Molecular Mechanism Of Copper Amine Oxidase And Its Transcription Factor MYB In Response To Auxin Signaling In Arabidopsis

Biogenic amines are nitrogen-containing compounds that are divided into monoamines and polyamines.More than 20 polyamines are found in plants,which are divided into diamines,triamines and tetramines,based on the number of amidogen.Polyamines mainly include:putrescine(Put),cadaverine(Cad),spermine(Spe)and spermidine(Spd)in plants.In plants,polyamines regulate biological stress,such as cell development,seed germination,aging,and abiotic stress,such as drought,chilling injury and salt damage.Polyamines are metabolized by amine oxidase which include copper-binding diamine oxidases(CuAO)and polyamine oxidases(PAO).Plant CuAOs mainly catalyze the oxidation of Put and Cad,while PAOs mainly catalyze Spm and Spd.The metabolites of polyamines mainly include NH3 and H2O2,which have important role in the growth and development of plants.The CuAO family in Arabidopsis is composed of ten genes,CuAO?1,CuAO?2,CuAO?3,CuAO?,CuAO?1,CuAO?2,CuAO?1,CuAO?2,CuAO? and CuAO? According to preliminary work of this experiment,the density and length of lateral root in WT are significantly higher than cuao? mutant after exogenous IB A treatment,and the CuAO?exhibits higher experssion in cortical and endordemal layers in primary root.To further explore the molecular mechanism of the lateral root development of CuAO? in response to auxin signaling,we performed the following experiments.Through tobacco transient expression experiments,we observed that CuAO? protein was localized in peroxisome.Exogenous IBA treatment increased the expression of CuAO?.Knockout of CuAO? reduced the activity of CuAOs protein.Using the ROS sensor and DAB staining,we observed that the content of H2O2 of cortical layer significantly increased in WT and COM1 after exogenous IBA treatment,but not in the cuao? mutant.The expression of PIN2 which transported auxin displayed high level in WT and COM1,but not in the cuao? mutant.Konckout of PIN2 resulted in obvious inhibition of IBA and H2O2-induced lateral root development.The above results indicate that the CuAO?-derived H2O2 regulate the expression of PIN2 protein to mediate the distribution and polarity transport of auxin to regulate lateral root development.The 5'UTR of CuAO? is a key sequence to regulate its expression at root tip,suggesting that 5'UTR may be regulated by transcription factors.By yeast one-hybrid,we screened out two putative transcription factors MYB and GATA2 of CuAO?.We generated MYB promoter:GUS fusion(PMYB:GUS)transgenic plants.GUS staining showed that MYB expressed in all tissues,and stronger express in the tip of primary and lateral root.Exogenous IBA treatment increased the expression of MYB.We crossed the PCuAO?:CuAO?-GFP with myb mutant.The results of qRT-PCR and confocal assay showed that the expression of CuAO? in myb mutant was significantly higher than WT under control conditions;the expression of CuAO? significantly increased in WT after exogenous IBA treatment,but not in myb mutant.Together,we suggest that MYB mainly regulates the expression of CuAO? in response to auxin singal and other transcription factors may be also involved in regulation of CuAO? expression.