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Molecular and biochemical identification of inositol 1,3,4,5,6-pentakisphosphate 2-kinase encoding mRNA variants in castor bean (Ricinus communis L.) seeds

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Abstract

During seed development, phytic acid (PA) associated with mineral cations is stored as phytin and mobilized following germination in support of seedling growth. Two parallel biosynthetic pathways for PA have been proposed; yet the pathway is still poorly understood in terms of its regulation and the enzymes involved. Here, the castor bean (Ricinus communis L.) gene for inositol 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1) has been identified. This encodes the enzyme implicated in catalyzing the final reaction in PA biosynthesis, and its expression is enhanced in isolated germinated embryos by application of phosphate and myo-inositol (Ins). Even though only one copy of the RcIPK1 gene is present in the genome, numerous RNA variants are present, most likely due to alternative splicing. These are translated into six closely related protein isoforms according to in silico analysis. Functional analyses using yeast ipk1Δ revealed that only three of the mRNA variants can rescue a temperature-sensitive growth phenotype of this strain. High-performance liquid chromatography (HPLC) analysis of the synthesized inositol phosphates demonstrated that the ability to complement the missing yeast IPK1 enzyme is associated with the production of enzyme activity. The three active isoforms possess unique conserved motifs important for IPK1 catalytic activity.

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Abbreviations

PAIP 6 :

Phytic acid myo-inositol 1,2,3,4,5,6-hexakisphosphate

RcIPK1 :

Ricinus communis inositol 1,3,4,5,6-pentakisphosphate 2-kinase

Ins:

Myo-inositol

Lpa:

Low phytic acid

PtdIns:

Phosphatidylinositol

HAI:

Hours after imbibition

HPLC:

High-performance liquid chromatography

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Acknowledgments

This research was supported by Natural Sciences and Engineering Research Council of Canada Discovery grants (106265) to JSG and (044191) JDB.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada

    Jaeju Yu, John S. Greenwood & J. Derek Bewley

  2. Medical Research Council, Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK

    Adolfo Saiardi

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  1. Jaeju Yu
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  2. Adolfo Saiardi
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  4. J. Derek Bewley
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Correspondence to John S. Greenwood.

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Yu, J., Saiardi, A., Greenwood, J.S. et al. Molecular and biochemical identification of inositol 1,3,4,5,6-pentakisphosphate 2-kinase encoding mRNA variants in castor bean (Ricinus communis L.) seeds. Planta 239, 965–977 (2014). https://doi.org/10.1007/s00425-014-2028-z

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