Brassinosteroids are growth-promoting hormones that are required for plant development. Most investigations have concentrated on either brassinosteroid biosynthesis or on their response pathways. In contrast, the Neff group studies brassinosteroid inactivation and has demonstrated the importance of this process with regard to plant development. To gain a more comprehensive understanding, the Neff group has expanded their studies to include all five potential brassinosteroid inactivating enzymes. The following hypotheses will be tested in this study: 1) The five different brassinosteroid-inactivating pathways have redundant as well as distinct roles in development. The Neff lab will use molecular, genetic and biochemical analysis to examine the potential overlap between each of these brassinosteroid-inactivating pathways. 2) The five different brassinosteroid-inactivating pathways have overlapping and distinct regulation mechanisms. The Neff group has identified a transcription factor that modulates both CYP734A1 expression and plant responses to the most-active brassinosteroid, brassinolide. They will expand these molecular/genetic studies to include all five brassinosteroid-inactivating genes in Arabidopsis. 3) CYP72C1 uses inactive brassinolide precursors as a substrate for regulating hormone levels. Early attempts at identifying CYP72C1 products via over-expression in plants or in heterologous expression systems involved feeding studies with brassinolide. However, the Neff group has recently discovered that CYP72C1 does not use brassinolide as a substrate, instead preferentially binding biochemical precursors. They have established international collaborations with hormone biochemists and will use multiple expression systems coupled with biochemical analysis to identify the product of CYP72C1 activity. Broader impact of the proposed activity: This work will likely impact multiple areas of plant research including studies of: plant hormones, gene regulation, and genetic interactions during development. These studies will contribute directly to the education of two Ph.D. students as well as undergraduate students in the Neff group. As in the past, results from these studies will be presented in peer-reviewed publications, in national and international meetings as well as in local public schools.