2007 Annual Report 1a.Objectives (from AD-416) Elucidate the biochemical and molecular basis of Or in controlling carotenoid accumulation. The three specific objectives are:. 1)to examine whether Or is a gain-of-function or a dominant negative mutation;. 2)to identify proteins that interact with OR; and. 3)to functionally study OR-protein association in regulating carotenoid accumulation. 1b.Approach (from AD-416) Objective 1: Investigating the nature of Or mutation. The approaches will include to generate Arabidopsis and cauliflower Or RNAi transgenic lines and transformants overexpressing the wild type or gene, and to examine the phenotype and carotenoid levels of these transformants. Also, we will generate transformants overexpressing the Or variants and examine the role of the individual Or transcript in inducing carotenoid accumulation. Objective 2: Isolating OR-associated proteins. The experimental strategies will be to employ blue native gel electrophoresis (BN-PAGE) followed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF MS) to identify protein components associated with OR, or to use yeast two-hybrid and GST-tagged pull-down assays to isolate proteins that physically interact with OR. Objective 3: Functionally investigating OR-protein interaction. This will involve using molecular and biochemical approaches to examine the correlation of gene expression in RNAi or T-DNA insertion lines and analyze the functional relevance of OR-interacting protein in regulating carotenoid accumulation. 3.Progress Report This report documents research conducted under a Reimbursable Agreement between ARS and Cornell University. Additional details of research can be found in the report for the in-house associated project 1907-21000-025-00D "Genomics approaches for improving nutritional quality of food crop species.” The main objective of this project is to elucidate the biochemical and molecular basis of a cauliflower Or gene in controlling carotenoid accumulation. Specific activities during this period included to examine whether Or is a gain-of-function or a dominant negative mutation and to identify proteins that interact with OR protein via a yeast two hybrid approach. We have generated cauliflower or dsRNAi and overexperssing transgenic lines and showed that those transformants exhibit no orange mutant phenotype. These results suggest that the cauliflower Or gene is likely a gain-of-function mutant. By screening an Arabidopsis expression library using the OR protein as a bait, we have isolated a protein that interacts with the OR protein through a yeast two hybrid approach. This protein has been demonstrated to physically associate with the OR protein by a protein pull-down assay. Currently, we are in the process to characterize the OR-interacting protein to examine how such interaction affects on the metabolic processes affected by the OR protein. In addition, we are also using proteomics approach to identify proteins that are specifically associated with carotenoid accumulating structures in the orange cauliflower mutant to gain a better understanding the basis of the Or gene in inducing carotenoid accumulation. The Reimbursable Agreement is monitored via regular meetings with the Cornell cooperators.