Project Number: 1907-21000-025-05
Project Type: Reimbursable

Start Date: Dec 01, 2006
End Date: Nov 30, 2009

Objective:
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.

Approach:
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.