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In Vitro Assay for ABA 8′-Hydroxylase: Implications for Improved Assays for Cytochrome P450 Enzymes

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Seed Dormancy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 773))

Abstract

In vitro assays for cytochrome P450 enzymes developed from plant-derived microsomal extracts have not been used extensively for the characterization and quantification of enzyme activities in plant tissues. We describe here an in vitro assay for abscisic acid (ABA) 8′-hydroxylase that was developed using microsomes extracted from (+)-ABA-induced corn suspension cultures. This assay may be useful for further characterization and monitoring of ABA 8′-hydroxylase activities in germinating seeds, seedlings, and other tissues. Additionally, the optimization protocols provided here may be adapted towards improving in vitro enzyme assays for other cytochrome P450 enzymes expressed in plants.

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Acknowledgments

The authors would like to thank Drs. Garth Abrams and Patricia Rose for their invaluable help in development of the corn ABA 8′-hydroxylase in vitro assay, Sandra Gillett for help with the tobacco in vitro assay, and Ning Zhou for the northern blot analysis. This is National Research Council of Canada publication No. 50161.

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Authors and Affiliations

  1. Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, SK, Canada

    Joan E. Krochko & Adrian J. Cutler

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  1. Joan E. Krochko
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  2. Adrian J. Cutler
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Corresponding author

Correspondence to Joan E. Krochko .

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Editors and Affiliations

  1. Dept. Biological Sciences, Simon Fraser Unviersity, University Drive 8888, Burnaby, V5A 1S6, British Columbia, Canada

    Allison R. Kermode

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Krochko, J.E., Cutler, A.J. (2011). In Vitro Assay for ABA 8′-Hydroxylase: Implications for Improved Assays for Cytochrome P450 Enzymes. In: Kermode, A. (eds) Seed Dormancy. Methods in Molecular Biology, vol 773. Humana Press. https://doi.org/10.1007/978-1-61779-231-1_8

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  • DOI: https://doi.org/10.1007/978-1-61779-231-1_8

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-230-4

  • Online ISBN: 978-1-61779-231-1

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