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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kim GT, Tsukaya H (2002) Regulation of the biosynthesis of plant hormones by cytochrome P450s. J Plant Res 115:169–77
Ehlting J, Sauveplane V, Olry A, Ginglinger JF, Provart NJ, Werck-Reichhart D (2008) An extensive (co-)expression analysis tool for the cytochrome P450 superfamily in Arabidopsis thaliana. BMC Plant Biol 8:47
Yinghong P, Michael TP, Hudson ME, Kay SA, Chory J, Schuler MA (2009) Cytochrome P450 monooxygenases as reporters for circadian-regulated pathways. Plant Physiol 150:858–878
Krochko JE, Abrams GD, Loewen MK, Abrams SR, Cutler AJ (1998) (+)-Abscisic acid 8′-hydroxylase is a cytochrome P450 monooxygenase. Plant Physiol 118:849–860
Cutler AJ, Rose PA, Squires TM, Loewen MK, Shaw AC, Quail JW, Krochko JE, Abrams SR (2000) Inhibitors of abscisic acid 8′-hydroxylase. Biochemistry 39:13614–13624
Kushiro T, Okamoto M, Nakabayashi K, Yamagishi K, Kitamura S, Asami T, Hirai N, Koshiba T, Kamiya Y, Nambara E (2004) The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′-hydroxylases: key enzymes in ABA metabolism. EMBO J 23:1647–1656
Saito S, Hirai N, Matsumoto C, Ohigashi H, Ohta D, Sakata K, Mizutani M (2004) Arabidopsis CYP707As encode (+)-abscisic acid 8′-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid. Plant Physiol 134:1439–1449
Umezawa T, Okamoto M, Kushiro T, Nambara E, Oono Y, Seki M, Kobayashi M, Koshiba T, Kamiya Y, Shinozaki K (2006) CYP707A3, a major ABA 8′-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana. Plant J 46:171–182
Yang SH, Choi D (2006) Characterization of genes encoding ABA 8′-hydroxylase in ethylene-induced stem growth of deepwater rice (Oryza sativa L.). Biochem Biophys Res Commun 350:685–690
Okamoto M, Tanaka Y, Abrams SR, Kamiya Y, Seki M, Nambara E (2009) High humidity induces abscisic acid 8′-hydroxylase in stomata and vasculature to regulate local and systemic abscisic acid responses in Arabidopsis. Plant Physiol 149:825–834
Millar AA, Jacobsen JV, Ross JJ, Helliwell CA, Poole AT, Scofield G, Reid JB, Gubler F (2006) Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8′-hydroxylase. Plant J 45:942–954
Okamoto M, Kuwahara A, Seo M, Kushiro T, Asami T, Hirai N, Kamiya Y, Koshiba T, Nambara E (2006) CYP707A1 and CYP707A2, which encode abscisic acid 8′-hydroxylases, are indispensable for proper control of seed dormancy and germination in Arabidopsis. Plant Physiol 141:97–107
Chiang GCK, Barua D, Kramer EM, Amasino RM, Donohue K (2009) Major flowering time gene, Flowering Locus C, regulates seed germination in Arabidopsis thaliana. Proc Natl Acad Sci USA 106:11661–11666
Liu Y, Shi L, Ye N, Liu R, Jia W, Zhang J (2009) Nitric oxide-induced rapid decrease of abscisic acid concentration is required in breaking seed dormancy in Arabidopsis. New Phytol 183:1030–1042
Liu Y, Zhang J (2009) Rapid accumulation of NO regulates ABA catabolism and seed dormancy during imbibitions in Arabidopsis. Plant Signal Behav 4:905–907
Matakiadis T, Alboresi A, Jikumaru Y, Tatematsu K, Pichon O, Renou JP, Kamiya Y, Nambara E, Truong HN (2009) The Arabidopsis abscisic acid catabolic gene CYP707A2 plays a key role in nitrate control of seed dormancy. Plant Physiol 149:949–960
Werck-Reichhart D, Hehn A, Didierjean L (2000) Cytochrome P450 for engineering herbicide tolerance. Trends Plant Sci 5:116–123.
Schuhegger R, Nafisi M, Mansourova M, Petersen BL, Olsen CE, Svatos A, Halkier BA, Glawischnig E (2006) CYP71B15 (PAD3) catalyzes the final step in camalexin biosynthesis. Plant Physiol 141:1248–1254
Kandel S, Sauveplane V, Compagnon V, Franke R, Millet Y, Schreiber L, Werck-Reichhart D, Pinot F (2007) Characterization of a methyl jasmonate and wounding responsive cytochrome P450 of Arabidopsis thaliana catalyzing dicarboxylic fatty acid formation in vitro. FEBS J 274:5116–5127
Böttcher C, Westphal L, Schmotz C, Prade E, Scheel D, Glawischnig E (2009) The multifunctional enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) converts cysteine-indole-3-acetonitrile to camalexin in the indole-3-acetonitrile metabolic network of Arabidopsis thaliana. Plant Cell 21:1830–1845
Ludwig SR, Somers DA, Peterson WL, Pohlmann BF, Zarovitz MA, Gengenbach BG, Messing J (1985) High-frequency callus formation from maize protoplasts. Theor Appl Genet 71:344–350
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Bradford MM (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem 72:248–254
Babiano MJ (1995) Metabolism of [2-14C]abscisic acid by a cell-free system from embryonic axes of Cicer arietinum L. seeds. J Plant Physiol 145:374–376
van der Hoeven TA (1981) Isolation of hepatic microsomes by polyethylene glycol 6000 fractionation of the post mitochondrial fraction. Anal Biochem 115:398–402
Hamilton RL, Moorehouse A, Lear SR, Wong JS, Erickson SK (1999) A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum. J Lipid Res 40:1140–1147
Alden PG, Plumb RS, Jones MD, Rainville PD, Shave D (2009) A rapid ultra-performance liquid chromatography/tandem mass spectrophotometric methodology for the in vitro analysis of Pooled and Cocktail cytochrome P450 assays. Rapid Commun Mass Spectrom 24:147–154
Cutler AJ, Squires TM, Loewen MK, Balsevich JJ (1997) Induction of (+)-abscisic acid 8′-hydroxylase by (+)-abscisic acid in cultured maize cells. J Exp Bot 48:1787–1795
Bonnafous JC, Fonzes L, Mousseron-Canet M (1971) Synthese d’acide abscisique radioactive. II. Marquage au tritium. Bull Soc Chim Fr 1971:4552–4554
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-61779-231-1_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-230-4
Online ISBN: 978-1-61779-231-1
eBook Packages: Springer Protocols