Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol.

Ludmila Včelařová,Martin F Kubeš,Ondřej Novák,Aleš Pěnčík,Vladimír Skalický,Ivo Chamrád,René Lenobel

doi:10.3390/ijms22179370

Abstract

The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various “omics” technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

Highlights

The endoplasmic reticulum (ER) is a highly dynamic, variable, and extensive nuclear membrane-bound network in eukaryotic cells
We have focused on auxin, a key phytohormone regulating a variety of crucial growth and developmental processes, and related metabolites
Upon comparing the relative abundances of free indole-3-acetic acid (IAA) and auxin metabolites at the beginning and end of the incubation, we observed that the proportion of free IAA increased slightly from 2.3% to 2.8%, while that of IAA-glc decreased slightly from 2.1% to 1.8% (Figure 5a)

Summary

Introduction

The endoplasmic reticulum (ER) is a highly dynamic, variable, and extensive nuclear membrane-bound network in eukaryotic cells. It consists of two parallel membranes that form a tangled system of tubules and cisternae, and plays key roles in lipid metabolism and the biosynthesis and sorting of proteins within the cell. Recent findings indicate that the ER plays a central role in maintaining subcellular auxin homeostasis by regulating its biosynthesis, subcellular distribution, and metabolism, and probably regulates its signalling [9]. In this way, the ER significantly affects the tightly

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Conclusion