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Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC

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Plant Lipids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2295))

Abstract

The phosphate esters of myo-inositol (Ins) occur ubiquitously in biology. These molecules exist as soluble or membrane-resident derivatives and regulate a plethora of cellular functions including phosphate homeostasis, DNA repair, vesicle trafficking, metabolism, cell polarity, tip-directed growth, and membrane morphogenesis. Phosphorylation of all inositol hydroxyl groups generates phytic acid (InsP6), the most abundant inositol phosphate present in eukaryotic cells. However, phytic acid is not the most highly phosphorylated naturally occurring inositol phosphate. Specialized small molecule kinases catalyze the formation of the so-called myo-inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8. These molecules are characterized by one or several “high-energy” diphosphate moieties and are ubiquitous in eukaryotic cells. In plants, PP-InsPs play critical roles in immune responses and nutrient sensing. The detection of inositol derivatives in plants is challenging. This is particularly the case for inositol pyrophosphates because diphospho bonds are labile in plant cell extracts due to high amounts of acid phosphatase activity. We present two steady-state inositol labeling-based techniques coupled with strong anion exchange (SAX)-HPLC analyses that allow robust detection and quantification of soluble and membrane-resident inositol polyphosphates in plant extracts. These techniques will be instrumental to uncover the cellular and physiological processes controlled by these intriguing regulatory molecules in plants.

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Acknowledgments

The authors thank L. Schlüter and D. Herrmann for technical assistance. This work was funded by grants from the Deutsche Forschungsgemeinschaft (research fellowship LA 4541/1-1 to D.L., and SCHA 1274/4-1 and Research Training Group GRK 2064 to G.S.). D.L. further acknowledges the MRC/UCL Laboratory for Molecular Cell Biology University Unit (MC_UU_12018/4) for the support of the Saiardi Lab.

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Authors and Affiliations

  1. Department of Biochemistry, Indian Institute of Science, Bangalore, India

    Debabrata Laha

  2. Department of Plant Nutrition, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-University Bonn, Bonn, Germany

    Marília Kamleitner, Philipp Johnen & Gabriel Schaaf

  3. BASF SE, Limburgerhof, Germany

    Philipp Johnen

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  1. Debabrata Laha
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  2. Marília Kamleitner
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  3. Philipp Johnen
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  4. Gabriel Schaaf
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Correspondence to Gabriel Schaaf .

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Editors and Affiliations

  1. Institute of Molecular Physiology and Biotechnology of Plants (IMBIO), University of Bonn, Bonn, Germany

    Dorothea Bartels

  2. IMBIO Institut, Universität Bonn, Bonn, Nordrhein-Westfalen, Germany

    Peter Dörmann

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Laha, D., Kamleitner, M., Johnen, P., Schaaf, G. (2021). Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC. In: Bartels, D., Dörmann, P. (eds) Plant Lipids. Methods in Molecular Biology, vol 2295. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1362-7_20

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