Project Summary The plant hormone auxin regulates virtually all aspects of plant growth and development. We and others have demonstrated that auxin promotes degradation of transcriptional repressors called Aux/IAA proteins, via a family E3 ligases called SCFTIR1/AFB. Auxin is perceived by a co-receptor consisting of a TIR1/AFB protein, the F- box subunit of the E3, and the Aux/IAA protein. The formation of this complex promotes degradation of the Aux/IAA protein and transcription by ARF transcription factors. Although this basic pathway is well established, it is not clear how auxin regulates such a wide range of developmental and physiological processes. Currently in the second year of our RO1 grant, we are investigating this broad question using two complementary genetic systems; the flowering plant Arabidopsis thaliana, and the basal land plant Physcomitrella patens. Our focus has been on the function of the TIR1/AFB proteins, regulation of Aux/IAA level, and the architecture of the auxin signaling network. We recently demonstrated that 5 of the 6 member TIR1/AFB family in Arabidopsis act in an overlapping fashion. The 6th member, AFB1, functions in a novel transcription-independent auxin response pathway. Further, our recent ChIPseq experiments indicate that TIR1/AFB proteins (excluding AFB1) are recruited by auxin to chromatin adjacent to auxin-regulated genes. We speculate that this mechanism permits rapid de-repression of auxin responsive genes. We also made significant advances in our understanding of Aux/IAA proteins and their regulation. We recently discovered that Aux/IAA are substrates of E3 ligases called CRL3-BPM, in addition to SCFTIR1/AFB. The BPMs are orthologs of the human SPOP proteins. In addition, we showed that the Aux/IAA genes are regulatory nodes that integrate environmental signals with the auxin gene regulatory network. For example, we have shown that certain Aux/IAA proteins confer drought tolerance by regulating the levels of secondary products called glucosinolates. These compounds promote stomatal closure and drought tolerance. In the next 5 years we will continue to investigate the molecular basis of auxin signaling and to characterize the auxin-based regulatory networks that control plant growth and development. We are very interested in the novel rapid auxin response pathway and its role in growth. We will also investigate the regulation of Aux/IAA levels by both SCFTIR1/AFB and CRL3-BPM. One of our long-term goals is understand the specificity of the AFB, Aux/IAA, and ARF proteins in collaboration with Joe Ecker's lab at the Salk Institute. This effort will include the investigation of AFB recruitment to chromatin. These studies address a number of key issues in cellular regulation and will have important implications for human health.