAM11 was up-regulated;The IAA conjugated genes EcGH3.8 and EcUDP were significantly up-regulated,and the genes EcGH3.1 and EcGH3.5 gene were up-regulated,gene EcGH3.3 was down-regulated,IAA oxidative gene EcDAO was up-regulated.In the two resistant biotypes,especially the foliar of the SSXB-R2 biotype,the expression of IAA conjugated gene EcUDP was significantly up-regulated,and the expression of IAA conjugated genes EcGH3.1,EcGH3.5 and EcUDP was significantly up-regulated in the roots.The expression levels of most of the auxin-related genes did not have a significant change after the quinclorac treatment.This study found there existed a change in gene expression during IAA homeostasis,especially in sensitive biotypes,which involved significant changes in gene expression,and the range of change was higher than that of resistant biotypes.This indicates that the sensitive biotypes are more easily affected after quinclorac treatment.Adjusting the expression levels of auxin-related genes respectively in the foliar and roots,the auxin content remain at a high level.while in resistant biotypes,the expression of most auxin genes can maintain at a relatively stable level,and small changes in auxin content was measured,the final degree of damage was slightly.After the quinclorac stress,the mechanism of the action of quinclorac involves the auxin homeostasis,and there exist a varity change between the resistant and sensitive biotypes and differences also exist in the foliar and roots.The activity of POD and IAAO change a lot in sensitive biotypes,there was slightly change in resistant biotypes.In other words,the activity of these two enzymes is relatively stable in resistant biotypes.The resistant biotype show more more resistance to quinclorac treatment.At the same time,this regulation mechanism may also differ in the foliar and roots parts of the seedlings,and that may explain the differences in enzyme activities between foliar and roots.Considering the changes of auxin-related gene expression and the changes of enzyme activity in the foliar and roots,a stronger auxin regulation mechanism may exist in resistant biotypes,which can maintain a relatively stable environment.it may be one of the resistant mechanism to quinclorac in E.crusgalli var.zelayensis.And as for the same genes were vary expressed between foliar and roots,as well as the enzymes,there may exist a different regulation mechanism after quinclorac treatment.