A Colorimetric Method for Measuring Iron Content in Plants

Summary

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Abstract

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.

Introduction

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-d....

Protocol

1. Plant Material and Growth Conditions

1. 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 °C. Irrigate with tap water until water drains from the pot.
2. After 50±5 days, start Fe treatments in the irrigation, according to the concentrations suitable for the experiment. For example, we used a range of iron concentrations from 0 to 6 mM, supplemented by a soluble Fe chelator (Fe EDDHA). Irrigate the plants with the appropriate solution every two days (to avoid dehydration) f....

Results

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.......

Discussion

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 tha.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.