Identification And Characterization Of Auxin Deficient Mutants Ckrc2 And Ckrc3

The phytohormone auxin participated in regulating many processes of plant growth and development,such as gametogenesis,embryogenesis,organogenesis and stress responses.However,lack of proper mutants has so far prevented the detailed analysis of auxin synthesis and regulation.As is well known,by using different precursors plants utilize two main strategies to synthesize IAA,the Trp-dependent(TD)pathway and the Trp-independent(TI)pathway.Four postulated TD pathways,namely the indole-3-acetamide(IAM)pathway,the indole-3-pyruvic acid(IPyA)pathway,the tryptamine(TAM)pathway and the indole-3-acetaldoxime(IAOx)pathway,have been identified biochemically.However,the genes and mutants involved in the TI pathway have not been identified clearly to date..IPyA pathway is the most clearly characterized auxin biosynthesis pathway based on the identification and investigation of the genes and their encoded enzymes involved in this pathway.In the IPyA pathway,TAAI(for TRYPTOPHAN AMINOTRANSFERAS E OF ARABIDOPSIS1,also called SAV3,WE18,TIR2 or CKRC1)catalyzes the formation of IPyA from L-Trp.Then,the IPyA was converted to IAA under the catalyzation of YUCCA(flavin monooxygenase).The YUCCA gene family has 11 predicted members,namely YUCCA1-YUCCA11.Through the auxin deficient mutants screening system our lab established,we obtained two mutants with the curling root on medium containing 0.1 ?M tZ,namely ckrc2(CK induced root curling 2)and ckrc3.We cloned the CKRC2 gene by map-based cloning and whole genome resequencing,and indentified that the ckrc2-1 is an allele of YUCCA8(AT4G28720).ckrc2-I exhibited shorter roots and hypocotyls,and was insensitive to cytokinin treatment.Our LC-MRM-MS results showed that the content of both free IAA and its metabolites decreased obviously in ckrc2-1.The transgenetic and genetic analysis revealed that the CKRC2 and CKRC1 function in the same pathway and that the CKRC2 encodes a rate-limiting enzyme.The phenotype observation showed that the ckrc2-1/yucca8 is the only one in yucca which can be screened from our system.In addition,we analyzed the relative expression levels of all YUCCA genes in roots with or without CK treatment.Our data suggest that the curling roots of ckrc2 are resulted from the high expression of CKRC2 in roots and its upregulation by CK treatment.We showed that the CK regulated auxin biosynthesis in roots was depended on CKRC1/TAA1-CKRC2/YUCCA8 pathway via AHK3-ARR1/12 signaling pathway.Moreover,high temperature could also up-regulate the CKRC1/TAA1-CKRC2/YUCCA8 expression to increase the endogenous auxin level.In addition to its role in the positive effect of temperature on auxin biosynthesis,our results indicated that the transcript factor PIF4 was also needed for CK to promote auxin production in Arabidopsis roots.By map-based cloning and whole genome resequencing,we identified that CKRC3 was AT5G58450,which encodes the auxiliary subunit of the NatB N-a-acetyltransferase complex.ckrc3 exhibited shorter roots,hypocotyls and petioles.Our data showed that the content of both free IAA and its metabolites decreased obviously in ckrc3.Our results suggest that the loss-of-function mutant(nbc-1)of AT1G03150,a gene encoded catalytic subunit of NatB N-a-acetyltransferase complex,exhibited the similar phenotypes on tZ meidum to ckrc3,consistent with the statement that CKRC3 and NBC formed the NatB N-a-acetyltransferase complex.To sum up,it is effective and efficient to screen auxin deficiency mutants by the system we established.Studies on ckrc2-1/yucca8 will shed light on the mechanism how CK and temperature regulate auxin biosynthsis in roots,while the investigation on ckrc3 might lead to the discovery of a new mechanism by which auxin biosynthesis was regulated on post transcriptional level.