Abstract
Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione-S-transferase:JAR1 fusion protein produced in Escherichia coli, including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas chromatography/mass spectrometry.
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References
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Acknowledgments
This research has been supported in part by funds from the Hatch Act and additionally by the National Science Foundation (Award IOS-0744758).
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Rowe, M.L., Staswick, P.E. (2013). Jasmonic Acid–Amino Acid Conjugation Enzyme Assays. In: Goossens, A., Pauwels, L. (eds) Jasmonate Signaling. Methods in Molecular Biology, vol 1011. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-414-2_12
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DOI: https://doi.org/10.1007/978-1-62703-414-2_12
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Publisher Name: Humana Press, Totowa, NJ
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