Determination of Fe(III) Reduction Activity

The FRA was determined as described by García et al. [56 (link)]. Previously to determine FRA, plants were subjected to a pre-treatment for 30 min in plastic vessels with 50 mL of a nutrient solution without micronutrients, pH 5.5. Then they were transferred into 50 mL of a Fe (III) reduction assay solution for 1 h. This assay solution consisted of nutrient solution without micronutrients, 100μM Fe(III)-EDTA and 300 μM Ferrozine, pH was adjusted to 5.0 with KOH. The environmental conditions during the measurement of Fe (III) reduction were the same as the growth conditions described above. FRA was determined spectrophotometrically by measuring the absorbance (562 nm) of the Fe(II)-Ferrozine complex and by using an extinction coefficient of 29.800 M⁻¹ cm⁻¹. After that, roots were excised and weighed, and the results were expressed on a root fresh weight basis. Also, SPAD values (as a proxy of the chlorophyll concentration in leaf) were measured daily with a portable chlorophyllmeter (SPAD 502 Minolta Camera Co., Osaka, Japan).