2008 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
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 the functional role of the isolated OR-interacting protein (OIP). While this protein belongs to a three member protein family and is involved in protein metabolism, its physiological function remains unknown. Comparison of expression patterns of At-OR and Oip shows that both genes are expressed throughout development although the abundance of their transcripts varies in different tissues and organs. Moreover, we show that overexpression or down regulation of the Oip affects the plant’s response to plant hormones resulting in short roots and delayed flowering. These results provide a new functional role of this isolated protein and helps explain the polytrophic effect of Or in affecting plant growth. This work fits into Component I: Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement, and Component 2: Biological Processes that Improve Crop Productivity and Quality, of the NP302 Action Plan. Management and Communication: Project participants including project leaders and key technicians, postdocs and graduate students involved in the project meet semi-annually in person or via conference calls. Phone conversations and email are used on a frequent basis as needed to address questions, technical problems, or to plan next steps when milestones are reached. Project ARS and Cornell participants work together on a daily basis which insures constant communication.
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