Project Number: 3640-21000-024-03
Project Type: Reimbursable

Start Date: Jun 25, 2007
End Date: Oct 24, 2008

Objective:
1. Use state-of-the-art genomic technologies and the forthcoming whole genome sequence of soybean to identify genes potentially involved in IDC efficiency or inefficiency. 2. Assess the gene expression and biological activity of specific proteins (organic acid transporters, iron reductases, and other enzymes) in soybeans having efficient and inefficient responses to iron stress (IDC). 3. Develop molecular markers that have diagnostic potential in identifying IDC efficient genotypes, extend the usefulness of markers to a broader range of soybean germplasm, and make the markers available to soybean.

Approach:
Evaluate global gene expression in near isogenic lines differing in IDC responsiveness grown under Fe-sufficient and Fe-deficient conditions. Using RNA isolated from plants grown under Fe-sufficient and Fe-deficient conditions we will probe the Affymetrix soybean microarray chip to identify differentially expressed genes. Our emphasis will be on the identification of differentially expressed genes in roots and stems, complementing the effort focused on leaves in Dr. Shoemaker's laboratory. We will also emphasize defining the functional role of genes identified through microarray analysis. Selected genes that respond to Fe identified through Affymetrix microarray analysis will be verified by semiquantitative RT-PCR. Due to their postulated role in the IDC response, we will focus on genes involved in organic acid metabolism and transport, MATE genes, and transcription factors (bHLHs and WRKYs). Root, stem, and leaf genes identified as being involved with IDC will be further characterized (sequence, tissue expression, developmental stage, other nutrient stresses) and will be forwarded to Drs. Shoemaker and McClean for use as molecular markers. Two genes that we are particularly interested in are Frd3 (a citrate efflux transporter) and Fit1 (a bHLH transcription factor). We will identify the soybean orthologs for these genes and assess their expression levels and tissue expression patterns. Should they show IDC responsiveness, we will isolate the promoters for Frd3 and Fit1, prepare promoters:GUS constructs, and transform these constructs into soybean. GUS activity will be assayed in plants grown under a variety of stress conditions. Dr. Clemente at the University of Nebraska has agreed to perform soybean transformations. Should time and finances allow, we will prepare constructs for overexpression of Frd3 and transform soybean.