Name: a colorimetric method for measuring iron content in plants
Uploaded: 2018-09-07T13:30:00
Description: Watch this Scientific Journal Video about A Colorimetric Method for Measuring Iron Content in Plants at JoVE.com

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).

1. Plant Material and Growth Conditions
<ol>
	<li>Seed one tobacco (cultivar Samsun) seed per 5 cm x 5 cm pot filled with standard pot medium. Place the pots on trays. Grow the plants in a growth room under long day conditions (16/8 h light/dark) at a constant temperature of 23 &#176;C. Irrigate with tap water until water drains from the pot.</li>
	<li>After 50&#177;5 days, start Fe treatments in the irrigation, according to the concentrations suitable for the experiment. For example, we used a range of iron concentrat...

<ol>
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R., Rodriguez-Ramiro, I., Fairweather-Tait, S., Uauy, C., Balk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+expression+of+a+vacuolar+iron+transporter+doubles+iron+content+in+white+wheat+flour.">Altering expression of a vacuolar iron transporter doubles iron content in white wheat flour.</a> bioRxiv. , 1-25 (2017).</li><li>de la Fuente, V., Rufo, L., Rodr&#237;guez, N., Franco, A., Amils, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+iron+localization+in+wild+plants+and+hydroponic+cultures+of+Imperata+cylindrica+(L.)+P.+Beauv.">Comparison of iron localization in wild plants and hydroponic cultures of Imperata cylindrica (L.) P. Beauv.</a> Plant Soil. 418 (1-2), 25-35 (2017).</li><li>Hsiao, P. Y., Cheng, C. P., Koh, K. W., Chan, M. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+correlation+coefficient:+An+overview.">The correlation coefficient: An overview.</a> Critical Reviews in Analytical Chemistry. 36 (1), 41-59 (2006).</li><li>. Analytical Chemistry. Calibration Curves Available from: <a target="_blank" href="https://www.jove.com/science-education/10188/calibration-curves">https://www.jove.com/science-education/10188/calibration-curves</a> (2018)</li></ol>

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 &#60; 1.0) to allow iron solubility. The burning process was performed to release all forms of iron and to ensure tha...

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&#8226;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&#175; + 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57408/57408fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/57408/57408fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.

<table>
	<tbody>
		<tr>
			<td>Potassium Hexacyanoferrate(II)</td>
			<td>Fisher Chemical</td>
			<td>14459-95-1</td>
			<td>Reagent for the Pussian Blue</td>
		</tr>
		<tr>
			<td>Millex Syringe Filter Unit, Vial Vent 0.22 &#956;m</td>
			<td>Millec</td>
			<td>SLGP033RS</td>
			<td>Filter used to filter the ashes + 4% HCl Solution</td>
		</tr>
		<tr>
			<td>Scintillation Vials</td>
			<td>Fisherbrand</td>
			<td>03-337-4</td>
			<td>Used to keep the dry powdered plant material during the burning procedure.</td>
		</tr>
		<tr>
			<td>Disposable Syringe 10 ml</td>
			<td>Medi-Plus</td>
			<td>1931</td>
			<td>Syringe used during the filtration</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>231-595-7</td>
			<td>Used in the 4% HCl solution to dilute the ashes and clean the materials</td>
		</tr>
		<tr>
			<td>Tobacco, Nicotiana tabacum cv. Samsun NN</td>
			<td>Obtained from Prof. Simon Barak and routinely used in the Zaccai Lab</td>
			<td>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.</td>
			<td>Tobacco cultivar used in this protocol</td>
		</tr>
		<tr>
			<td>Glass Wool (Rock Wool)</td>
			<td>Sigma-Aldrich</td>
			<td>659997-17-3</td>
			<td>Used in the procedure of burning samples in the furnace.</td>
		</tr>
	</tbody>
</table>

a colorimetric method for measuring iron content in plants

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.

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 diffe...

Watch this Scientific Journal Video about A Colorimetric Method for Measuring Iron Content in Plants at JoVE.com

A Colorimetric Method for Measuring Iron Content in Plants

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 ...

Just medically it can help answer a key question in biology field. Such as how can we easily quantify iron in biology core samples. The main venture of this technique is that it's a easy to produce, and a very precise colorimetric technique.While preferring this technique, you should avoid iron contamination. Visual demonstration of this method will help you to understand its different steps. To begin this procedure, prepare five centimeter by five centimeter pots by filling them with standard pot medium.Plant one tobacco seed in each pot. Transfer the plants to a growth room that is at a constant temperature, under long day conditions of 23 degrees Celsius. Irrigate with tap water until water drains from the pot, and grow the plants for approximately 50 days.After this time, start iron treatments in the irrigation at concentrations appropriate for the experiment. Irrigate the plants with this solution every two days for six to eight days. Then, detach the leaves from the stem by hand, making sure not to use any metal equipment.Using a spray bottle filled with double distilled water, clean each leaf. Dry the leaves on a paper towel, and then transfer them into a paper bag. Place the leaf filled bags in an oven, set to a constant temperature of 80 degrees Celsius, for two to three days.Clean the mortar and pestle twice, with a 4%hydrochloric acid solution. And use a filter paper to dry. Using the mortar and pestle, crush the dry leaves to a powder.Then, transfer the powder to sterile 15 milliliter plastic tubes. First, weigh a new, sealed 20 milliliter scintillation vial without its lid. Either note the weight or tare the scale.Then add the crushed leaves. Weigh the sample in the vial, and note the weight. Use rock wool to close the vial.Weigh three additional vials without adding samples, for use as controls, making note of each weight. Next, transfer the sample and control vials to a furnace. Burn the leaves as outlined in the text protocol.After this, let the samples cool to approximately 100 degrees Celsius. Using heavy gloves and tweezers, remove the samples from the furnace, making sure to hold the vial exteriorly. Place the vials on a flat surface.Remove the rock wool, and close the vials with their original lids. Then, weigh the three control vials, and calculate their average weight gain. If the weight gain is equal to or above 1%of the ash weight, use this value as an estimate of the measurement error.Weigh a 15 milliliter plastic tube. Either record the weight or tare the scale. Then transfer the ashes to the tube.Record this value, which is the ash weight. Next, add five milliliters of the one molar hydrochloric acid solution to the ashes. Filter the ashes through a 22 micrometer filter.Then add an additional five milliliters of the one molar hydrochloric acid solution through the same filter, leading to a final sample volume of 10 milliliters. After this, remove four milliliters from each sample for measurement by atomic spectroscopy. And determine the iron concentration per gram of ash as outlined in the text protocol.To begin, add four grams of potassium ferrocyanide to 100 milliliters of double distilled water, to prepare the Prussian blue solution. Vortex to mix, and store at 4 degrees Celsius until ready to use. When ready to use, mix 50 milliliters of Prussian blue solution with 50 milliliters of one molar hydrochloric acid to serve as the blank solution.Then, use a pipette to mix 0.5 milliliters of a previously obtained sample, with 0.5 milliliters of Prussian blue solution. Let this mixture rest for at least one minute, but for less than five minutes. Transfer this mixture to a cuvette, and use a spectrophotometer to measure the optical density at 715 nanometers.Then, determine the optical density per gram of ash, and plot the linear regression between the iron concentrations, as outlined in the text protocol. The representative results from 21 tobacco leaf samples show that the iron concentration in the irrigation water greatly affected the iron content in the leaf. The spectra of all 21 representative samples are then tested with the Prussian blue method.As seen here, the absorbance at 715 nanometers is the optimal wavelength while measuring solutions containing different concentrations of iron two and iron three. A linear regression curve, which is obtained by plotting iron concentration values obtained by atomic spectroscopy, versus the absorbance values, obtained by the Prussian blue method, allows for new samples of the same plant type to be analyzed. Don't forget that working with HCL can be extremely hazardous.And precaution, such as eye protection, should always be taken while performing this procedure. In the linear regression, it's important to verify that the results are in a realistic range. After its development, this technique can help biology researcher explore additional plants for food production or bioremediation.

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