Title: The root iron reductase assay: an examination of key factors that must be respected to generate meaningful assay results Author
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 1, 2010
Publication Date: June 26, 2010
Citation: Grusak, M.A. 2010. The root iron reductase assay: an examination of key factors that must be respected to generate meaningful assay results [abstract]. 15th International Symposium on Iron Nutrition and Interactions in Plants. p. 84. Technical Abstract: Plant iron researchers have been quantifying root iron reductase activity since the 1970's, using a simple spectrophotometric method based on the color change of a ferrous iron chromophore. The technique was used by Chaney, Brown, and Tiffin (1972) to demonstrate the obligatory reduction of ferric iron, prior to ferrous iron uptake, in soybean. Subsequently, researchers have used this technique to assess a host of plant species, using plants maintained on iron-sufficient, iron-limiting, or iron-deficient conditions. The general method is to immerse roots in a solution containing a ferric iron chelate, along with a ferrous iron complexing agent (either bathophenanthroline disulfonic acid [BPDS] or ferrozine). After a fixed amount of time, an aliquot of the assay solution is collected for spectrophotometric analysis, the plant roots are weighed (usually fresh weight), and a reductase rate is calculated based on the assay solution volume, the solution's absorbance reading, the assay time, the root weight, and the appropriate extinction coefficient for the chromophore. This rate is generally defined to be derived from membrane-localized reductase activity, or is also referred to as activity resulting from one of the FRO proteins. The methods for this assay seem to have been passed down through the literature, or from lab to lab, but without a full, critical assessment of the various contributing and confounding factors that must be addressed to yield a valid, reliable reductase measurement. In this contribution, we will present an analysis of the following factors relevant to root iron reduction: (1) handling the roots (2) using whole root systems versus root sections (3) intact plant versus excised roots (4) collecting assay aliquots (5) optimal assay volume (6) root size (7) weighing roots (8) test of assay time-dependent linearity, to determine proper assay duration (9) test for soluble reductants (10) test for change in assay iron concentration. We will discuss the impact of these factors on the root iron reductase measurement and what steps the investigator should take to meet basic assumptions behind these factors. Based on this examination, we will offer new recommendations for how one should approach and conduct this central assay for plant iron research.