The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

• Keywords
• auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
• MeSH
• Arabidopsis drug effects   metabolism   ultrastructure   MeSH
• Cell Nucleus drug effects   metabolism   ultrastructure   MeSH
• Cell Culture Techniques MeSH
• Centrifugation methods   MeSH
• Cell Fractionation instrumentation   methods   MeSH
• Mass Spectrometry MeSH
• Homeostasis physiology   MeSH
• Indoles metabolism   pharmacology   MeSH
• Indoleacetic Acids metabolism   MeSH
• Protoplasts chemistry   MeSH
• Flow Cytometry MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Cells drug effects   metabolism   ultrastructure   MeSH
• Nicotiana drug effects   metabolism   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• indole MeSH Browser
• Indoles MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH

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.

• Keywords
• auxin, density gradient centrifugation, endoplasmic reticulum, mass spectrometry, subcellular fractionation,
• MeSH
• Arabidopsis cytology   metabolism   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Metabolome MeSH
• Metabolomics methods   MeSH
• Arabidopsis Proteins analysis   metabolism   MeSH
• Proteomics methods   MeSH
• Plant Cells MeSH
• Seedlings cytology   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• indoleacetic acid MeSH Browser
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH