USE OF SYNCHROTRON X-RAY FLUORESCENCE MICROTOMOGRAPHY TO CHARACTERIZE THE GENES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our research combines established genomic techniques, such as gene deletion and overexpression, with synchrotron x-ray fluorescence (SXRF) microspectroscopy and microtomography to characterize the function of genes involved in metal ion homeostasis in plants. We pioneered the use of SXRF in gene characterization with a gene critical for vacuolar iron (Fe) transport in seed, which has far reaching implications for the development of Fe enriched grain and the reduction in Fe deficiency anemia in humans. While mature seed represents an ideal sample for SXRF analysis, seed-expressed genes are only a subset of the plant genome. To characterize genes that encode for metal(loid) transporters in roots, leaves and developing seed, fresh or live plant imaging is preferred, due to the elemental and distributional artifacts that can result from dehydration, resin infiltration and sectioning. Constraints on live or fresh plant imaging range from the sensitivity of the detector, the motion of the stepping motors, as well as a range of issues with sample drying. We have conducted preliminary studies at BL2-3 that have allowed us to collect tomograms of Fe and other important elements from the excised stem of a 2 day old Arabidopsis seedling in approximately one hour, analysis that would have taken 10 hours at other environmental microprobe beamlines. By comparing the wild type tomogram with that of a mutant lacking a vacuolar iron transporter (VIT1) we have shown clear distributional contrasts that have shed new light on the function of the gene. We wish to conduct fresh- and live-plant imaging studies at BL2-3 to help characterize genes involved in metal ion homeostasis. Our current projects involve further characterizing genes that encode for vacuolar Fe transporter, VIT1, and transporters responsible for the transport of arsenic within the plant and into the seed.