Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific.

Department of Life Sciences Imperial College London London SW7 2AZ United Kingdom

Laboratory of Growth Regulators and Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Institute of Experimental Botany AS CR and Faculty of Science of Palacký University Šlechtitelů 27 CZ 78371 Olomouc Czech Republic

Umeå Plant Science Centre Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences SE 901 83 Umeå Sweden

Umeå Plant Science Centre Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences SE 901 83 Umeå Sweden Department of Life Sciences Imperial College London London SW7 2AZ United Kingdom

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Plant Cell. 2015 Jul;27(7):1819. doi: 10.1105/tpc.15.00553. PubMed

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Aloni R., Aloni E., Langhans M., Ullrich C.I. (2006). Role of cytokinin and auxin in shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism. Ann. Bot. (Lond.) 97: 883–893. PubMed PMC

Aremu A.O., Bairu M.W., Novák O., Plačková L., Zatloukal M., Doležal K., Finnie J.F., Strnad M., Van Staden J. (2012). Physiological responses and endogenous cytokinin profiles of tissue-cultured ‘Williams’ bananas in relation to roscovitine and an inhibitor of cytokinin oxidase/dehydrogenase (INCYDE) treatments. Planta 236: 1775–1790. PubMed

Bielach A., Podlesáková K., Marhavy P., Duclercq J., Cuesta C., Müller B., Grunewald W., Tarkowski P., Benková E. (2012). Spatiotemporal regulation of lateral root organogenesis in PubMed PMC

Birnbaum K., Jung J.W., Wang J.Y., Lambert G.M., Hirst J.A., Galbraith D.W., Benfey P.N. (2005). Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nat. Methods 2: 615–619. PubMed

Birnbaum K., Shasha D.E., Wang J.Y., Jung J.W., Lambert G.M., Galbraith D.W., Benfey P.N. (2003). A gene expression map of the PubMed

Bishopp A., Help H., El-Showk S., Weijers D., Scheres B., Friml J., Benková E., Mähönen A.P., Helariutta Y. (2011). A mutually inhibitory interaction between auxin and cytokinin specifies vascular pattern in roots. Curr. Biol. 21: 917–926. PubMed

Brady S.M., Orlando D.A., Lee J.Y., Wang J.Y., Koch J., Dinneny J.R., Mace D., Ohler U., Benfey P.N. (2007). A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318: 801–806. PubMed

Bürkle L., Cedzich A., Döpke C., Stransky H., Okumoto S., Gillissen B., Kühn C., Frommer W.B. (2003). Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of PubMed

Caesar K., Thamm A.M., Witthöft J., Elgass K., Huppenberger P., Grefen C., Horak J., Harter K. (2011). Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum. J. Exp. Bot. 62: 5571–5580. PubMed PMC

Cedzich A., Stransky H., Schulz B., Frommer W.B. (2008). Characterization of cytokinin and adenine transport in PubMed PMC

Dello Ioio R., Linhares F.S., Scacchi E., Casamitjana-Martinez E., Heidstra R., Costantino P., Sabatini S. (2007). Cytokinins determine PubMed

Dello Ioio R., Nakamura K., Moubayidin L., Perilli S., Taniguchi M., Morita M.T., Aoyama T., Costantino P., Sabatini S. (2008). A genetic framework for the control of cell division and differentiation in the root meristem. Science 322: 1380–1384. PubMed

Dinneny J.R., Long T.A., Wang J.Y., Jung J.W., Mace D., Pointer S., Barron C., Brady S.M., Schiefelbein J., Benfey P.N. (2008). Cell identity mediates the response of PubMed

Dobrev P.I., Kamínek M. (2002). Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J. Chromatogr. A 950: 21–29. PubMed

Drake G.A. (1979). Electrical coupling, potentials, and resistances in oat coleoptiles: effects of azide and cyanide. J. Exp. Bot. 30: 719–725.

El-Showk S., Ruonala R., Helariutta Y. (2013). Crossing paths: cytokinin signalling and crosstalk. Development 140: 1373–1383. PubMed

Gajdosová S., et al. (2011). Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants. J. Exp. Bot. 62: 2827–2840. PubMed

Galuszka P., Popelková H., Werner T., Frébortová J., Pospíšilová H., Mik V., Köllmer I., Schmülling T., Frébort I. (2007). Biochemical characterization of cytokinin oxidases/dehydrogenases from

Gaudinová A., Dobrev P.I., Šolcová B., Novák O., Strnad M., Friedecký D., Motyka V. (2005). The involvement of cytokinin oxidase/dehydrogenase and zeatin reductase in regulation of cytokinin levels in pea (

Gilroy S., Hughes W.A., Trewavas A.J. (1989). A comparison between quin-2 and aequorin as indicators of cytoplasmic calcium levels in higher plant cell protoplasts. Plant Physiol. 90: 482–491. PubMed PMC

Hirose N., Makita N., Yamaya T., Sakakibara H. (2005). Functional characterization and expression analysis of a gene, OsENT2, encoding an equilibrative nucleoside transporter in rice suggest a function in cytokinin transport. Plant Physiol. 138: 196–206. PubMed PMC

Ishihama Y., Rappsilber J., Mann M. (2006). Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J. Proteome Res. 5: 988–994. PubMed

Jones B., Gunnerås S.A., Petersson S.V., Tarkowski P., Graham N., May S., Doležal K., Sandberg G., Ljung K. (2010). Cytokinin regulation of auxin synthesis in PubMed PMC

Kiba T., Takei K., Kojima M., Sakakibara H. (2013). Side-chain modification of cytokinins controls shoot growth in PubMed

Ko D., et al. (2014). PubMed PMC

Köllmer I., Novák O., Strnad M., Schmülling T., Werner T. (2014). Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from PubMed

Kuroha T., Tokunaga H., Kojima M., Ueda N., Ishida T., Nagawa S., Fukuda H., Sugimoto K., Sakakibara H. (2009). Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in PubMed PMC

Lomin S.N., Krivosheev D.M., Steklov M.Y., Osolodkin D.I., Romanov G.A. (2012). Receptor properties and features of cytokinin signaling. Acta Naturae 4: 31–45. PubMed PMC

Mähönen A.P., Bishopp A., Higuchi M., Nieminen K.M., Kinoshita K., Törmäkangas K., Ikeda Y., Oka A., Kakimoto T., Helariutta Y. (2006). Cytokinin signaling and its inhibitor AHP6 regulate cell fate during vascular development. Science 311: 94–98. PubMed

Marhavý P., Duclercq J., Weller B., Feraru E., Bielach A., Offringa R., Friml J., Schwechheimer C., Murphy A., Benková E. (2014). Cytokinin controls polarity of PIN1-dependent auxin transport during lateral root organogenesis. Curr. Biol. 24: 1031–1037. PubMed

Matsumoto-Kitano M., Kusumoto T., Tarkowski P., Kinoshita-Tsujimura K., Václavíková K., Miyawaki K., Kakimoto T. (2008). Cytokinins are central regulators of cambial activity. Proc. Natl. Acad. Sci. USA 105: 20027–20031. PubMed PMC

Miyawaki K., Matsumoto-Kitano M., Kakimoto T. (2004). Expression of cytokinin biosynthetic isopentenyltransferase genes in PubMed

Miyawaki K., Tarkowski P., Matsumoto-Kitano M., Kato T., Sato S., Tarkowska D., Tabata S., Sandberg G., Kakimoto T. (2006). Roles of PubMed PMC

Moreira S., Bishopp A., Carvalho H., Campilho A. (2013). AHP6 inhibits cytokinin signaling to regulate the orientation of pericycle cell division during lateral root initiation. PLoS One 8: e56370. PubMed PMC

Motyka V., Faiss M., Strand M., Kamínek M., Schmülling T. (1996). Changes in cytokinin content and cytokinin oxidase activity in response to derepression of PubMed PMC

Müller B., Sheen J. (2008). Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature 453: 1094–1097. PubMed PMC

Nishimura C., Ohashi Y., Sato S., Kato T., Tabata S., Ueguchi C. (2004). Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. Plant Cell 16: 1365–1377. PubMed PMC

Pěnčík A., et al. (2013). Regulation of auxin homeostasis and gradients in PubMed PMC

Petersson S.V., Johansson A.I., Kowalczyk M., Makoveychuk A., Wang J.Y., Moritz T., Grebe M., Benfey P.N., Sandberg G., Ljung K. (2009). An auxin gradient and maximum in the PubMed PMC

Petricka J.J., Schauer M.A., Megraw M., Breakfield N.W., Thompson J.W., Georgiev S., Soderblom E.J., Ohler U., Moseley M.A., Grossniklaus U., Benfey P.N. (2012). The protein expression landscape of the PubMed PMC

Rappsilber J., Ishihama Y., Mann M. (2003). Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75: 663–670. PubMed

Rappsilber J., Mann M., Ishihama Y. (2007). Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2: 1896–1906. PubMed

Romanov G.A., Lomin S.N., Schmülling T. (2006). Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay. J. Exp. Bot. 57: 4051–4058. PubMed

Růžička K., Simásková M., Duclercq J., Petrásek J., Zazímalová E., Simon S., Friml J., Van Montagu M.C., Benková E. (2009). Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proc. Natl. Acad. Sci. USA 106: 4284–4289. PubMed PMC

Sakakibara H. (2006). Cytokinins: activity, biosynthesis, and translocation. Annu. Rev. Plant Biol. 57: 431–449. PubMed

Schaller G.E., Bishopp A., Kieber J.J. (2015). The yin-yang of hormones: cytokinin and auxin interactions in plant development. Plant Cell 27: 44–63. PubMed PMC

Schmitz R.Y., Skoog F. (1972). Cytokinins: synthesis and biological activity of geometric and position isomers of zeatin. Plant Physiol. 50: 702–705. PubMed PMC

Spanswick R.M., Miller A.G. (1977). Measurement of the cytoplasmic pH in PubMed PMC

Spíchal L., Rakova N.Y., Riefler M., Mizuno T., Romanov G.A., Strnad M., Schmülling T. (2004). Two cytokinin receptors of PubMed

Spíchal L. (2012). Cytokinins–recent news and views of evolutionarily old molecules. Funct. Plant Biol. 39: 267–284. PubMed

Spiess L.D. (1975). Comparative activity of isomers of zeatin and ribosyl-zeatin on PubMed PMC

Svačinová J., Novák O., Plačková L., Lenobel R., Holík J., Strnad M., Doležal K. (2012). A new approach for cytokinin isolation from PubMed PMC

Swarup R., Kramer E.M., Perry P., Knox K., Leyser H.M., Haseloff J., Beemster G.T., Bhalerao R., Bennett M.J. (2005). Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal. Nat. Cell Biol. 7: 1057–1065. PubMed

Takei K., Ueda N., Aoki K., Kuromori T., Hirayama T., Shinozaki K., Yamaya T., Sakakibara H. (2004). AtIPT3 is a key determinant of nitrate-dependent cytokinin biosynthesis in PubMed

Tucker E.B. (1993). Azide treatment enhances cell-to-cell diffusion in staminal hairs of

Turnbull C.G.N., Hanke D.E. (1985). The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins. Planta 165: 366–376. PubMed

Wang J., Ma X.-M., Kojima M., Sakakibara H., Hou B.-K. (2013). Glucosyltransferase UGT76C1 finely modulates cytokinin responses via cytokinin N-glucosylation in PubMed

Werner T., Motyka V., Laucou V., Smets R., Van Onckelen H., Schmülling T. (2003). Cytokinin-deficient transgenic PubMed PMC

Wulfetange K., Lomin S.N., Romanov G.A., Stolz A., Heyl A., Schmülling T. (2011). The cytokinin receptors of PubMed PMC

Yonekura-Sakakibara K., Kojima M., Yamaya T., Sakakibara H. (2004). Molecular characterization of cytokinin-responsive histidine kinases in maize. Differential ligand preferences and response to PubMed PMC

Zatloukal M., Gemrotová M., Doležal K., Havlícek L., Spíchal L., Strnad M. (2008). Novel potent inhibitors of PubMed

Zhang K., Novák O., Wei Z., Gou M., Zhang X., Yu Y., Yang H., Cai Y., Strnad M., Liu C.J. (2014). PubMed

Zhang W., Swarup R., Bennett M., Schaller G.E., Kieber J.J. (2013). Cytokinin induces cell division in the quiescent center of the PubMed

Zhang W., To J.P.C., Cheng C.Y., Schaller G.E., Kieber J.J. (2011). Type-A response regulators are required for proper root apical meristem function through post-transcriptional regulation of PIN auxin efflux carriers. Plant J. 68: 1–10. PubMed

Zürcher E., Tavor-Deslex D., Lituiev D., Enkerli K., Tarr P.T., Müller B. (2013). A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network PubMed PMC

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