Functional Analysis Of Arabidopsis FKBP15-1 And FKBP15-2 Genes On Lateral Root Development

Root system,which involved in anchoring,nutrient uptake and resistance to biotic and abiotic stresses,is an indispensable part of plant.Lateral root is one component of root system,and its development affects the capabilities that mentioned above.Previous studies have revealed that phytohormone and the nutrients including carbohydrate,nitrogen,phosphorus and potassium regulate lateral root growth through different signalling pathways.However,the mechanism of lateral root development is not entirely clear.FK506 binding proteins(FKBPs)function in protein folding in organisms,and play important roles in plant growth,biotic and abiotic stresses resistance.FKBPs are found to participate in lateral root development and auxin signalling.Among them,FKBP42 affects shootward root auxin reflux by interacting with ABCB transporters in Arabidopsis(Arabidopsis thaliana).Our previous studies suggested that FKBP15-1 and FKBP15-2 are likely to regulate lateral root development,thus a thorough investigation on the topic was performed to investigate the regulatory mechanism of them.To analyze the function of FKBP15-1 and FKBP15-2 in lateral root development,phenotype analysis,expression profiles analysis and protein-protein interactions analysis were adopted and we illustrated the mechanism of FKBP15-1 and FKBP15-2 on lateral root development.The main results are the followings:(1)FKBP15-1 and FKBP15-2 cooperatively and negatively regulate the lateral root development.Compared with wild-type(Col-0),the number of lateral root in fkbp15-1fkbp15-2 and fkbp15-1fkbp15-2RNAi increased significantly by more than 20% while no obvious change in single mutants(fkbp15-1,fkbp15-2 and fkbp15-2RNAi).The overexpression lines of FKBP15-1 or FKBP15-2 showed no difference from the Col-0.(2)FKBP15-1 and FKBP15-2 express in the root pericycle,and their transcriptional levels are not affected by the addition of auxin.Expression profile analysis revealed that FKBP15-1 and FKBP15-2 mainly expressed in the root pericycle and vascular bundle,and strong signals were detected in the basal meristem of root tip.At cellular level,FKBP15-1 and FKBP15-2 proteins located in endoplasmic reticulum.Exogenous application of auxin did not change the difference between double mutant and Col-0 in the number of lateral root.(3)FKBP15-1 and FKBP15-2 repress the activity of VIN2 in Col-0.Using the method of immunoprecipitation and mass spectrometry-based proteomics,41 proteins were found to be associated with FKBP15-1 and FKBP15-2.KEGG pathway analysis in the KEGG database revealed that the 41 proteins were assigned to 21 pathways.The most enriched pathway groups included starch and sucrose metabolism,ribosome,and RNA transport.Further studies showed that the enzyme activity of the VIN2,which is involved in the metabolism of starch and sucrose,was significantly higher in the fkbp15-1fkbp15-2 and fkbp15-1fkbp15-2RNAi roots compared to Col-0.(4)The double mutant fkbp15-1fkbp15-2 increase sucrose usage efficiency,FKBP15-1 and FKBP15-2 acts genetically downstream of VIN2 to regulate lateral root development.Compared to Col-0,more lateral roots were detected in fkbp15-1fkbp15-2 under higher sucrose condition.While under low sucrose or various concentrations of glucose and fructose condition,the double mutant fkbp15-1fkbp15-2 failed to produce more lateral roots.Moreover,the triple mutant fkbp15-1fkbp15-2vin2 has the same number of lateral roots as the Col-0,indicated that VIN2 performs hereditary epistasis.In summary,FKBP15-1 and FKBP15-2 were found to negatively regulate lateral root development in Arabidopsis.FKBP15-1 and FKBP15-2 decrease the utilization efficiency of sucrose by repressing the enzyme activity of VIN2.This study advances our knowledge about the roles of immunophilin in plant organ development,also provides a paradigm of function in regulatory function of molecular chaperone.