Critical evaluation of three indirect assays for quantifying phytosiderophores released by the roots of Poaceae

Indirect assays are commonly used to measure phytosiderophore (PS) concentrations in the root exudates of grasses. The Cu-mobilizing, Cu-CAS and Fe-binding assays are three commonly used assays but there have been few published attempts to validate or calibrate them rigorously. Thus, we undertook to compare, validate and, where appropriate, to improve and/or optimize each assay. Ethylenediamine-N,N'-diacetic acid (EDDA) and N-2-hydroxyethylethylenediamine-N,N',N'-triacetic acid (HEDTA) bracket deoxymugineic acid for binding strength with both Fe and Cu and were thus used as model ligands. For the Cu-mobilizing assay, reasonable regressions (as R-2) were obtained between measured and known ligand concentrations for all resin combinations tested. However, the relationship was non-stoichiometric; less than 5% of the ligand present was recovered as a Cu-complex. Testing of the Cu-CAS assay yielded good 1:1 stoichiometric relationships (slopes of 0.97 and 1.08 for EDDA and HEDTA, respectively) with little scatter (R-2 = 0.97). However, the limits of quantification were approximately 30 mu M, which is greater than concentrations generally found in root-exudate collections. Thus, the Cu-CAS assay is best suited to situations where large concentrations of PSs are expected. Results from the original Fe-binding assay indicated a lack of linearity and considerable scatter (R-2 = 0.57 for EDDA; R-2 = 0.28 for HEDTA). Thus, as published, use of the original Fe-binding assay is not recommended. The effects of filter paper, FeCl3 reagent concentration and increase in the reduction time were tested and a revised Fe-binding assay developed with both good stoichiometry and great precision (R-2 >= 0.99). We suggest the use of the revised Fe-binding assay in most situations where PS concentration would be expected to be < 30 mu M.