Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei
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 me...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/22/12369 |
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| author | Vladimír Skalický Tereza Vojtková Aleš Pěnčík Jan Vrána Katarzyna Juzoń Veronika Koláčková Michaela Sedlářová Martin F. Kubeš Ondřej Novák |
| author_facet | Vladimír Skalický Tereza Vojtková Aleš Pěnčík Jan Vrána Katarzyna Juzoń Veronika Koláčková Michaela Sedlářová Martin F. Kubeš Ondřej Novák |
| author_sort | Vladimír Skalický |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T05:25:50Z |
| format | Article |
| id | doaj.art-a03906fcc2064901822cd000d1688849 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T05:25:50Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-a03906fcc2064901822cd000d16888492023-11-22T23:41:13ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-11-0122221236910.3390/ijms222212369Auxin Metabolite Profiling in Isolated and Intact Plant NucleiVladimír Skalický0Tereza Vojtková1Aleš Pěnčík2Jan Vrána3Katarzyna Juzoń4Veronika Koláčková5Michaela Sedlářová6Martin F. Kubeš7Ondřej Novák8Laboratory 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 RepublicInstitute of Experimental Botany of the Czech Academy of Sciences, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, CZ-77900 Olomouc, Czech RepublicDepartment of Biotechnology, The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Krakow, PolandInstitute of Experimental Botany of the Czech Academy of Sciences, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, CZ-77900 Olomouc, Czech RepublicDepartment of Botany, 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 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.https://www.mdpi.com/1422-0067/22/22/12369subcellular fractionationflow cytometrynucleusauxinauxin metabolism |
| spellingShingle | Vladimír Skalický Tereza Vojtková Aleš Pěnčík Jan Vrána Katarzyna Juzoń Veronika Koláčková Michaela Sedlářová Martin F. Kubeš Ondřej Novák Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei International Journal of Molecular Sciences subcellular fractionation flow cytometry nucleus auxin auxin metabolism |
| title | Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei |
| title_full | Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei |
| title_fullStr | Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei |
| title_full_unstemmed | Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei |
| title_short | Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei |
| title_sort | auxin metabolite profiling in isolated and intact plant nuclei |
| topic | subcellular fractionation flow cytometry nucleus auxin auxin metabolism |
| url | https://www.mdpi.com/1422-0067/22/22/12369 |
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