A Functional Study Of The Copper Amine Oxidase CuAO5 In Main Root Development Of Arabidopsis Thaliana

As one of the plant's nutrient organs,the root system plays important functions such as absorbing nutrients,fixing and supporting.Some roots can form metamorphosis roots which store large amounts of nutrients,in addition the root system is important for responding to abiotic and biotic stresses.Therefore,the molecular study of the root development is necessary for crop breeding and the trait improvements to breed new cultivars with high stress resistance and efficient absorption of nutrients.Root apical meristem(RAM)is a source of plant subsurface parts.Reactive oxygen species(ROS)and auxin both play critical roles in regulating RAM development.ROS are in a dynamic equilibrium but poorly defined state in RAM,where H2O2 promotes stem cell differentiation and O2-maintains stem cell activity.ROS and many downstream key genes cooperatively regulate RAM;moreover,auxin is distributed in a gradient in the root system,dominating RAM development by regulating key genes such as downstream PLT1,PLT2 and WOX5.The interplay between different signaling pathways integrates external and internal factors to form a complex network regulation,which collectively regulates stem cell maintenance,proliferation and differentiation.Copper amine oxidases(CuAOs)can catalyze the degradation of diamines to produce small molecules such as ?-aminobutyric acid(GABA),NH3,and ROS.Many studies have reported the involvement of CuAOs in plant vascularature,lateral and adventitious root development,and stomatal movement.Nevertheless,our knowledge on the function of CuAOs is still very limited.Our previous work shown that,the Arabidopsis CuAO5 loss of function mutant,cuao5-2 has a smaller apical meristematic zone,abnormal primary root development,and slower growth which resulting in a short-root phenotype.Additionally,CuAO5 is abundantly expressed in Arabidopsis vascular vessels,implying that CuAO5 may regulate the primary root development.To further investigate the molecular mechanism by which CuAO5 regulates the root development,we studied the potential roles of ROS and auxin in the CuAO5-mediated root development using genetics,biochemistry,molecular biology and plant physiology.The main results and conclusions obtained experimentally are as follows:1.By observing the GUS and GFP signals at the seedling stage of pCuAO5:GUS-GFP transgenic plants,it was found that CuAO5 is abundantly expressed in the primary root vessels while not expressed in the root apical meristem;there are eight members of the Arabidopsis CuAOs gene family that are abundantly expressed in vessels,whereas CuAO6 and are expressed in the RAM.2.The CuAO5 loss of function mutant cuao5-2 exhibited significantly fewer cortex cells than the wild-type plant showing smaller RAM and shorter primary root length.The p35S:CuAO5 was transferred into cuao5-2 to test its complementary ability.We observed that the transgenic plants partially complemented the defective root of the mutant,indicating that CuAO5 is involved in primary root development.No root developmental abnormalities were observed in other loss-of-function of CuAOs mutants.3.The apical ROS homeostasis was disrupted in cuao5-2 showing,compared with the wild type,the accumulation of H2O2 increased while the content of O2-decreased,but the exogenous H2O2 scavengers KI(potassium iodide),CAT(catalase),and ASA(ascorbic acid),as well as MT(melatonin),RGF1p,GABA,and D-Arg(D-arginine,an inhibitor of polyamine synthesis),all failed to complement the short root phenotype of cuao5-2,implying that regulation of primary root growth by CuAO5 may not directly related to the level of ROS and GABA.4.Comparison of the DR5:GUS signal in cuao5-2 mutants and wild-type plants revealed that the DR5:GUS signal in cuao5-2 was attenuated,suggetsing that CuAO5mediated primary root development depends on auxin.5.Comparison of pPLT1:PLT1-GFP,pPLT2:PL T2-EGFP and pPLT2:GFP signals in cuao5-2 mutants and wild-type plants showed that CuA05-regulated root development required PLT1 and PLT2.6.Comparison of the QC25:GUS and WOX5:GUS signals in cuao5-2 mutants and wild-type plants indicated that the expression of QC25 and WOX5 was unaffected in cuao 5-2,implying that the loss-of-function of CuAO5 did not affect QC morphogenesis.In summary,we analyzed the phenotypes of various loss-of-function mutants of CuAOs by a combination of biochemical,molecular and genetic approaches.We found that the loss-of-function of CuAO5 caused the disruption of ROS homeostasis,reduced auxin content,and decreased expression of downstream key genes,PLT1 and PLT2.We thus initially established a CuAO5-mediated regulatory network in Arabidopsis primary root growth and development.Nevertheless,the relationship between ROS and auxin in CuAO5-mediated root development needs to be further explored.