|Punshon, Tracy -|
|Tappero, Ryan -|
|Ricachenevsky, Felipe -|
|Hirschi, Kendal -|
Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 2, 2013
Publication Date: October 10, 2013
Citation: Punshon, T., Tappero, R., Ricachenevsky, F.K., Hirschi, K., Nakata, P.A. 2013. Contrasting calcium localization and speciation in leaves of Medicago trunculata mutant COD5 analyzed via synchrotron X-ray techniques. Plant Journal. 76:627-633. Interpretive Summary: Oxalate is an antinutrient found in foods that binds calcium in a form that prevents humans from absorbing this essential mineral. Thus, efforts have been made to try and find ways to reduce the amount of oxalate in edible plants. Recently, we reported the identification of a genetically modified plant from the model legume, Medicago truncatula. Compared to the parent plant this modified plant had dramatically reduced oxalate amounts but the same amount of calcium. Animal feeding studies revealed that the modified plant was a better nutritional source of calcium than the original parent plant. Thus, the modified plant was nutritional improved. In this study we show using cutting edge biophysical technologies that it is possible to visually detect where the calcium is found within the plant leaves and what is bound to it. These technologies allowed us to see in the modified plant that the calcium was redistributed within of the leaves and was no longer bound to oxalate. Thus, this report not only highlights the use of these new technologies for the study of mineral distributions but also advances our knowledge about how an alteration in the distribution and storage of calcium affects its nutritional value. Such information helps us in our efforts to design strategies aimed at improving the nutritional value of plant foods through the redistribution and storage of minerals.
Technical Abstract: Oxalate-producing plants accumulate calcium oxalate crystals (CaOx(C)) in the range of 3-80%(w/w) of their dry weight, reducing calcium (Ca) bioavailability. The calcium oxalate deficient 5 (cod5) mutant of Medicago truncatula has been previously shown to contain similar Ca, but lower oxalate and CaOx(C) concentrations than wild type (WT) plants. In feeding studies, cod5 has appreciably higher Ca bioavailability. We imaged the Ca distribution in both freeze-dried, whole and fresh, cryo-sectioned WT and cod5 leaves via synchrotron X-ray fluorescence (SXRF) mapping and found a striking contrast in Ca localization in cod5 and WT leaves, manifested as a lack of Ca in the crystal idioblast cells that flank the secondary veins of cod5. X-ray microdiffraction of cryo-sectioned Medicago leaves confirmed X-ray crystalline CaOx(C) (whewellite; CaC2O4 H2O) in WT idioblast cells only. Together with mXRD, microbeam Ca K-edge X-ray absorption near-edge structure (uXANES) spectroscopy suggests that of the forms of CaOx (crystalline or amorphous), only the X-ray amorphous CaOx form was found in cod5. This study is novel in studying both the localization and speciation of the macronutrient Ca in WT and a mutant of a model plant using synchrotron techniques as means of characterizing the function of a gene.