Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol
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 inv...
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MDPI AG
2021-08-01
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| Series: | International Journal of Molecular Sciences |
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| author | Ludmila Včelařová Vladimír Skalický Ivo Chamrád René Lenobel Martin F. Kubeš Aleš Pěnčík Ondřej Novák |
| author_facet | Ludmila Včelařová Vladimír Skalický Ivo Chamrád René Lenobel Martin F. Kubeš Aleš Pěnčík Ondřej Novák |
| author_sort | Ludmila Včelařová |
| collection | DOAJ |
| description | 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 <i>Arabidopsis thaliana</i> 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. |
| first_indexed | 2024-03-10T08:10:56Z |
| format | Article |
| id | doaj.art-9027bcfeb5fa47b989ca5d22356dff66 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T08:10:56Z |
| publishDate | 2021-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-9027bcfeb5fa47b989ca5d22356dff662023-11-22T10:42:23ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-08-012217937010.3390/ijms22179370Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation ProtocolLudmila Včelařová0Vladimír Skalický1Ivo Chamrád2René Lenobel3Martin F. Kubeš4Aleš Pěnčík5Ondřej Novák6Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences, and Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech RepublicLaboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences, and Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech RepublicLaboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences, and Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech RepublicLaboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences, and Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech RepublicLaboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences, and Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech RepublicLaboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences, and Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech RepublicLaboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences, and Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech RepublicThe 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 <i>Arabidopsis thaliana</i> 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.https://www.mdpi.com/1422-0067/22/17/9370endoplasmic reticulumauxinsubcellular fractionationdensity gradient centrifugationmass spectrometry |
| spellingShingle | Ludmila Včelařová Vladimír Skalický Ivo Chamrád René Lenobel Martin F. Kubeš Aleš Pěnčík Ondřej Novák Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol International Journal of Molecular Sciences endoplasmic reticulum auxin subcellular fractionation density gradient centrifugation mass spectrometry |
| title | Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol |
| title_full | Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol |
| title_fullStr | Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol |
| title_full_unstemmed | Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol |
| title_short | Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol |
| title_sort | auxin metabolome profiling in the arabidopsis endoplasmic reticulum using an optimised organelle isolation protocol |
| topic | endoplasmic reticulum auxin subcellular fractionation density gradient centrifugation mass spectrometry |
| url | https://www.mdpi.com/1422-0067/22/17/9370 |
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