The plant hormone auxin is required for diverse aspects of plant growth and development. We have shown that auxin is requires conjunction of the ubiquitin-like protein RUB to one or more cellular proteins. In Arabidopsis, the RUB-activation enzyme AXR1-ECR1 and the RUB- conjugating enzyme RCE1 participate in RUB conjugation. Recessive mutations in the AXR1 gene or expression of a dominant-negative version of ECR1, result in reduced auxin response. In plants, fungi and animals, RUB is conjugated to members of the cullin family of proteins, including Cdc53 in S. cerevisiae and AtCUL1 in Arabidopsis. Our studies indicate that RUB modification affects the function of the cullin rather that its stability. In the case of Arabidopsis, we propose the RUB-modification of AtCUL1 affects the function of SCFtiri, a ubiquitin-protein ligase required for auxin response. In this project we will continue our studies of auxin action by investigating the function of these isoforms both in vitro and in vivo. In addition, there are at least two divergent RUB proteins. The function of these two molecules in auxin response will be determined. The second aim is to confirm that RUB-modified AtCUL1 is required for auxin response. There are at least two additional cullins in Arabidopsis and the functions of these proteins will be investigated. The third aim is to determine the effects of RUB-modification on AtCUL1 function using a variety of experimental approaches. The fourth aim is to isolate and characterize the SAR1 and AXR4 genes. Genetic studies indicate that both of these genes function in the AXR1-TIR1 pathway. In the fifth and last specific aim we will conduct the genetic and phenotype characterization of several novel suppressors of the axr1 mutation. Based on the results of these studies we will isolate and characterize one or more of these new genes. We expect the results of these studies to provide important new information on the mechanism of auxin response. In addition, our studies will provide new insight into the function of the highly conserved RUB- conjugated pathway in all eukaryote.