A UPLCMS/MS method for quantification of metabolites in the ethylene biosynthesis pathway and its biological validation in Arabidopsis

Author:

Cao Da1ORCID,Depaepe Thomas1ORCID,Sanchez‐Muñoz Raul1ORCID,Janssens Hilde2ORCID,Lemière Filip3,Willems Tim4ORCID,Winne Johan2ORCID,Prinsen Els4ORCID,Van Der Straeten Dominique1ORCID

Affiliation:

1. Laboratory of Functional Plant Biology, Department of Biology, Faculty of Sciences Ghent University 9000 Ghent Belgium

2. Department of Organic Chemistry, Polymer Chemistry Research Group and Laboratory for Organic Synthesis Ghent University 9000 Ghent Belgium

3. Department of Chemistry, Biomolecular and Analytical Mass Spectrometry University of Antwerp 2020 Antwerp Belgium

4. Integrated Molecular Plant Physiology Research, Department of Biology University of Antwerp 2020 Antwerp Belgium

Abstract

Summary The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1‐aminocyclopropane‐1‐carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl‐ACC (MACC), glutamyl‐ACC (GACC), and jasmonyl‐ACC (JA‐ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra‐high‐performance liquid chromatography coupled to tandem mass spectrometry (UPLC‐MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild‐type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA‐ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene‐independent role of ACC.

Funder

Fonds Wetenschappelijk Onderzoek

Publisher

Wiley

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