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.

Department of Biotechnology The Franciszek Górski Institute of Plant Physiology Polish Academy of Sciences Niezapominajek 21 30 239 Krakow Poland

Department of Botany Faculty of Science Palacký University Šlechtitelů 27 CZ 78371 Olomouc Czech Republic

Institute 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 Republic

Laboratory 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 Republic

School of Life Sciences University of Warwick Coventry CV4 7AL UK

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Brumos J., Robles L.M., Yun J., Vu T.C., Jackson S., Alonso J.M., Stepanova A.N. Local Auxin Biosynthesis is a key regulator of plant development. Dev. Cell. 2018;47:306–318. doi: 10.1016/j.devcel.2018.09.022. PubMed DOI

Di Mambro R., De Ruvo M., Pacifici E., Salvi E., Sozzani R., Benfey P.N., Busch W., Novak O., Ljung K., Di Paola L., et al. Auxin minimum triggers the developmental switch from cell division to cell differentiation in the Arabidopsis root. Proc. Natl. Acad. Sci. USA. 2017;114:E7641–E7649. doi: 10.1073/pnas.1705833114. PubMed DOI PMC

Grones P., Majda M., Doyle S.M., Van Damme D., Robert S. Fluctuating auxin response gradients determine pavement cell-shape acquisition. Proc. Natl. Acad. Sci. USA. 2020;117:16027–16034. doi: 10.1073/pnas.2007400117. PubMed DOI PMC

Skalický V., Kubeš M., Napier R., Novák O. Auxins and Cytokinins—The role of subcellular organization on homeostasis. Int. J. Mol. Sci. 2018;19:3115. doi: 10.3390/ijms19103115. PubMed DOI PMC

Tanaka H., Dhonukshe P., Brewer P.B., Friml J. Spatiotemporal asymmetric auxin distribution: A means to coordinate plant development. Cell. Mol. Life Sci. 2006;63:2738–2754. doi: 10.1007/s00018-006-6116-5. PubMed DOI PMC

Barbez E., Kubeš M., Rolčík J., Béziat C., Pěnčík A., Wang B., Rosquete M.R., Zhu J., Dobrev P.I., Lee Y., et al. A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants. Nature. 2012;485:119–122. doi: 10.1038/nature11001. PubMed DOI

Dindas J., Scherzer S., Roelfsema M.R.G., Von Meyer K., Müller H.M., Al-Rasheid K.A.S., Palme K., Dietrich P., Becker D., Bennett M.J., et al. AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling. Nat. Commun. 2018;9:1–10. doi: 10.1038/s41467-018-03582-5. PubMed DOI PMC

Fendrych M., Akhmanova M., Merrin J., Glanc M., Hagihara S., Takahashi K., Uchida N., Torii K.U., Friml J. Rapid and reversible root growth inhibition by TIR1 auxin signalling. Nat. Plants. 2018;4:453–459. doi: 10.1038/s41477-018-0190-1. PubMed DOI PMC

Retzer K., Singh G., Napier R. It starts with TIRs. Nat. Plants. 2018;4:410–411. doi: 10.1038/s41477-018-0196-8. PubMed DOI

Simonini S., Mas P.J., Mas C.M.V.S., Østergaard L., Hart D.J. Auxin sensing is a property of an unstructured domain in the Auxin Response Factor ETTIN of Arabidopsis thaliana. Sci. Rep. 2018;8:1–11. doi: 10.1038/s41598-018-31634-9. PubMed DOI PMC

Kubeš M., Napier R. Non-canonical auxin signalling: Fast and curious. J. Exp. Bot. 2019;70:2609–2614. doi: 10.1093/jxb/erz111. PubMed DOI PMC

Mravec J., Skůpa P., Bailly A., Hoyerová K., Křeček P., Bielach A., Petrášek J., Zhang J., Gaykova V., Stierhof Y.-D., et al. Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature. 2009;459:1136–1140. doi: 10.1038/nature08066. PubMed DOI

Bosco C.D., Dovzhenko A., Liu X., Woerner N., Rensch T., Eismann M., Eimer S., Hegermann J., Paponov I.A., Ruperti B., et al. The endoplasmic reticulum localized PIN8 is a pollen-specific auxin carrier involved in intracellular auxin homeostasis. Plant J. 2012;71:860–870. doi: 10.1111/j.1365-313X.2012.05037.x. PubMed DOI

Ding Z., Wang B., Moreno I., Dupláková N., Simon S., Carraro N., Reemmer J., Pěnčík A., Chen X., Tejos R., et al. ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis. Nat. Commun. 2012;3:941. doi: 10.1038/ncomms1941. PubMed DOI

Feraru E., Vosolsobě S., Feraru M.I., Petrášek J., Kleine-Vehn J. Evolution and structural diversification of PILS Putative Auxin carriers in plants. Front. Plant Sci. 2012;3:227. doi: 10.3389/fpls.2012.00227. PubMed DOI PMC

Ranocha P., Dima O., Nagy R., Felten J., Corratgé-Faillie C., Novák O., Morreel K., Lacombe B., Martinez Y., Pfrunder S., et al. Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis. Nat. Commun. 2013;4:2625. doi: 10.1038/ncomms3625. PubMed DOI PMC

Middleton A.M., Dal Bosco C., Chlap P., Bensch R., Harz H., Ren F., Bergmann S., Wend S., Weber W., Hayashi K.-I., et al. Data-driven modeling of intracellular auxin fluxes indicates a dominant role of the er in controlling nuclear auxin uptake. Cell Rep. 2018;22:3044–3057. doi: 10.1016/j.celrep.2018.02.074. PubMed DOI

Zhao Y. Auxin biosynthesis: A simple two-step pathway converts tryptophan to indole-3-acetic acid in plants. Mol. Plant. 2012;5:334–338. doi: 10.1093/mp/ssr104. PubMed DOI PMC

Kriechbaumer V., Wang P., Hawes C., Abell B.M. Alternative splicing of the auxin biosynthesis gene YUCCA4 determines its subcellular compartmentation. Plant J. 2012;70:292–302. doi: 10.1111/j.1365-313X.2011.04866.x. PubMed DOI

Ljung K. Auxin metabolism and homeostasis during plant development. Development. 2013;140:943–950. doi: 10.1242/dev.086363. PubMed DOI

Casanova-Sáez R., Mateo-Bonmatí E., Ljung K. Auxin metabolism in plants. Cold Spring Harb. Perspect. Biol. 2021;13:a039867. doi: 10.1101/cshperspect.a039867. PubMed DOI PMC

Jin S.-H., Ma X.-M., Kojima M., Sakakibara H., Wang Y.W., Hou B.-K. Overexpression of glucosyltransferase UGT85A1 influences trans-zeatin homeostasis and trans-zeatin responses likely through O-glucosylation. Planta. 2013;237:991–999. doi: 10.1007/s00425-012-1818-4. PubMed DOI

Staswick P.E., Serban B., Rowe M., Tiryaki I., Maldonado M.T., Maldonado M.C., Suza W. Characterization of an arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell Online. 2005;17:616–627. doi: 10.1105/tpc.104.026690. PubMed DOI PMC

Cano A., Sánchez-García A.B., Albacete A., González-Bayón R., Justamante M.S., Ibáñez S., Acosta M., Pérez-Pérez J.M. Enhanced conjugation of auxin by gh3 enzymes leads to poor adventitious rooting in carnation stem cuttings. Front. Plant Sci. 2018;9:566. doi: 10.3389/fpls.2018.00566. PubMed DOI PMC

Di Mambro R., Svolacchia N., Dello Ioio R., Pierdonati E., Salvi E., Pedrazzini E., Vitale A., Perilli S., Sozzani R., Benfey P.N., et al. The lateral root cap acts as an auxin sink that controls meristem size. Curr. Biol. 2019;29:1199–1205. doi: 10.1016/j.cub.2019.02.022. PubMed DOI

Sanchez Carranza A.P., Singh A., Steinberger K., Panigrahi K., Palme K., Dovzhenko A., Dal Bosco C. Hydrolases of the ILR1-like family of Arabidopsis thaliana modulate auxin response by regulating auxin homeostasis in the endoplasmic reticulum. Sci. Rep. 2016;6:1–11. doi: 10.1038/srep24212. PubMed DOI PMC

Fu Y., Yang Y., Chen S., Ning N., Hu H. Arabidopsis IAR4 modulates primary root growth under salt stress through ros-mediated modulation of auxin distribution. Front. Plant Sci. 2019;10:522. doi: 10.3389/fpls.2019.00522. PubMed DOI PMC

Porco S., Pěnčík A., Rashed A., Voß U., Casanova-Sáez R., Bishopp A., Golebiowska A., Bhosale R., Swarup R., Swarup K., et al. Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis. Proc. Natl. Acad. Sci. USA. 2016;113:11016–11021. doi: 10.1073/pnas.1604375113. PubMed DOI PMC

Zhang J., Lin J.E., Harris C., Campos Mastrotti Pereira F., Wu F., Blakeslee J.J., Peer W.A. DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA. 2016;113:11010–11015. doi: 10.1073/pnas.1604769113. PubMed DOI PMC

Novák O., Napier R., Ljung K. Zooming in on plant hormone analysis: Tissue- and cell-specific approaches. Annu. Rev. Plant Biol. 2017;68:323–348. doi: 10.1146/annurev-arplant-042916-040812. PubMed DOI

Pertoft H. Fractionation of cells and subcellular particles with Percoll. J. Biochem. Biophys. Methods. 2000;44:1–30. doi: 10.1016/S0165-022X(00)00066-X. PubMed DOI

Folta K.M., Kaufman L.S. Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Nat. Protoc. 2007;1:3094–3100. doi: 10.1038/nprot.2006.471. PubMed DOI

Lee Y.H., Tan H.T., Chung M.C.M. Subcellular fractionation methods and strategies for proteomics. Proteomics. 2010;10:3935–3956. doi: 10.1002/pmic.201000289. PubMed DOI

Saxena P.K., Fowke L.C., King J. An efficient procedure for isolation of nuclei from plant protoplasts. Protoplasma. 1985;128:184–189. doi: 10.1007/BF01276340. DOI

Zhang H.-B., Zhao X., Ding X., Paterson A.H., Wing R.A. Preparation of megabase-size DNA from plant nuclei. Plant J. 1995;7:175–184. doi: 10.1046/j.1365-313X.1995.07010175.x. DOI

Vertommen A., Panis B., Swennen R., Carpentier S.C. Challenges and solutions for the identification of membrane proteins in non-model plants. J. Proteomics. 2011;74:1165–1181. doi: 10.1016/j.jprot.2011.02.016. PubMed DOI

Zini N., Matteucci A., Squarzoni S., Galanzi A., Rizzoli R., Papa S. Electron microscopy microsampling of isolated nuclei sorted by flow cytometry. Cytometry. 1986;7:605–608. doi: 10.1002/cyto.990070617. PubMed DOI

Šimková H., Číhalíková J., Vrána J., Lysák M.A., Doležel J. Preparation of HMW DNA from plant nuclei and chromosomes isolated from root tips. Biol. Plant. 2003;46:369–373. doi: 10.1023/A:1024322001786. DOI

Zhang C., Barthelson R.A., Lambert G.M., Galbraith D.W. Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiol. 2008;147:30–40. doi: 10.1104/pp.107.115246. PubMed DOI PMC

Deal R.B., Henikoff S. A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell. 2010;18:1030–1040. doi: 10.1016/j.devcel.2010.05.013. PubMed DOI PMC

Deal R.B., Henikoff S. The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat. Protoc. 2011;6:56–68. doi: 10.1038/nprot.2010.175. PubMed DOI PMC

Seifertová D., Klíma P., Pařezová M., Petrášek J., Zažímalová E., Opatrný Z. Plant cell lines in cell morphogenesis research. Methods Mol. Biol. 2014;1080:215–229. doi: 10.1007/978-1-62703-643-6_18. PubMed DOI

Gigli-Bisceglia N., Engelsdorf T., Hamann T. Plant cell wall integrity maintenance in model plants and crop species-relevant cell wall components and underlying guiding principles. Cell Mol. Life Sci. 2020;77:2049–2077. doi: 10.1007/s00018-019-03388-8. PubMed DOI PMC

Xu F., Copeland C. Nuclear extraction from Arabidopsis thaliana. Bio Protoc. 2012;2:e306. doi: 10.21769/BioProtoc.306. DOI

Yoo S.-D., Cho Y.-H., Sheen J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2007;2:1565–1572. doi: 10.1038/nprot.2007.199. PubMed DOI

Koláčková V., Perničková K., Vrána J., Duchoslav M., Jenkins G., Phillips D., Turkosi E., Šamajová O., Sedlářová M., Šamaj J., et al. Nuclear disposition of alien chromosome introgressions into wheat and rye using 3D-FISH. Int. J. Mol. Sci. 2019;20:4143. doi: 10.3390/ijms20174143. PubMed DOI PMC

Perničková K., Koláčková V., Lukaszewski A.J., Fan C., Vrána J., Duchoslav M., Jenkins G., Phillips D., Šamajová O., Sedlářová M., et al. Instability of alien chromosome introgressions in wheat associated with improper positioning in the nucleus. Int. J. Mol. Sci. 2019;20:1448. doi: 10.3390/ijms20061448. PubMed DOI PMC

Antoniadi I., Plačková L., Simonovik B., Doležal K., Turnbull C., Ljung K., Novák O. Cell-type-specific cytokinin distribution within the Arabidopsis primary root apex. Plant Cell. 2015;27:1955–1967. doi: 10.1105/tpc.15.00176. PubMed DOI PMC

Petrovská B., Jeřábková H., Chamrád I., Vrána J., Lenobel R., Uřinovská J., Šebela M., Doležel J. Proteomic analysis of barley cell nuclei purified by flow sorting. Cytogenet. Genome Res. 2014;143:78–86. doi: 10.1159/000365311. PubMed DOI

Novák O., Hényková E., Sairanen I., Kowalczyk M., Pospíšil T., Ljung K. Tissue-specific profiling of the Arabidopsis thaliana auxin metabolome. Plant J. 2012;72:523–536. doi: 10.1111/j.1365-313X.2012.05085.x. PubMed DOI

Polanská L., Vičánková A., Nováková M., Malbeck J., Dobrev P.I., Brzobohatý B., Vaňková R., Macháčková I. Altered cytokinin metabolism affects cytokinin, auxin, and abscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure in transgenic tobacco. J. Exp. Bot. 2007;58:637–649. doi: 10.1093/jxb/erl235. PubMed DOI

Včelařová L., Skalický V., Chamrád I., Lenobel R., Kubeš M., Pěnčík A., Novák O. Auxin metabolome profiling in the Arabidopsis endoplasmic reticulum using an optimised organelle isolation protocol. Int. J. Mol. Sci. 2021;22:9370. doi: 10.3390/ijms22179370. PubMed DOI PMC

Šafář J., Noa-Carrazana J.C., Vrána J., Bartoš J., Alkhimova O., Sabau X., Šimková H., Lheureux F., Caruana M.-L., Doležel J., et al. Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome. Genome. 2004;47:1182–1191. doi: 10.1139/g04-062. PubMed DOI

Somerville C.R., Somerville S.C., Ogren W.L. Isolation of photosynthetically active protoplasts and chloroplastids from Arabidopsis thaliana. Plant Sci. Lett. 1981;21:89–96. doi: 10.1016/0304-4211(81)90073-0. DOI

Robert S., Zouhar J., Carter C.J., Raikhel N. Isolation of intact vacuoles from Arabidopsis rosette leaf–derived protoplasts. Nat. Protoc. 2007;2:259–262. doi: 10.1038/nprot.2007.26. PubMed DOI

Petersson S.V., Johansson A.I., Kowalczyk M., Makoveychuk A., Wang J.Y., Moritz T., Grebe M., Benfey P.N., Sandberg G., Ljung K. An Auxin gradient and maximum in the Arabidopsis root apex shown by high-resolution cell-specific analysis of IAA distribution and synthesis. Plant Cell. 2009;21:1659–1668. doi: 10.1105/tpc.109.066480. PubMed DOI PMC

McKeown P., Pendle A.F., Shaw P.J. Preparation of arabidopsis nuclei and nucleoli. Methods Mol. Biol. 2008;463:67–75. doi: 10.1007/978-1-59745-406-3_5. PubMed DOI

Tarkowská D., Novák O., Floková K., Tarkowski P., Turečková V., Grúz J., Rolčík J., Strnad M. Quo vadis plant hormone analysis? Planta. 2014;240:55–76. doi: 10.1007/s00425-014-2063-9. PubMed DOI

Pěnčík A., Casanova-Sáez R., Pilařová V., Žukauskaitė A., Pinto R., Luis Micol J., Ljung K., Novák O. Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis. J. Exp. Bot. 2018;69:2569–2579. doi: 10.1093/jxb/ery084. PubMed DOI PMC

Béziat C., Barbez E., Feraru M.I., Lucyshyn D., Kleine-Vehn J. Light triggers PILS-dependent reduction in nuclear auxin signalling for growth transition. Nat. Plants. 2017;3:17105. doi: 10.1038/nplants.2017.105. PubMed DOI PMC

May M.J., Leaver C.J. Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures. Plant Physiol. 1993;103:621–627. doi: 10.1104/pp.103.2.621. PubMed DOI PMC

Nagata T., Nemoto Y., Hasezawa S. Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants. Int. Rev. Cytol. 1992;132:1–30. doi: 10.1016/S0074-7696(08)62452-3. DOI

Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. PubMed DOI

Wolf P.G., Karol K.G., Mandoli D.F., Kuehl J., Arumuganathan K., Ellis M.W., Mishler B.D., Kelch D.G., Olmstead R.G., Boore J.L. The first complete chloroplast genome sequence of a lycophyte, Huperzia lucidula (Lycopodiaceae) Gene. 2005;350:117–128. doi: 10.1016/j.gene.2005.01.018. PubMed DOI

Thibivilliers S., Anderson D., Libault M. Isolation of plant root nuclei for single cell RNA sequencing. Curr. Protoc. Plant Biol. 2020;5:e20120. doi: 10.1002/cppb.20120. PubMed DOI

On the trail of auxin: Reporters and sensors