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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Autores principales:	Vladimír Skalický, Tereza Vojtková, Aleš Pěnčík, Jan Vrána, Katarzyna Juzoń, Veronika Koláčková, Michaela Sedlářová, Martin F. Kubeš, Ondřej Novák
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	subcellular fractionation flow cytometry nucleus auxin auxin metabolism Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/a03906fcc2064901822cd000d1688849
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spelling	oai:doaj.org-article:a03906fcc2064901822cd000d16888492021-11-25T17:55:47ZAuxin Metabolite Profiling in Isolated and Intact Plant Nuclei10.3390/ijms2222123691422-00671661-6596https://doaj.org/article/a03906fcc2064901822cd000d16888492021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12369https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067The 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.Vladimír SkalickýTereza VojtkováAleš PěnčíkJan VránaKatarzyna JuzońVeronika KoláčkováMichaela SedlářováMartin F. KubešOndřej NovákMDPI AGarticlesubcellular fractionationflow cytometrynucleusauxinauxin metabolismBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12369, p 12369 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	subcellular fractionation flow cytometry nucleus auxin auxin metabolism Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	subcellular fractionation flow cytometry nucleus auxin auxin metabolism Biology (General) QH301-705.5 Chemistry QD1-999 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
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.
format	article
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
title	Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei
title_short	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_sort	auxin metabolite profiling in isolated and intact plant nuclei
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/a03906fcc2064901822cd000d1688849
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Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei

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