Identification of Auxin Metabolites in Brassicaceae by Ultra-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry.

Panagiota-Kyriaki Revelou,Violetta Constantinou-Kokotou,Maroula G Kokotou

doi:10.3390/molecules24142615

Abstract

Auxins are signaling molecules involved in multiple stages of plant growth and development. The levels of the most important auxin, indole-3-acetic acid (IAA), are regulated by the formation of amide and ester conjugates with amino acids and sugars. In this work, IAA and IAA amide conjugates with amino acids bearing a free carboxylic group or a methyl ester group, along with some selected IAA metabolites, were studied in positive and negative electrospray ionization (ESI) modes, utilizing high-resolution mass spectrometry (HRMS) as a tool for their structural analysis. HRMS/MS spectra revealed the fragmentation patterns that enable us to identify IAA metabolites in plant extracts from eight vegetables of the Brassicaceae family using a fast and reliable ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) method. The accurate m/z (mass to charge) ratio and abundance of the molecular and fragment ions of the studied compounds in plant extracts matched those obtained from commercially available or synthesized compounds and confirmed the presence of IAA metabolites.

Highlights

Phytohormones are natural products that regulate all physiological and developmental processes occurring in plants
indole-3-acetic acid (IAA) biosynthesis occurs via two major routes—tryptophan (Trp)-independent and Trp-dependent pathways
The high-resolution mass spectrometry (HRMS) spectra were recorded on an Agilent 6530 Quadrupole Time of Flight LC-MS system (UPLC-quadrupole time-of-flight (QToF)-MS), with an electrospray ionization (ESI) source, coupled with Agilent 1290 Infinity UPLC system and an autosampler (Agilent Technologies, Santa Clara, CA, USA)

Summary

Introduction

Phytohormones are natural products that regulate all physiological and developmental processes occurring in plants. Auxins were the first class of endogenous plant hormones discovered by Charles and Francis Darwin [1]. The most important auxin, indole-3-acetic acid (IAA), is critically involved in different stages during plant growth, development, and metabolic homeostasis [2], 4-chloroindole-3-acetic acid (4-Cl-IAA), a naturally occurring chlorinated derivative of IAA, displays notable auxin activity [3]. IAA biosynthesis occurs via two major routes—tryptophan (Trp)-independent and Trp-dependent pathways. Trp-dependent auxin biosynthesis pathways include the indole-3-pyruvic acid (IPyA) pathway, the tryptamine (TAM) pathway, the indole-3-acetaldoxime (IAOx) pathway, and the indole-3-acetamide (IAM) pathway [4,5] (Figure 1a). Biosynthesis of IAA through the IAOx pathway has been observed primarily in Cruciferae [6]

Methods

Results

Conclusion