Materials and Reagents

Butter paper
Tissue paper/Kimwipes
15 mL screw cap conical tubes (Sarstedt, catalog number: NC1377856)
96-well microplate (Greiner CELLSTAR^®, catalog number: 655180)
Nitric acid (65%) (Merck, catalog number: 1.00456.1000, store at room temperature)
Hydrogen peroxide solution (30%) (Merck, catalog number: 1.07209.1000, store at room temperature)
ddH₂O
Bathophenanthrolinedisulfonic acid disodium salt hydrate (Sigma, catalog number: B1375-5G, store at room temperature)
Sodium acetate (Sigma, catalog number: S8750-500G, store at room temperature)
Hydroxylamine hydrochloride (Alfa Aesar, catalog number: A15398.36, store at room temperature)
Iron (III) chloride hexahydrate (Merck, catalog number: 1.03943.0250, store at room temperature inside a dry cabinet)
Assay solution (see Recipes)
Fe standard solutions (see Recipes)

Equipment

Vannas scissors
Drying oven (Hot air oven, PREMA^®)
Vortex mixer (Vortex-Genie 2, Scientific Industries)
Heat block (Elite Dry Bath Incubator, Major Science)
Microplate spectrophotometer (Power Wave XS2, BioTek Instruments, Agilent Technologies)

Software

Gen5^TM BioTek Instruments (Agilent Technologies)
GraphPad Prism version 9

Procedure

Sampling
1. Sow seeds on Estelle and Somerville (ES) nutrient media (Estelle and Somerville, 1987), and stratify for at least two days in the dark at 4°C, before transferring to a growth chamber. Grow seedlings at 21–22°C under continuous illumination (50 μmol m²s⁻¹) for two weeks.
2. To separate seedlings into shoots and roots for sample harvesting, use Vannas scissors to cut seedlings on the media at hypocotyl junctions.
3. For shoot samples, collect 25–30 shoots of two-week-old seedlings in butter paper.
4. For root samples, collect 30–36 roots of two-week-old seedlings in butter paper. Before placing roots into butter paper, make sure that the roots don’t contain any residual media, which also contains Fe. Remove any media from the roots with tissue paper/Kimwipes, followed by washing twice with ddH₂O. Then gently dry roots with tissue paper/Kimwipes.
5. Dry samples in an oven for at least two days at 60–65°C.
6. Determine the dry weight of each sample.
7. After weighing, transfer the samples into 15 mL conical tubes.
Sample digestion
1. Add 225 µL of nitric acid (65%) into each tube and screw the caps tightly.
2. Incubate for 6 h at 95°C on a heat block, vortexing the tubes every 1–2 h. Make sure that samples do not stick to the walls of the tubes and no precipitates are present, as these could affect measurements.
3. Carefully unscrew the caps of the tubes, add 150 µL of H₂O₂(30%) into each tube, and screw the caps back tightly.
4. Incubate for 2 h at 56°C on a heat block, and vortex every 30–60 min to make sure no precipitates are formed, as this could also affect measurements.
5. Add 225 µL of ddH₂O. Fully digested samples should be pale yellow in color, and ideally should have no white precipitates.
6. For the ease of pipetting in the following steps, carefully transfer the digested samples into 1.5 mL microcentrifuge tubes. At this point, samples are ready to be immediately used for the Fe quantification in procedure C, or can be stored at 4°C in the dark for up to 1 month for later analysis.
Standard and sample preparation, and spectroscopic reading
1. Prepare the assay solution (see Recipe 1) and Fe standard solutions (see Recipe 2).
2. Add 5 µL of standards/samples in microplate wells. Run three technical replicates for each standard/sample.
3. Add 245 µL of assay solution to each well with standard/sample.
4. Cover the microplate and keep it in the dark to incubate at room temperature for 5 min before spectrophotometric measurement. Following incubation, the standards will turn pink/red, with increasing color saturation as the Fe level increases (Figure 1A), and the samples will turn light yellowish-pink/orange-red depending on the Fe content.
5. Measure absorbance at 535 nm. Use the 0 Fe standards as blanks to zero the plate reader.

Data analysis

Create a standard curve in an Excel sheet by plotting the average blank-corrected 535 nm absorbance values vs. the concentration of standards. Make sure that the coefficient of determination (R²) value is ≥0.990.
Use the equation derived from the standard curve to calculate the Fe concentrations in the samples. Table 1 shows the calculation of Fe concentrations in root samples.
Divide the Fe concentrations of the samples by their respective dry weight (DW), to obtain the final Fe concentration per g DW (Table 1).
Plot the final graph in Excel or any suitable software. The representative plot shown in Figure 1B was generated using GraphPad Prism 9. The sample data shown only represents one biological replicate, with three technical replicates. A complete experiment should have at least three biological replicates.

Table 1. Calculations to quantify Fe in root samples.
Spectrophotometric readings of the standards were used to plot a calibration curve. The Fe concentrations in the samples were calculated using the equation derived from the slope. Wild-type (Col-0) plants, a coumarin-deficient mutant (f6’h1-1; Schmid et al., 2014), and an Fe over-accumulating genotype (IMA2ox; Gautam et al., 2021) grown on nutrient media containing 50 µM Fe-EDTA are used to represent Fe quantification. The data represent the standard deviation of three technical replicates from the spectrophotometer reading. The microplate layout used to make spectrophotometric readings is shown at the top. STD, standard; S, sample; Abs., absorbance; DW, dry weight; Avg., average; SD, standard deviation.

Figure 1. Fe content-dependent color pattern of Fe²⁺(BPDS)₃ complexes and their spectrophotometric quantification. A. Representative figure showing the change in color of Fe²⁺(BPDS)₃ complex across an increasing Fe content gradient. Equal volume of assay solution was added in a 96 well plate microplate containing different amounts of Fe (0, 2.5, 5, 10, 20, and 40 µg) in three technical repeats. The microplate was kept in the dark to incubate at room temperature for 5 min, a photo was captured immediately afterwards. B. Graph showing the Fe concentrations in different samples (genotypes). Wild-type (Col-0) plants, a coumarin-deficient mutant (f6’h1-1; Schmid et al., 2014), and an Fe over-accumulating genotype (IMA2ox) (Gautam et al., 2021) grown on nutrient media containing 50 µM of Fe-EDTA are used to represent the Fe quantification. The data represent standard deviation of three technical replicates from the spectrophotometer reading. DW, dry weight.

Notes

This method can be used to quantify Fe in samples grown on both soil and nutrient media.
Steps B to C need to be carried out inside a fume hood with proper protection, including corrosion resistant gloves, safety glasses, lab coat, and shoes.
The sample volume should be reduced or the reagent volumes should be adjusted for complete digestion of samples.
Use only high-grade acid-resistant falcon, PTFE, or Teflon^TM tubes for acid digestion. Make sure to tightly cap the tubes during the wet acid digestion steps.
Although the volume of the digested sample is only 600 µL, a 15 mL Falcon tube is used for safety reasons, and to avoid damaging the tubes due to pressure from gas build-up during the heat assisted digestion steps.

Recipes

Assay solution (for 100 mL)

No.	Reagent	Working concentration	Amount to add
1.	Bathophenanthrolinedisulfonic acid disodium salt hydrate (MW: 590.53 g/mol)	1 mM	0.06 g
2.	Sodium acetate (MW: 82.03 g/mol)	0.6 M	4.92 g
3.	Hydroxylamine hydrochloride (MW: 69.49 g/mol)	0.48 M	3.34 g
4.	ddH₂O	-	adjust to 100 mL

Note: Prepare fresh assay solution in a light-shield bottle and store at 4°C until use.

Fe standard solutions

Prepare a 50 mM FeCl₃stock solution (50 mM FeCl₃ contains 2.79 µg/µL of Fe). With this stock solution, prepare Fe standards in 1.5 mL microcentrifuge tubes as follows:

Standards	Blank	STD1	STD2	STD3	STD4	STD5	STD6
Fe content (µg)	0	1.25	2.5	5.0	10.0	20.0	40.0
Amount of stock to add (µL)	0	0.45	0.90	1.79	3.58	7.17	14.34
Nitric acid (65%)	225 µL into each tube
H₂O₂(30%)	150 µL into each tube
ddH₂O	225 µL into each tube

Acknowledgments

This work was supported by a grant from the Ministry of Science and Technology to W.S. (grant No.: 108-2311-B-001-033-MY3). The data used to create the representative graph and table has been extracted from previously published work (Gautam et al., 2021).

Competing interests

The authors declare no conflict of interest.

References

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