Background

Iron (Fe) is an essential micronutrient, which is involved in numerous biochemical and physiological processes in plants. A deficiency or an excess of Fe in plants limit plant growth and cause severe losses in crop yield and quality. Imbalances of Fe levels in plants also affect the homeostasis of other nutrients (Schmidt et al., 2020). Therefore, the determination of Fe concentrations in plant tissues is mandatory for the assessment of the nutritional status of the plant and serves as an important parameter for studying Fe-related genes. Currently, inductively coupled plasma (ICP) spectrometry-based methods are considered the gold standard for determining Fe levels. Although these methods are highly sensitive and reliable, their accessibility is rather limited due to costly equipment, specialized training required for the operation of such equipment, and the requirement of generally large amounts of material, which may render the analysis difficult in cases where sample size is limited. Therefore, a simple, reliable, and easily accessible method to measure Fe levels at high accuracy is a valuable alternative to ICP-based methods.

Here, we describe a method for quantifying total Fe in roots and shoots of plant seedlings. The method is based on a protocol established for the analysis of Fe concentration in Plantago lanceolata L. leaves (Schmidt, 1996), which we have adapted and optimized for convenient and routine analysis of the more commonly used model plant Arabidopsis thaliana. The method involves wet acid digestion of dried plant samples with nitric acid and hydrogen peroxide, to solubilize Fe and reduction of Fe(III) complexes by hydroxylammonium chloride, followed by a colorimetric assay based on the ability of the chelating agent bathophenanthroline disulfonate (BPDS) to form red-colored complexes with ferrous Fe. The resulting Fe²⁺(BPDS)₃ complex has a distinct absorbance band at 535nm under acid conditions, allowing for the quantification of Fe concentrations by interpolation from a standard curve. BPDS has been widely used for the determination of Fe levels in a variety of samples (Pré and Benlatrèche, 1977; Tangerås, 1983; Hirayama and Nagasawa, 2017; Freinbichler et al., 2020). The present protocol was optimized for Arabidopsis thaliana samples, and has been used for quantifying Fe concentrations as low as 50 μg/g dry weight sample or less (Tsai et al., 2018; Gautam et al., 2021), but can be modified and applied to determine Fe levels in samples from other plant species.