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

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.

Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei

“…The maintenance of auxin homeostasis and its distribution within the plant cell remains elusive because only a few previous studies were focused on subcellular auxin analysis in chloroplasts [51], vacuoles [16], and recently ER [52], but none in nuclei. The nucleus represents a key organelle because it contains the process of canonical auxin signaling.…”

“…We should also mention the roles of other organelles in auxin homeostasis. For example, Ranocha et al [16] detected IAA, its precursors and metabolites in vacuoles, and Včelařová et al [52] recently in ER. Nevertheless, the biological importance and function of compartmentation in the nucleus and other organelles remains elusive and needs to be further studied.…”

Fluorescence‐activated multi‐organelle mapping of subcellular plant hormone distribution

“…After completing the gate design (Figure S2a), we performed several tests to confirm the identity of the FAmOS‐sorted organelle populations (Figure S3). The identity of enriched organelle fractions was initially assessed by determining the presence of protein markers by immunoblot analysis (Včelařová et al., 2021). While the results confirmed substantial enrichment in chloroplast and nuclear fractions, no antibody signal was detected in either the ER or the mitochondrial fraction (Figure S3a).…”

“…The time would increase both due to the longer isolation process that vacuoles require and to longer sorting time. While auxin and/or CK levels have been previously measured in chloroplasts (Polanská et al., 2007), vacuoles (Jiskrová et al., 2016; Ranocha et al., 2013), ER (Včelařová et al., 2021) and nuclei (Skalický et al., 2021), sample preparation processes and the divergent normalization methods used did not allow comparison of the data between the organelles. Due to the different sizes, shapes and abundance of organelles per plant cell, data normalization was essential for comparison of auxin and CK levels between organelles.…”

“…(Chamr ad et al, 2014). The acquired MS data were processed for protein identification using MaxQuant software version 1.6.10.43 (Tyanova et al, 2016) with an instrument parameter setting "Bruker QTOF" (Beck et al, 2015) and Andromeda search engine (Cox et al, 2011). The data were searched against the Arabidopsis thaliana (cv.…”