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Raman Spectroscopy Detection of Phytic Acid in Plant Seeds Reveals the Absence of Inorganic Polyphosphate

2015; Elsevier BV; Volume: 8; Issue: 5 Linguagem: Inglês

10.1016/j.molp.2015.01.015

1674-2052

Bernadett Kolozsvári, Steven Firth, Adolfo Saiardi,

Selenium in Biological Systems

The fully phosphorylated myo-inositol ring, inositol hexakisphosphate (IP6) is commonly known as phytic acid. It is the most abundant form of organic phosphate in soil (Raboy, 2003Raboy V. myo-Inositol-1,2,3,4,5,6-hexakisphosphate.Phytochemistry. 2003; 64: 1033-1043Crossref PubMed Scopus (348) Google Scholar). Phytic acid is able to regulate a broad range of intracellular processes, controlling many aspects of cell physiology both directly or as its derivative inositol pyrophosphates (Wilson et al., 2013Wilson M.S. Livermore T.M. Saiardi A. Inositol pyrophosphates: between signalling and metabolism.Biochem. J. 2013; 452: 369-379Crossref PubMed Scopus (181) Google Scholar). One such process is phosphate homeostasis, as IP6 regulates the level of inorganic polyphosphate (polyP), a linear polymer of phosphate groups that buffers cellular phosphate by regulation of its synthesis/degradation (Saiardi, 2012Saiardi A. How inositol pyrophosphates control cellular phosphate homeostasis?.Adv. Biol. Regul. 2012; 52: 351-359Crossref PubMed Scopus (48) Google Scholar). The quantification of seed phosphate levels, and the identification and localization of phosphate-containing molecular species, is of fundamental importance for the biotechnology industry. Several studies have reported the biochemical extraction and analysis of the levels of phytic acid in plant seeds (Thavarajah, 2014Thavarajah P. Thavarajah D. Inaccuracies in phytic acid measurement: implications for mineral biofortification and bioavailability.Am. J. Plant Sci. 2014; 5: 29-34Crossref Google Scholar). Direct experimental tools to visualize the phytic acid cellular distribution have not yet been developed, although DAPI fluorescence shift has been used to visualize the cellular presence of polyP. Recently it was demonstrated that phytic acid is also able to induce DAPI fluorescence shift at 550 nm, identically to polyP, questioning this assay's specificity (Kolozsvari et al., 2014Kolozsvari B. Parisi F. Saiardi A. Inositol phosphates induce DAPI fluorescence shift.Biochem. J. 2014; 460: 377-385Crossref PubMed Scopus (22) Google Scholar). Our aim was to develop a method that can distinguish between phytic acid and polyP signals in seeds without the need for biochemical extraction. Raman microscopy, based on chemical vibrations of molecules, allows the non-destructive, specific visualization of spatially resolved chemical information of any biological sample. Raman microscopy has previously been successfully used to visualize polyP in human tissues (Omelon et al., 2014Omelon S. Georgiou J. Variola F. Dean M.N. Colocation and role of polyphosphates and alkaline phosphatase in apatite biomineralization of elasmobranch tesserae.Acta Biomater. 2014; 10: 3899-3910Crossref PubMed Scopus (34) Google Scholar), therefore we decided to test if this technique allows analysis of the presence of polyP and phytic acid in plant seeds. The different molecular architecture of IP6 and polyP (Figure 1A and 1C ) suggests different vibration patterns and thus quite dissimilar Raman spectra. We initially investigated the Raman spectra of phytic acid (D-myo-inositol 1,2,3,4,5,6-hexakisphosphate, dodecasodium salt; Sichem) and polyP (polyP as sodium phosphate glass, T45; Sigma-Aldrich) from solid samples. Raman spectra were acquired using a Renishaw Ramanscope with a Leica DMLM optical microscope. Excitation (ca. 10 mW at 633 nm) was provided by a helium/neon laser focused onto the sample with a 50×/0.75 objective. Spectral intensities were recorded at room temperature from samples for 50 s in total, subsequently subtracting the background. GRAMS/32 software was used to measure Raman shifts for phytic acid and polyP. The Raman spectrum of polyP (Figure 1A) is dominated by the PO2− (phosphate) symmetrical stretching vibration of the ester chain at 1154 cm−1 while the less intense peak at 682 cm−1 can be assigned to the P-O-P stretching vibration of the phosphate ester bond. This is consistent with previously reported Raman spectra of polyP (Omelon et al., 2014Omelon S. Georgiou J. Variola F. Dean M.N. Colocation and role of polyphosphates and alkaline phosphatase in apatite biomineralization of elasmobranch tesserae.Acta Biomater. 2014; 10: 3899-3910Crossref PubMed Scopus (34) Google Scholar). The structure of phytic acid is more complex and contains several types of bond, therefore the spectral region between 700 and 1400 cm−1 shows more bands (Figure 1C). The phosphate symmetric stretching vibrations appear as a group of five sharp bands between 917 and 1043 cm−1. Significant intensities are caused by the P-O-C bending vibrations at 749–826 cm−1 and antisymmetric P-O stretches at 1061–1147 cm−1 with C-H bending vibrations at 1210–1320 cm−1 (Figure 1C). Recording Raman spectra from solutions of polyP (100 mM) (Figure 1B) and phytic acid (10 mM) (Figure 1D) (at pH 7.0–8.5) showed the same patterns in the phosphate stretching regions as observed for solid samples. Phytic acid is of interest to the biotechnology industry (Raboy, 2002Raboy V. Progress in breeding low phytate crops.J. Nutr. 2002; 132: 503S-505SPubMed Google Scholar); the aim is to reduce its accumulation in seeds. Due to the chelating properties of phytic acid, a high grain diet has been linked to human iron deficiency. Very little is known about polyP in plants (van Voorthuysen et al., 2000van Voorthuysen T. Regierer B. Springer F. Dijkema C. Vreugdenhil D. Kossmann J. Introduction of polyphosphate as a novel phosphate pool in the chloroplast of transgenic potato plants modifies carbohydrate partitioning.J. Biotechnol. 2000; 77: 65-80Crossref PubMed Scopus (20) Google Scholar) and we are unaware of any report confirming or denying the presence of this phosphate storage polymer in seeds. The absence of polyP studies could be a consequence of the typical phytic acid extraction procedure, involving the boiling of crushed plant seeds in strong acid. While phytic acid is stable in this condition, polyP will be promptly degraded. Alternative, mild extraction methods at 4°C (Raboy et al., 2000Raboy V. Gerbasi P.F. Young K.A. Stoneberg S.D. Pickett S.G. Bauman A.T. Murthy P.P. Sheridan W.F. Ertl D.S. Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1.Plant Physiol. 2000; 124: 355-368Crossref PubMed Scopus (363) Google Scholar) would preserve polyP, but these extracts are often subjected to wet ashing prior to phosphate analysis, which would also destroy any polyP present. Therefore, we were interested in using Raman spectroscopy on plant seeds for two reasons: to detect phytic acid in a biological sample, but also to investigate the possible presence of polyP in plant seeds. Wheat grains were fixed in 6% glutaraldehyde in 0.025 M phosphate buffer (pH 6.8–7.0) for 36–48 h at 0–4°C. The fixed tissue was washed in phosphate buffer for 2–3 h and placed in liquid nitrogen for 5–10 min before sectioning by microtome (Leica CM1850). The 10–20 μm sections were mounted on quartz slides (Agar Scientific). Phytase treatment was performed on seed sections mounted on quartz slides in sodium citrate buffer (pH 4.0) for 1 h at 30°C adding 10 units/section of phytase (Sigma-Aldrich). The samples were washed twice in water and analyzed by Raman spectroscopy. Previous biochemical separation studies in monocotyledon seeds localize the majority of phytic acid (∼80%) in phytin globoids inside the protein storage vacuoles of the aleurone cells, the outer layer of the endosperm, with the remainder mainly in the germ (Raboy et al., 2014Raboy V. Cichy K. Peterson K. Reichman S. Sompong U. Srinives P. Saneoka H. Barley (Hordeum vulgare L.) low phytic acid 1-1: an endosperm-specific, filial determinant of seed total phosphorus.J. Hered. 2014; 105: 656-665Crossref PubMed Scopus (17) Google Scholar). The inner endosperm contains little phytic acid. We recorded Raman spectra from the three different layers of wheat seeds, as shown in the confocal image, namely the aleurone cells, inner endosperm cells, and pericarp (Figure 1E). We detected a strong phosphate symmetrical stretching vibration at 1003 cm−1 (Figure 1G in red) demonstrating the presence of phytic acid in the aleuronic cell layer. Furthermore, a weaker but clearly present peak at 1001 cm−1 is observable in endosperm cells (Figure 1H, in red), while the spectrum of the pericarp layer lacks the phytic acid Raman signature (Figure 1F, in red). To verify the nature of the phytic acid Raman signal, we treated the wheat seeds with phytase, a phosphohydrolase which degrades phytic acid. The disappearance of the 1003 cm−1 and 1001 cm−1 peaks in the Raman spectra acquired from these samples confirms our identification of the phytic acid signals (Figure 1G and 1H, in black). We failed to detect any consistent and reproducible signal around 1152 cm−1, the Raman signature of polyP. While the failure to detect polyP in wheat seeds by Raman spectroscopy is not definitive proof of its absence, it certainly indicates that, if present, polyP is not a major phosphate storage molecule in plant seeds. This report demonstrates the possibility of localizing and studying phytic acid in biological specimens by Raman spectroscopy. We analyzed the presence of phytic acid in plant seeds where it is known to accumulate; the high level of phytic acid might have facilitated its detection by Raman spectroscopy, but our results are supportive of further development of Raman microscopy techniques to visualize phytic acid in different cell types and organisms. The implementation of enhanced Raman methods, such as surface-enhanced Raman spectroscopy, will potentially improve signal sensitivity. Furthermore, the ability of Raman spectroscopy to simultaneously study phytic acid and polyP within a biological sample will help in decoupling the relevant signaling pathways regulating their metabolic connection, improving our understanding of how seed phosphate accumulation is achieved. This work was supported by the Medical Research Council core support of the Laboratory for Molecular Cell Biology University Unit (MC_UU_1201814).