Project Number: 6250-21520-042-00
Project Type: Appropriated

Start Date: May 21, 2004
End Date: Apr 30, 2009

Objective:
1) Identify and characterize plant genes whose products are involved in root iron acquisition or phloem-to-seed delivery of iron, and assess their overall contribution to seed iron content; 2) Determine the biophysical mechanisms and molecular processes occurring within pods that regulate calcium movement into developing legume seeds; 3) Develop cost-effective methods for the intrinsic stable isotope labeling of plants for use in bioavailability studies; 4) Identify cod [calcium oxalate defective] mutant genes in Medicago truncatula by positional cloning; 5) Determine whether multiple pathways of oxalate biosynthesis exist in plants; 6) Determine the influence of light on calcium oxalate formation in plants; 7) Demonstrate the ability to alter nutrient uptake in plants by expression of CAX2 [calcium exchanger 2] variants in Arabidopsis and tomato; 8) Elucidate the regulation of CAX2; and 9) Characterize plants perturbed in CAX2 expression; 10) Identify and characterize Arabidopsis CAX2 homologs.

Approach:
1) Use diverse pea germplasm to assess the correlation between whole-plant iron partitioning, root iron reductase activity, and seed iron concentration; measure seed mineral concentrations in recombinant inbred lines of Arabidopsis thaliana and Medicago truncatula to identify quantitative trait loci associated with elevated iron content in seeds; conduct global gene expression studies with Medicago truncatula plants bearing mutations in known iron genes to identify new genes involved in iron homeostasis; 2) Measure and characterize calcium accretion in developing seeds of chickpea and measure individual components of calcium transit from pod wall to seeds; 3) Analyze selenate influx kinetics in wheat and broccoli roots and the timing of selenate administration, in order to optimize isotopic selenate incorporation in edible tissues; generate plants labeled with isotopic selenate, or with deuterium oxide, for collaborative bioavailability studies; 4) Use genetic mapping technologies coupled with molecular and transgenic complementation analysis to map and identify genes required for calcium oxalate formation; 5) Analyze metabolic precursors of oxalate, using mutants with altered oxalate levels, to determine the biosynthetic pathway of oxalate in Medicago truncatula; 6) Measure biomass, chlorophyll, starch, and oxalate levels of plants grown under differing light intensities, and use cytochemical investigations to assess the role of oddly shaped plastids present in oxalate accumulating cells; 7) Engineer modified versions of the Arabidopsis broad substrate specificity vacuolar antiporter CAX2 into Arabidopsis and tomato; measure mineral concentrations in tissues; measure transport capacity of variant proteins in both yeast and plant systems; 8) Identify novel regulatory proteins that interact with the N-terminal autoinhibitory sequence of CAX2; 9) Measure phenotypic changes in plants demonstrating an altered expression of the native CAX2 transporter; and 10) Clone and characterize CAX2 homologs in an attempt to understand functional and regulatory diversity among transporters.