Summary of Study ST002810

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001757. The data can be accessed directly via it's Project DOI: 10.21228/M8M13Z This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002810
Study Title	GAS2 encodes a 2-oxoglutarate dependent dioxygenase involved in ABA catabolism
Study Summary	Liu et al. [1] recently reported the characterization of Arabidopsis thaliana GAS2 (Gain of Function in ABA-modulated Seed Germination 2), which was described as an enzyme that catalyzes the stereospeciﬁc hydration of GA12 to produce GA12 16, 17-dihydro-16α-ol (DHGA12). A second paper describes the conversion of GA12 to an unidentified product by GAS2 and also reports that this enzyme does not convert ABA [2]. However, as previously reported [3], we did not find any conversion of [17-14C]-labeled or [1-,7-,12-,18-14C4]-labeled GA12 by GAS2. Instead, we present here data showing that the recombinant GAS2 enzyme is capable of catabolising abscisic acid (ABA) to phaseic acid (PA) and further to a second product, putative 8’-carboxy-ABA (compound A; Fig. 1a) [4]. References: [1] Liu, H. et al. Biosynthesis of DHGA12 and its roles in Arabidopsis seedling establishment. Nat. Commun. 10, 1768 (2019). [2] Xiong, W. et al. The dioxygenase GIM2 functions in seed germination by altering gibberellin production in Arabidopsis. J. Integr. Plant Biol. 60, 276-291 (2018). [3] Lange, T. & Pimenta Lange, M. J. The Multifunctional Dioxygenases of Gibberellin Synthesis. Plant Cell Physiol. 61, 1869-1879 (2020). [4] Lange, T., Atiq, N., Pimenta Lange. GAS2 encodes a 2-oxoglutarate dependent dioxygenase involved in ABA catabolism. bioRxiv, doi: 10.1101/2022.11.16.516706 (2022).
Institute	Technische Universität Braunschweig
Department	Biochemie und Physiology der Pflanzen
Laboratory	AG Lange
Last Name	Lange
First Name	Theo
Address	Mendelssohnstr. 4
Email	theo.lange@tu-bs.de
Phone	00495313915880
Submit Date	2023-08-01
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	GC-MS
Release Date	2023-10-11
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP002923
Sampleprep Summary:	3’,5’,5’,7’,7’,7’-d6-labelled ABA and 17,17-d2-labelled GA12 were purchased from OlChemIm, Czech Republic. PA and 7’,7’,7’-d3-PA were gifts from Professor Eiji Nambara (University of Toronto, Canada). Preparations of E. coli cell lysates were incubated in a total volume of 100 µl containing 100 mM Tris-HCl, pH 7.0 at 30°C for 16 h with 2-oxoglutarate and ascorbate (100mM each, final concentrations), FeSO4 (0.5 mM), catalase (1mg/ml), and the substrates (5 µl in methanol for ABA, 3’,5’,5’,7’,7’,7’-d6-labeled ABA (500 ng), PA (500 ng), and 7’,7’,7’-d3-labeled PA (50 ng), and 2 µl in methanol per 5 ng 17,17-d2-labelled GA12. Variations to the standard incubation conditions are indicated in the individual experiments. Incubation products were extracted and analyzed by reverse-phase HPLC as described previously12 using gradients of increasing methanol in water, containing 1% acetic acid, at 1 ml.min-1 as follows: 50% methanol, followed by five 0.5-min steps to 57.4%, 60.6%, 61.8%, 62.3%, 62.5%, one 5-min step to 62.7%, one 1-min step to 63.2% and seven 2-min steps to 64.3%, 67.2%, 70.5%, 74.9%, 81%, 89% and 100% methanol. PA and compound A eluted between 3 and 6 min, and ABA between 6 and 9 min. The dried HPLC-fractions were redissolved in 100 µl methanol and methylated with 100 µl ethereal diazomethane.

Sampleprep ID:

SP002923

Sampleprep Summary:

3’,5’,5’,7’,7’,7’-d6-labelled ABA and 17,17-d2-labelled GA12 were purchased from OlChemIm, Czech Republic. PA and 7’,7’,7’-d3-PA were gifts from Professor Eiji Nambara (University of Toronto, Canada). Preparations of E. coli cell lysates were incubated in a total volume of 100 µl containing 100 mM Tris-HCl, pH 7.0 at 30°C for 16 h with 2-oxoglutarate and ascorbate (100mM each, final concentrations), FeSO4 (0.5 mM), catalase (1mg/ml), and the substrates (5 µl in methanol for ABA, 3’,5’,5’,7’,7’,7’-d6-labeled ABA (500 ng), PA (500 ng), and 7’,7’,7’-d3-labeled PA (50 ng), and 2 µl in methanol per 5 ng 17,17-d2-labelled GA12. Variations to the standard incubation conditions are indicated in the individual experiments. Incubation products were extracted and analyzed by reverse-phase HPLC as described previously12 using gradients of increasing methanol in water, containing 1% acetic acid, at 1 ml.min-1 as follows: 50% methanol, followed by five 0.5-min steps to 57.4%, 60.6%, 61.8%, 62.3%, 62.5%, one 5-min step to 62.7%, one 1-min step to 63.2% and seven 2-min steps to 64.3%, 67.2%, 70.5%, 74.9%, 81%, 89% and 100% methanol. PA and compound A eluted between 3 and 6 min, and ABA between 6 and 9 min. The dried HPLC-fractions were redissolved in 100 µl methanol and methylated with 100 µl ethereal diazomethane.