Inositol phosphates are essential for cell development and signaling in all living organisms. Inositol hexakisphosphate (InsP6) is the most abundant phosphoinositol in both plants and animals. While the concentration of inorganic phosphorous (Pi) is often limited in soil, some plants overcome this limitation by creating a phosphate reservoir that serves as a source of Pi during phosphate deficiency. Although this strategy benefits plant development and signaling under adverse environmental conditions, excessive accumulation of Pi in crop plants has raised serious concerns about its toxicity and ill effects on human health. Consumption of crop plants with high InsP6 content or food products made from these crops is found to reduce nutrient intake significantly by way of chelating essential metal cations in human and livestock fed by such plants. Therefore, it is necessary to determine InsP6 contents in crop plants. Several methods have been developed for the screening and detection of InsP6 in plants. These detection methods however, are complex, labor-intensive, and often provide inaccurate results. We have developed a fast, reliable, and cost-effective method for the detection and quantification of InsP6 in plants using polyacrylamide gel electrophoresis (PAGE) with potential applications in industry, quality control labs, and research projects.
Phytic acid or phytate (inositol hexakisphosphate, InsP6), is one of the major components of seeds, leaves, and fruits in many plant species [1-3]. It is considered as the reservoir for phosphorus storage in plants and is used in case of phosphate deficiency in soil. Phytate is primarily presented as phytin in combination with K+, Ca2+, Mg2+ or Zn2+ and is deposited in plant cells together with proteins [
toxicity and negatively affect nutrition uptake and as a result cause several health problems including nutrient deficiencies, digestive problems and tooth decay.
All of these factors suggest that a reliable and efficient method for the quantitative detection of phytate in crop plants is of utmost importance. This will facilitate identification of high-phytate crop plants and limit or prevent their application in the food industry. The methods that are currently used for detection of phytate are complex, labor-intensive and involve enzymatic assays, high-performance liquid chromatography (HPLC), and radioactive labeling. Enzymatic assays involve extraction and phytase treatment of InsP6 followed by measurement of inorganic phosphorous (Pi) liberated during the enzymatic reaction. These assays are not reliable and do not give an accurate insight into the total phytate content in the tested samples. The HPLC method is more accurate but is complicated and difficult to perform because it is labor-intensive and often requires radioactive labeling. Metabolic radio-labeling of plant materials is expensive, cumbersome, and hazardous. Therefore, a simple, easy to perform method that is fast and reliable giving more accurate quantitation is needed to establish phytate contents in crop plants.
In this study, we have used polyacrylamide gel electrophoresis (PAGE) to separate and quantitatively visualize the InsP6 contents in plant samples. This method can be used as a tool for rapid screening of high-phytate crop plants. Our approach in developing this method is especially valuable because it can precisely detect and quantify InsP6 molecules in plant samples directly without dealing with complications of hydrolysis with phytase and then determine the Pi or the labor-intensive HPLC method involving the risks of working with radio-hazard materials. The simplicity and accuracy of this method suggest its potential as an effective alternative method for the detection and quantification of phytic acid in various plant tissues. To our knowledge, this is the first attempt to implement and quantify the phytate content in plants using PAGE.
Tomato (cv. Micro-Tom), rice (cv. Millie), and tobacco (cv. Petit Havana) plants were germinated in pots containing 25% sand and 75% Sun Gro Redi-earth “Plug and Seedling” Mix (Sun Gro Horticulture, Bellevue, WA) in a growth chamber with light intensity of 600 µmol∙m−2∙s−1 for 16 h light (28˚C) and 8 h dark (22˚C). Tissue samples were collected from the plants 1 month after germination and used for the detection of InsP6 using PAGE.
Tissue samples were prepared using a modified version of the phosphoinositol extraction method described by Stevenson-Paulik et al. [
Phytate was detected on a 8 × 8.5 × 0.1 cm polyacrylamide gel prepared by modification of the method originally described by Losito et al. [
Phytate concentration was quantified by using the gel images in a GelQuant.NET software (biochemlabsolutions.com). The phytate concentration was expressed in nmol InsP6 per 20 µL of tissue extract based on the standard curve. Volume Density − 115.36 = nmol InsP6 × 59271 obtained over a dynamic range of 0 - 5 nmol InsP6 (R2 = 0.994). The values were then converted to nmol∙g−1 fresh weight of tissue.
Phosphorous is an essential element for plants. It plays a critical role in the formation of multiple cell components [
Losito et al. [
We optimized this method for the extraction of phytate in tomato, rice, and tobacco samples. All of these plant species have significance as model crops in both agriculture and research. Our results demonstrated that phytate can be accurately visualized and quantified by using this new method.
As a first step, we validated the technique by application of different concentrations of standard InsP6 (0 - 5 nmol) and successfully visualized on the gel (
In the next step, phytate was extracted from leaves of tomato, rice, and tobacco plants and was separated using PAGE as described in materials and methods. Gel images were captured using a gel imaging system after staining the gels with toluidine blue. The images clearly revealed the phytate content of the tested samples and demonstrated differences in phytate levels in the three different crop plants that were studied (
The identity of the visualized band as phytate was confirmed by its sensitivity to phytase. To achieve this goal, tissue extracts and standard InsP6 were treated with phytase and incubated at 37˚C for 1 hour prior to loading on the gel and were compared with the samples that were not treated with the enzyme (
result in disappearance of the phytate band on the gel. Results revealed that observed bands on the gel were indeed related to InsP6 as treatment with the enzyme led to degradation of InsP6 and disappearance of the band (
Finally, gel images were used to determine the quantity of phytate in various plant samples. Data analysis using the standard curve showed that phytate contents vary among plant species and thus this method can be used to quantitate phytate contents in various crop plants (
In conclusion, the phytate detection method described in this study can effectively facilitate the screening of crop plants for their InsP6 content. The detection of phytate by PAGE has several advantages over other methods which would make it a preferred choice for research studies and also for the mass detection and quantification of phytate in crop plants in quality test laboratories. This method is rapid, cost-effective, and reliable. It is also safer to use and more environmentally-friendly for it does not involve radioactive materials.
Further preparatory modifications in the phytate extraction method to minimize salt and protein contents are likely to minimize distortions in the bands on the gel. The PAGE-based method can also be an ideal substitute for the detection of InsP6 and other inositol polyphosphates in organisms including animal and human cells where InsP6 is present at much lower concentration. This would require an improved staining procedure which is more sensitive than toluidine blue. Other cationic dyes that have higher affinity for InsP6 and that fluoresce might enhance the sensitivity of detection.