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Assay for abscisic acid 8′-hydroxylase activity of cloned plant cytochrome P450 oxidases in Saccharomyces cerevisiae

Author:
Eggels, Stella, Avramova, Viktoriya, Schön, Chris-Carolin, Poppenberger, Brigitte, Rozhon, Wilfried
Source:
Analytical biochemistry 2018 v.553 pp. 24-27
ISSN:
0003-2697
Subject:
Saccharomyces cerevisiae, abscisic acid, cell culture, complementary DNA, cytochrome P-450, enzyme activity, gas chromatography-mass spectrometry, homeostasis, metabolism, molecular cloning, oxidoreductases, phaseic acid, rapid methods, yeasts
Abstract:
The plant hormone abscisic acid (ABA) regulates many processes, including response to drought, seed dormancy and abscission of leaves and fruits. For maintenance of ABA homeostasis, catabolism of ABA by 8′-hydroxylation and subsequent cyclisation to phaseic acid (PA) is crucial. However, detection of ABA 8′-hydroxylation activity is tedious. We present a simple and rapid method for detection of ABA 8′-hydroxylase activity by cloning cDNAs of interest and expressing the respective protein in yeast. Upon addition of ABA, PA is formed and subsequently quantified in the yeast cell culture supernatant by heart cutting 2D-HPLC or GC-MS.
Agid:
5969426