We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.
Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.
Iron (Fe) is an important micronutrient in all living organisms. In plants, it is an essential micronutrient1 because of its involvement in basic processes, such as respiration, photosynthesis and chlorophyll biosynthesis. High accumulation of free iron ions is harmful to plant cells due to reactions leading to the release of free radicals causing oxidative stress. To maintain iron homeostasis within the plant cell, ions are stored in vacuoles and sequestered within ferritins, protein cages directly involved in iron homeostasis2 and the principal storage structure of iron in all living organisms. At the same time, iron-deficiency anemia affects a significant proportion of the human population, resulting in an increasing need for plant Fe biofortification. Due to the unique properties of plant ferritin, food enrichment with ferritin-iron offers a promising strategy to fight this problem of malnutrition3.
Iron ions are mainly found in two oxidation states, namely the ferrous (divalent Fe2+ or iron (II)) and ferric (trivalent Fe3+ or iron (III)) forms. Several other forms of iron, such as iron clusters4, are also found in cells. Fe is stored as iron oxide within the cell and naturally forms hematites (Fe2O3) and ferryhidrites ((Fe3+)2O3•0.5H2O) under physiological conditions5. The hydroxides formed in these reactions, especially the ferric form, have very low solubility. Iron retention is consequently affected by the pH of the solution and is largely in a solid state above pH 56.
Considering the poor solubility and high reactivity of Fe, its transfer among plant tissues and organs must be associated with suitable chelating molecules. Moreover, its redox states between the ferrous and ferric forms1 must be controlled. Within leaves, about 80% of the iron is found in photosynthetic cells, due to its essential roles in the electron transport system, in the biosynthesis of cytochromes, chlorophyll and other heme molecules, and in the formation of Fe-S clusters7. In the case of iron excess within the cell, the surplus is translocated into the vacuole where the metal is stored in ferritin molecules8.
Iron can be measured in plant tissues by several methods, including flame atomic absorption spectroscopy9 (FAAS) or colorimetric assays10, the former being far more precise than the latter. FAAS is a highly accurate technique that enables one to determine the elemental composition of a sample on the basis of the electromagnetic emission of the individual elements. FAAS converts metal ions to atomic states by flame-heating of the sample, leading to ion excitation and emission of a specific wavelength when a given ion returns to its ground state. The emissions from the different ions are separated by a monochromator and detected by an absorption sensor11. FAAS thus serves to directly quantify iron concentrations. Other techniques for visualizing iron in biological tissues are, however, available. Inductively-coupled plasma-mass spectroscopy (ICP-MS)12 is a very precise technique for measuring iron and other trace elements but the lack of equipment, both for FAAS and ICP-MS, is a common problem. On the other hand, iron measurement by thiocyanate colorimetry13 lacks precision and fails to detect small variations between samples. Prussian blue staining14,15,16,17 is an indirect method based on the reaction of potassium ferric ferrocyanide (K4Fe(CN)6) with Fe cations, producing a strong blue color, and is used for qualitative iron detection in histological sections of animal and plant tissues.
Metallic (zero-valent) iron is rare in the lithosphere. The dominant non-complexed ionic form of iron in the environment is mostly dictated by the amount of oxygen in the surroundings, with ferrous iron being relatively more abundant in anoxic environments and ferric iron predominating in aerobic sites. This latter form is also dominant in extremely acidic environments, although the causative agents of ferrous iron oxidation often differ in anoxic and acidic surroundings18. When iron is solubilized in 4% HCl (pH 0) in an aerobic environment, the major part of the diluted iron exists as the ferric form (Fe3+)19,20.
The reactions between Fe ions and K4Fe(CN)6 are as follows:
Fe3+: FeCl3 + K4Fe(CN)6 = KFe(III)Fe(II)(CN)6¯ + 3KCl
Fe2+: 4 FeCl2 + 2 K4Fe(CN)6 = Fe4(Fe(CN)6)2 + 8 KCl
In the present study, we asked whether Prussian blue staining can be useful for measuring iron levels in solution.
Initially, we verified the correlation between the concentration of Fe in aqueous solution and Prussian blue staining. The Fe (as FeCl2, FeCl3 or a 1:1 mixture of the two) concentration in aqueous solutions was measured both by atomic spectroscopy and by absorbance (OD) after addition of Prussian blue. Figure 1 shows the linear regression curves for measurements obtained by each method. We concluded that the Prussian blue method can be used for quantitative analysis of iron concentration in solution.
Figure 1: Linear regressions between Fe concentration measured by FAAS and light absorbance (OD, 715 nm) obtained by the Prussian blue method. The blue squares and line represent the Fe2+ solution, the red squares and line represent the Fe3+ solution and the black squares and line represent a 1:1 mixture between Fe2+ and Fe3+. The following regressions were obtained: [Fe2+] = 3 + 123 x OD, r = 0.996, R2 = 0.989; [Fe3+] = 1 + 292 x OD, r = 0.999, R2 = 0.997; and [Fe2+/3+] = 11 + 146 x OD, r = 0.983, R2 = 0.956. The Fe2+ donor was FeCl2 and the Fe3+ donor was FeCl3. Please click here to view a larger version of this figure.
To adapt the colorimetric Prussian blue method for quantitative iron analysis of plant tissues, the iron content of tobacco leaf ashes was measured by flame absorption atomic spectroscopy and Prussian blue staining. There was good correlation between the results from by the two techniques.
1. Plant Material and Growth Conditions
2. Preparing the Leaves for Iron Measurement
Notes: All materials to be used must be iron-free so as to reduce the risk of iron contamination. Clean the mortar and pestle twice with 4% HCl solution and dry with filter paper each time before use. If any material is reused, clean it twice with 4% HCl solution and dry with filter paper.
3. Burning the Leaves to Ash
Notes: The use of a low pH (close to 0) solution of HCl is meant to increase iron solubility. The rock wool is used to prevent the gases from escaping the vial during burning.
4. Preparing the Ashes for Iron Measurement
Notes: The final iron concentration in the initial sample is calculated as the weight of the ashes divided by the added volume of HCl.
5. Measuring Fe Concentration by FAAS
6. Preparing the Prussian Blue Staining Solution
7. Generating a Calibration Curve for the Prussian Blue Method Using FAAS Results
NOTE: Calculate the iron concentration in the ashes using the following formula
Equation 1
C: concentration, V: sample volume, W: ash weight (g).
8. Using the Prussian Blue Method for Determining Iron Levels in Other Samples from the Same Plant Type
Notes: Since a calibration curve has already been established for this type of plant, iron concentration in any new samples from the same plant type can be directly calculated using the linear regression formula.
When this protocol is carried out correctly, one should get excellent correlation between the results obtained by the Prussian blue and atomic spectroscopy methods. Therefore, the Prussian blue method can be easily used to obtain an accurate measurement of iron concentration in plant samples, as reflected in the following experiment.
Tobacco plants were grown as described in the protocol and irrigated with water containing different iron concentrations (0, 1, 2, 3, 4, 5, or 6 mM) over 7 days. The plants were then harvested, cleaned and dried for 3 days at 80 °C. The next steps (i.e., from step 2.3) of the protocol were followed as described. Since the control vials showed a variation of less than 1% from the ash weight, this value was not added to further calculations.
Iron concentration was measured by FAAS. Table 1 shows representative results of these measurements. Data obtained from 21 samples (7 concentrations in 3 replicates) were used to generate a calibration curve.
Treatment (mM iron in irrigation) | Plant | Iron concentration in HCl solution (ppm) |
0 | A | 7.1 |
1 | B | 16.6 |
2 | C | 23.4 |
3 | D | 31.2 |
4 | F | 47.4 |
5 | G | 50.7 |
6 | H | 41.6 |
Table 1: Iron concentration in the ash solutions of tobacco leaves from plants irrigated with water containing different iron levels.
The iron concentration in the ashes of the above mentioned 21 samples was calculated using values obtained by FAAS (see note in step 7). The results showed that iron concentration in the irrigation water greatly affected leaf iron content (Figure 2).
Figure 2: Effect of iron supplied in the irrigation on iron concentration in tobacco leaves. Bars represent standard deviations (n=3). Please click here to view a larger version of this figure.
In a preliminary experiment (not shown), the absorbance spectra of solutions containing different concentrations of Fe2+ and Fe3+ were measured and the best peak was obtained at 715 nm. All spectra of the 21 samples were also tested with the Prussian blue method. It was clear that absorbance at 715 nm was also the optimal wavelength here as well (Figure 3). Accordingly, this wavelength was used in all experiments.
A linear regression curve was plotted between iron concentration values obtained by FAAS and the absorbance (OD) values obtained using the Prussian blue method for the samples represented in Figure 3 (Figure 4). The following regression was obtained: [Fe] = 0.32 + 25.3 x OD, r = 0.994 and R2= 0.988.
Figure 3: Absorbance wavelength spectra of tobacco ashes mixed with Prussian blue solution as described in the protocol. The wavelength spectra were divided by the concentration of ash. Please click here to view a larger version of this figure.
Figure 4: Linear regressions of Fe concentration in tobacco ash measured by FAAS and light absorbance (OD, 715 nm) obtained by the Prussian Blue method. The following regression was obtained: [Fe] = 0.32 + 25.3 x OD, r = 0.994, R2= 0.988. Please click here to view a larger version of this figure.
The obtained linear regression can now be used for new samples from the same plant type, as stated in step 8.
Iron measurement in plant tissues is very important for evaluating the effects of irrigation or other environmental conditions. Here, we described an easy and accurate colorimetric method for Fe content measurement in tobacco leaves, which can be readily adapted to other plant species and tissues.
In optimizing conditions for the colorimetric method, we used a low pH medium (pH < 1.0) to allow iron solubility. The burning process was performed to release all forms of iron and to ensure that no contaminants present in the samples would modify the results. Regarding iron contamination, one should keep in mind that 6.7% of the Earth's crust comprises iron oxide species (FeOn)21 and that their concentrations can reach 0.009% of plant dry weight22. Therefore, one should consider the risk of contamination, since dust, which can contain as much as 3-7% iron (depending on the region)23, can greatly affect results. It is also imperative to carefully check the iron content of all equipment used in the experiment and to take all necessary precautions to avoid contamination. It is recommended to clean all equipment which had been in contact with air or which is being reused in the experiment with 4% HCl solution. The use of any metallic tools, such as scissors or spoons, should be totally avoided, instead being substituted by glass or plastic versions. Critical steps to avoid iron contamination were highlighted in steps 3.1 and 4.1, for example.
Failing to obtain a signal from a sample could occur due to problems arising at different steps in the protocol. In this instance, it is recommended to check the reagents against solutions with a known iron concentration. If problems persist, fresh Prussian blue reagent should be prepared. Should the standard, however, show a signal, this indicates that the iron concentration in the sample was below the detection threshold and that the sample should be more concentrated. Large variations among replicates could stem from iron contamination and will require the use of new samples.
Calibration curves need to be generated every six months or every time a new batch of Prussian blue reagent is used. The initial calibration curve should be revalidated by measuring iron content in at least 6 samples and confirming that the linear regression is still accurate. In calibration curves, it is essential to obtain values close to +1 or -1 for r (the correlation coefficient) and close to 1 for R2 (the coefficient of determination)24,25,26. While a perfect line would have an R2 value of 1, most R2 values over 0.95 are acceptable for calibration curves.
Limitations of the protocol include the fact that different plant tissues and species may show different correlation curves using the described procedures for iron concentration determination. Therefore, it is recommended to perform a pilot test to ensure that the correlation curve is accurate for a given experiment. To use the curve for obtained results, the newly measured OD value should be substituted in the formula Y = a + bX. In the present case, the following formula was used: [Fe] = 0.32 + 25.3 x OD.
Another limitation arises from the need of a rather large amount of plant tissue. This can be circumvented to some extent by adapting the HCl volume used to dissolve the ashes (see step 5.3) so as to obtain an appropriate signal. Nonetheless, the method described here is a good option if costly equipment, such as that used for ICP-MS, or ready access to FAAS is not available and/or numerous samples of the same plant species are to be analyzed.
Possible additional applications of the Prussian blue method as an iron detection system include quantitative iron detection in any organic material that can be brought to ash, as well as in inorganic matter, such as soils.
The authors have nothing to disclose.
This work was supported by the Israel Ministry of Science, Technology and Spaceand by a grant from the Chief Scientist of the Israeli Ministry of Agriculture (#16-16-0003).
Potassium Hexacyanoferrate(II) | Fisher Chemical | 14459-95-1 | Reagent for the Pussian Blue |
Millex Syringe Filter Unit, Vial Vent 0.22 μm | Millec | SLGP033RS | Filter used to filter the ashes + 4% HCl Solution |
Scintillation Vials | Fisherbrand | 03-337-4 | Used to keep the dry powdered plant material during the burning procedure. |
Disposable Syringe 10 ml | Medi-Plus | 1931 | Syringe used during the filtration |
Hydrochloric acid | Sigma-Aldrich | 231-595-7 | Used in the 4% HCl solution to dilute the ashes and clean the materials |
Tobacco, Nicotiana tabacum cv. Samsun NN | Obtained from Prof. Simon Barak and routinely used in the Zaccai Lab | Barak S, Nejidat A, Heimer Y, Volokita M. Transcriptional and posttranscriptional regulation of the glycolate oxidase gene in tobacco seedlings. Plant Molecular Biology. 2001 Mar 1;45(4):399-407. | Tobacco cultivar used in this protocol |
Glass Wool (Rock Wool) | Sigma-Aldrich | 659997-17-3 | Used in the procedure of burning samples in the furnace. |