The development and activity of the procambium and cambium, which ensure vascular tissue formation, is critical for overall plant architecture and growth. However, little is known about the molecular factors affecting the activity of vascular meristems and vascular tissue formation. Here, we show that the His kinase CYTOKININ-INDEPENDENT1 (CKI1) and the cytokinin receptors ARABIDOPSIS HISTIDINE KINASE2 (AHK2) and AHK3 are important regulators of vascular tissue development in Arabidopsis thaliana shoots. Genetic modifications of CKI1 activity in Arabidopsis cause dysfunction of the two-component signaling pathway and defects in procambial cell maintenance. CKI1 overexpression in protoplasts leads to cytokinin-independent activation of the two-component phosphorelay, and intracellular domains are responsible for the cytokinin-independent activity of CKI1. CKI1 expression is observed in vascular tissues of inflorescence stems, and CKI1 forms homodimers both in vitro and in planta. Loss-of-function ahk2 and ahk3 mutants and plants with reduced levels of endogenous cytokinins show defects in procambium proliferation and an absence of secondary growth. CKI1 overexpression partially rescues ahk2 ahk3 phenotypes in vascular tissue, while the negative mutation CKI1H405Q further accentuates mutant phenotypes. These results indicate that the cytokinin-independent activity of CKI1 and cytokinin-induced AHK2 and AHK3 are important for vascular bundle formation in Arabidopsis.

Department of Functional Genomics and Proteomics Institute of Experimental Biology Faculty of Science Masaryk University CZ 61137 Brno Czech Republic

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Aloni, R. (1987). Differentiation of vascular tissues. Annu. Rev. Plant Physiol. Plant Mol. Biol. 38 179–204.

Altamura, M.M., Possenti, M., Matteucci, A., Baima, S., Ruberti, I., and Morelli, G. (2001). Development of the vascular system in the inflorescence stem of Arabidopsis. New Phytol. 151 381–389.

Baucher, M., El Jaziri, M., and Vandeputte, O. (2007). From primary to secondary growth: Origin and development of the vascular system. J. Exp. Bot. 58 3485–3501. PubMed

Brewer, P.B., Heisler, M.G., Hejátko, J., Friml, J., and Benkova, E. (2006). In situ hybridization for mRNA detection in Arabidopsis tissue sections. Nat. Protocols 1 1462–1467. PubMed

Cano-Delgado, A., Yin, Y., Yu, C., Vafeados, D., Mora-Garcia, S., Cheng, J.C., Nam, K.H., Li, J., and Chory, J. (2004). BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development 131 5341–5351. PubMed

Carlsbecker, A., and Helariutta, Y. (2005). Phloem and xylem specification: pieces of the puzzle emerge. Curr. Opin. Plant Biol. 8 512–517. PubMed

Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16 735–743. PubMed

D'Agostino, I.B., Deruere, J., and Kieber, J.J. (2000). Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiol. 124 1706–1717. PubMed PMC

Dortay, H., Mehnert, N., Burkle, L., Schmulling, T., and Heyl, A. (2006). Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana. FEBS J. 273 4631–4644. PubMed

Emery, J.F., Floyd, S.K., Alvarez, J., Eshed, Y., Hawker, N.P., Izhaki, A., Baum, S.F., and Bowman, J.L. (2003). Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes. Curr. Biol. 13 1768–1774. PubMed

Eriksson, M.E., Israelsson, M., Olsson, O., and Moritz, T. (2000). Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length. Nat. Biotechnol. 18 784–788. PubMed

Fisher, K., and Turner, S. (2007). PXY, a receptor-like kinase essential for maintaining polarity during plant vascular-tissue development. Curr. Biol. 17 1061–1066. PubMed

Friml, J., Vieten, A., Sauer, M., Weijers, D., Schwarz, H., Hamann, T., Offringa, R., and Jurgens, G. (2003). Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426 147–153. PubMed

Friml, J., et al. (2004). A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux. Science 306 862–865. PubMed

Fukuda, H. (2004). Signals that control plant vascular cell differentiation. Nat. Rev. Mol. Cell Biol. 5 379–391. PubMed

Gao, Z., Wen, C.K., Binder, B.M., Chen, Y.F., Chang, J., Chiang, Y.H., Kerris III, R.J., Chang, C., and Schaller, G.E. (2008). Heteromeric interactions among ethylene receptors mediate signaling in Arabidopsis. J. Biol. Chem. 283 23801–23810. PubMed PMC

Grefen, C., Städele, K., Ruzicka, K., Obrdlik, P., Harter, K., and Horák, J. (2008). Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene receptor family members. Mol. Plant 1 308–320. PubMed

Hartmann, U., Hohmann, S., Nettesheim, K., Wisman, E., Saedler, H., and Huijser, P. (2000). Molecular cloning of SVP: A negative regulator of the floral transition in Arabidopsis. Plant J. 21 351–360. PubMed

Hejátko, J., Pernisova, M., Eneva, T., Palme, K., and Brzobohaty, B. (2003). The putative sensor histidine kinase CKI1 is involved in female gametophyte development in Arabidopsis. Mol. Genet. Genomics 269 443–453. PubMed

Higuchi, M., et al. (2004). In planta functions of the Arabidopsis cytokinin receptor family. Proc. Natl. Acad. Sci. USA 101 8821–8826. PubMed PMC

Hirakawa, Y., Shinohara, H., Kondo, Y., Inoue, A., Nakanomyo, I., Ogawa, M., Sawa, S., Ohashi-Ito, K., Matsubayashi, Y., and Fukuda, H. (2008). Non-cell-autonomous control of vascular stem cell fate by a CLE peptide/receptor system. Proc. Natl. Acad. Sci. USA 105 15208–15213. PubMed PMC

Hirose, N., Makita, N., Yamaya, T., and 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

Hirose, N., Takei, K., Kuroha, T., Kamada-Nobusada, T., Hayashi, H., and Sakakibara, H. (2008). Regulation of cytokinin biosynthesis, compartmentalization and translocation. J. Exp. Bot. 59 75–83. PubMed

Horak, J., Grefen, C., Berendzen, K.W., Hahn, A., Stierhof, Y.D., Stadelhofer, B., Stahl, M., Koncz, C., and Harter, K. (2008). The Arabidopsis thaliana response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds. BMC Plant Biol. 8 77. PubMed PMC

Hutchison, C.E., Li, J., Argueso, C., Gonzalez, M., Lee, E., Lewis, M.W., Maxwell, B.B., Perdue, T.D., Schaller, G.E., Alonso, J.M., Ecker, J.R., and Kieber, J.J. (2006). The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling. Plant Cell 18 3073–3087. PubMed PMC

Hwang, I., and Sheen, J. (2001). Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413 383–389. PubMed

Jacobs, W.P. (1952). The role of auxin in differentiation of xylem around a wound. Am. J. Bot. 39 301–309.

Kakimoto, T. (1996). CKI1, a histidine kinase homolog implicated in cytokinin signal transduction. Science 274 982–985. PubMed

Kim, H.J., Ryu, H., Hong, S.H., Woo, H.R., Lim, P.O., Lee, I.C., Sheen, J., Nam, H.G., and Hwang, I. (2006). Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis. Proc. Natl. Acad. Sci. USA 103 814–819. PubMed PMC

Ko, J.H., Han, K.H., Park, S., and Yang, J. (2004). Plant body weight-induced secondary growth in Arabidopsis and its transcription phenotype revealed by whole-transcriptome profiling. Plant Physiol. 135 1069–1083. PubMed PMC

Kurakawa, T., Ueda, N., Maekawa, M., Kobayashi, K., Kojima, M., Nagato, Y., Sakakibara, H., and Kyozuka, J. (2007). Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445 652–655. PubMed

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

Mähönen, A.P., Bonke, M., Kauppinen, L., Riikonen, M., Benfey, P.N., and Helariutta, Y. (2000). A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root. Genes Dev. 14 2938–2943. PubMed PMC

Mähönen, A.P., Higuchi, M., Tormakangas, K., Miyawaki, K., Pischke, M.S., Sussman, M.R., Helariutta, Y., and Kakimoto, T. (2006. a). Cytokinins regulate a bidirectional phosphorelay network in Arabidopsis. Curr. Biol. 16 1116–1122. PubMed

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

Medford, J.I., Horgan, R., El-Sawi, Z., and Klee, H.J. (1989). Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. Plant Cell 1 403–413. PubMed PMC

Miyawaki, K., Matsumoto-Kitano, M., and Kakimoto, T. (2004). Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: Tissue specificity and regulation by auxin, cytokinin, and nitrate. Plant J. 37 128–138. PubMed

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

Nakamura, A., Kakimoto, T., Imamura, A., Suzuki, T., Ueguchi, C., and Mizuno, T. (1999). Biochemical characterization of a putative cytokinin-responsive His-kinase, CKI1, from Arabidopsis thaliana. Biosci. Biotechnol. Biochem. 63 1627–1630. PubMed

Nieminen, K., et al. (2008). Cytokinin signaling regulates cambial development in poplar. Proc. Natl. Acad. Sci. USA 105 20032–20037. PubMed PMC

Nishimura, C., Ohashi, Y., Sato, S., Kato, T., Tabata, S., and 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

Pernisova, M., Klima, P., Horak, J., Valkova, M., Malbeck, J., Soucek, P., Reichman, P., Hoyerova, K., Dubova, J., Friml, J., Zazimalova, E., and Hejatko, J. (2009). Cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux. Proc. Natl. Acad. Sci. USA 106 3609–3614. PubMed PMC

Pischke, M.S., Jones, L.G., Otsuga, D., Fernandez, D.E., Drews, G.N., and Sussman, M.R. (2002). An Arabidopsis histidine kinase is essential for megagametogenesis. Proc. Natl. Acad. Sci. USA 99 15800–15805. PubMed PMC

Prigge, M.J., Otsuga, D., Alonso, J.M., Ecker, J.R., Drews, G.N., and Clark, S.E. (2005). Class III homeodomain-leucine zipper gene family members have overlapping, antagonistic, and distinct roles in Arabidopsis development. Plant Cell 17 61–76. PubMed PMC

Sachs, T. (2000). Integrating cellular and organismic aspects of vascular differentiation. Plant Cell Physiol. 41 649–656. PubMed

Samuelson, M.E., Eliasson, L., and Larsson, C.M. (1992). Nitrate-regulated growth and cytokinin responses in seminal roots of barley. Plant Physiol. 98 309–315. PubMed PMC

Schaller, G.E., Ladd, A.N., Lanahan, M.B., Spanbauer, J.M., and Bleecker, A.B. (1995). The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer. J. Biol. Chem. 270 12526–12530. PubMed

Scheres, B., Dilaurenzio, L., Willemsen, V., Hauser, M.T., Janmaat, K., Weisbeek, P., and Benfey, P.N. (1995). Mutations affecting the radial organization of the Arabidopsis root display specific defects throughout the embryonic axis. Development 121 53–62.

Surette, M.G., Levit, M., Liu, Y., Lukat, G., Ninfa, E.G., Ninfa, A., and Stock, J.B. (1996). Dimerization is required for the activity of the protein histidine kinase CheA that mediates signal transduction in bacterial chemotaxis. J. Biol. Chem. 271 939–945. PubMed

Szekeres, M., Nemeth, K., Koncz-Kalman, Z., Mathur, J., Kauschmann, A., Altmann, T., Redei, G.P., Nagy, F., Schell, J., and Koncz, C. (1996). Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85 171–182. PubMed

Takei, K., Ueda, N., Aoki, K., Kuromori, T., Hirayama, T., Shinozaki, K., Yamaya, T., and Sakakibara, H. (2004). AtIPT3 is a key determinant of nitrate-dependent cytokinin biosynthesis in Arabidopsis. Plant Cell Physiol. 45 1053–1062. PubMed

To, J.P., Haberer, G., Ferreira, F.J., Deruere, J., Mason, M.G., Schaller, G.E., Alonso, J.M., Ecker, J.R., and Kieber, J.J. (2004). Type-A Arabidopsis response regulators are partially redundant negative regulators of cytokinin signaling. Plant Cell 16 658–671. PubMed PMC

Tomomori, C., et al. (1999). Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ. Nat. Struct. Biol. 6 729–734. PubMed

Vitha, S., Baluska, F., Braun, M., Samaj, J., Volkmann, D., and Barlow, P.W. (2000). Comparison of cryofixation and aldehyde fixation for plant actin immunocytochemistry: Aldehydes do not destroy F-actin. Histochem. J. 32 457–466. PubMed

Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C., Harter, K., and Kudla, J. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40 428–438. PubMed

Werner, T., Kollmer, I., Bartrina, I., Holst, K., and Schmulling, T. (2006). New insights into the biology of cytokinin degradation. Plant Biol. 8 371–381. PubMed

Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H., and Schmulling, T. (2003). Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15 2532–2550. PubMed PMC

Yamada, H., Suzuki, T., Terada, K., Takei, K., Ishikawa, K., Miwa, K., Yamashino, T., and Mizuno, T. (2001). The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol. 42 1017–1023. PubMed

Yang, J., Zhang, J., Wang, Z., Zhu, Q., and Wang, W. (2001). Hormonal changes in the grains of rice subjected to water stress during grain filling. Plant Physiol. 127 315–323. PubMed PMC

Ye, Z.H., Freshour, G., Hahn, M.G., Burk, D.H., and Zhong, R. (2002). Vascular development in Arabidopsis. Int. Rev. Cytol. 220 225–256. PubMed

Yokoyama, A., Yamashino, T., Amano, Y., Tajima, Y., Imamura, A., Sakakibara, H., and Mizuno, T. (2007). Type-B ARR transcription factors, ARR10 and ARR12, are implicated in cytokinin-mediated regulation of protoxylem differentiation in roots of Arabidopsis thaliana. Plant Cell Physiol. 48 84–96. PubMed

Zhao, C., Craig, J.C., Petzold, H.E., Dickerman, A.W., and Beers, E.P. (2005). The xylem and phloem transcriptomes from secondary tissues of the Arabidopsis root-hypocotyl. Plant Physiol. 138 803–818. PubMed PMC

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