BACKGROUND: When applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays [1]. Those showing comparable activities with N(6)-benzylaminopurine were further tested in planta. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants. CONCLUSIONS/SIGNIFICANCE: Tetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected hormonal effects in plant propagation techniques in the future.

Department of Biochemistry Faculty of Science Palacký University Olomouc Czech Republic

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Szüčová L, Spíchal L, Doležal K, Zatloukal M, Greplová J, et al. Synthesis, characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives. Bioorgan Med Chem. 2009;17:1938–1947. PubMed

West T, Preece JE. Effects of thidiazuron and nutrient salt formulations on micropropagation of hardy hibiscus (Hibiscus moscheutos L.). Acta Hortic. 2004;630:293–297.

Debnath SC. Zeatin-induced one-step in vitro cloning affects the vegetative growth of cranberry (Vaccinium macrocarpon Ait.) micropropagules over stem cuttings. Plant Cell Tiss Org. 2008;93:231–240.

Robert-Seilaniantz A, Navarro L, Bari R, Jones JD. Pathological hormone imbalances. Curr Opin Plant Biol. 2007;10:372–379. PubMed

Bairu MW, Fennell CW, van Staden J. The effect of plant growth regulators on somaclonal variation in Cavendish banana (Musa AAA cv. ‘Zelig’). Sci Hortic-Amterdam. 2006;108:347–351.

Galuszka P, Popelková H, Werner T, Frébortová J, Pospíšilová H, et al. Biochemical characterization and histochemical localization of cytokinin oxidases/dehydrogenases from Arabidopsis thaliana expressed in Nicotiana tabaccum L. J Plant Growth Regul. 2007;26:255–267.

Werbrouck SPO, Strnad M, van Onckelen HA, Debergh PC. Meta-topolin, an alternative to benzyladenine in tissue culture? Physiol Plantarum. 1996;98:291–297.

Bairu MW, Stirk WA, Doležal K, van Staden J. The role of topolins in micropropagation and somaclonal variation of banana cultivars ‘Williams’ and ‘Grand Naine’ (Musa spp. AAA). Plant Cell Tiss Org. 2008;95:373–379.

Valero-Aracama C, Kane ME, Wilson SB, Philman NL. Substitution of benzyladenine with meta-topolin during shoot multiplication increases acclimatization of difficult- and easy-to-acclimatize sea oats (Uniola paniculata L.) genotypes. Plant Growth Regul. 2010;60:43–49.

Holub J, Hanuš J, Hanke DE, Strnad M. Biological activity of cytokinins derived from ortho- and meta-hydroxybenzyladenine. Plant Growth Regul. 1998;26:109–115.

Mok MC, Martin RC, Dobrev PI, Vaňková R, Ho PS, et al. Topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin O-glucosyltransferase with position specificity related to receptor recognition. Plant Physiol. 2005;137:1057–1066. PubMed PMC

Spíchal L, Rakova NY, Riefler M, Mizuno T, Romanov GA, et al. Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol. 2004;45:1299–1305. PubMed

Werbrouck SPO, van der Jeugt B, Dewitte W, Prinsen E, van Onckelen HA. The metabolism of benzyladenine in Spathiphyllum floribundum ‘Schott Petite’ in relation to acclimatisation problems. Plant Cell Rep. 1995;14:662–665. PubMed

Auer CA. Cytokinin inhibition of Arabidopsis root growth: An examination of genotype, cytokinin activity, and N-6-benzyladenine metabolism. J Plant Growth Regul. 1996;15:201–206.

Cary AJ, Liu WN, Howell SH. Cytokinin action is coupled to ethylene in its effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings. Plant Physiol. 1995;107:1075–1082. PubMed PMC

Bertell G, Eliasson L. Cytokinin effects on root growth and possible interactions with ethylene and indole-3-acetic acid. Physiol Plantarum. 1992;84:255–261.

Růžička K, Ljung K, Vanneste S, Podhorská R, Beeckman T, et al. Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution. Plant Cell. 2007;7:2197–2212. PubMed PMC

Růžička K, Šimašková M, Duclercq J, Petrášek J, Zažímalová E, et al. Cytokinin regulates root meristem activity via modulation of the polar auxin transport. P Natl Acad Sci USA. 2009;106:4284–4289. PubMed PMC

Zhang R, Letham DS. Cytokinin biochemistry in relation to leaf senescence. III. The senescence-retarding activity and metabolism of 9-substituted 6-benzylaminopurines in soybean leaves. J Plant Growth Regul. 1989;8:181–197.

Corse J, Pacovsky RS, Lyman ML, Brandon DL. Biological activity of several 9-nonglycosidic-substituted natural cytokinins. J Plant Growth Regul. 1989;8:211–223.

Fox JE, Sood CK, Buckwalter B, McChesney JD. The metabolism and biological activity of a 9-substituted cytokinin. Plant Physiol. 1971;47:275–281. PubMed PMC

Pietraface WJ, Blaydes DF. Activity and metabolism of 9-substituted cytokinins during lettuce seed germination. Physiol Plantarum. 1981;53:249–254.

Sul IW, Korban SS. Effect of different cytokinins on axillary shoot proliferation and elongation of several genotypes of Sequoia sempervirens. In Vitro Cell Dev-Pl. 1994;30:131–135.

Weaver RJ, Overbeek RM, Pool RM. Induction of fruit set in Vitis vinifera L. by a kinin. Nature. 1965;206:952–953.

Letham DS, Summons RE, Entsch B, Gollnow BI, Parker CW, et al. Glycosylation of cytokinin analogues. Phytochemistry. 1978;17:2053–2057.

Suzuki T, Miwa K, Ishikawa K, Yamada H, Aiba H, et al. The Arabidopsis sensor His-kinase, AHK4, can respond to cytokinins. Plant Cell Physiol. 2001;42:107–113. PubMed

Yonekura-Sakakibara K, Kojima M, Yamaya T, Sakakibara H. Molecular characterization of cytokinin-responsive histidine kinases in maize. Differential ligand preferences and response to cis-zeatin. Plant Physiol. 2004;134:1654–1661. PubMed PMC

Gallie DR, Young TE. The ethylene biosynthetic and perception machinery is differentially expressed during endosperm and embryo development in maize. Mol Genet Genomics. 2004;271:267–281. PubMed

Suttle JC, Mornet R. Mechanism-based irreversible inhibitors of cytokinin dehydrogenase. J Plant Physiol. 2005;162:1189–1196. PubMed

Kopečný D, Briozzo P, Popelková H, Šebela M, Končitíková R, et al. Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study. Biochimie. 2010;92:1052–1062. PubMed

Romanov GA, Lomin SN, Schmülling T. 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. 2006;57:4051–4058. PubMed

Tarkowská D, Doležal K, Tarkowski P, Åstot C, Holub J, et al. Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus x canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography/frit-fast atom bombardment mass spectrometry. Physiol Plantarum. 2003;117:579–590. PubMed

Higuchi M, Pischke MS, Mähonen AP, Miyawaki K, Hashimoto Y, et al. In planta functions of the Arabidopsis cytokinin receptor family. P Natl Acad Sci USA. 2004;101:8821–8826. PubMed PMC

Hothorn M, Dabi T, Chory J. Structural basis for cytokinin recognition by Arabidopsis thaliana histidine kinase 4. Nat Chem Biol. 2011;7:766–768. PubMed PMC

Kudo T, Kiba T, Sakakibara H. Metabolism and long-distance translocation of cytokinins. J Integr Plant Biol. 2010;52:53–60. PubMed

Saleem M, Lamkemeyer T, Schützenmeister A, Madlung J, Sakai H, et al. Specification of cortical parenchyma and stele of maize (Zea mays L.) primary roots by asymmetric levels of auxin, cytokinin and cytokinin-regulated proteins. Plant Physiol. 2010;152:4–18. PubMed PMC

Laplaze L, Benková E, Casimiro I, Maes L, Vanneste S, et al. Cytokinins act directly on lateral root founder cells to inhibit root initiation. Plant Cell. 2007;19:3889–3900. PubMed PMC

Mik V, Szüčová L, Šmehilová M, Zatloukal M, Doležal K, et al. N9-substituted derivatives of kinetin: Effective anti-senescence agents. Phytochemistry. 2011;72:821–831. PubMed

Vyroubalová Š, Václavíková K, Turečková V, Novák O, Šmehilová M, et al. Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels. Plant Physiol. 2009;51:433–447. PubMed PMC

Spíchal L, Werner T, Popa I, Riefler M, Schmülling T, et al. The purine derivative PI-55 blocks cytokinin action via receptor inhibition. FEBS J. 2009;276:244–253. PubMed

Nisler J, Zatloukal M, Popa I, Doležal K, Strnad M, et al. Cytokinin receptor antagonists derived from 6-benzylaminopurine. Phytochemistry. 2010;71:823–830. PubMed

Riefler M, Novák O, Strnad M, Schmülling T. Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. Plant Cell. 2006;18:40–54. PubMed PMC

Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, et al. 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. 2003;15:2532–2550. PubMed PMC

McGaw BA, Heald JK, Horgan R. Dihydrozeatin metabolism in radish seedlings. Phytochemistry. 1984;23:1373–1377.

McGaw BA, Horgan R, Heald JK. Cytokinin metabolism and the modulation of cytokinin activity in radish. Phytochemistry. 1985;24:9–13.

Winkler R, Sandermann H. N-Glucosyl conjugates of chlorinated anilines: spontaneous formation and cleavage. J Agr Food Chem. 1992;40:2008–2012.

Brzobohatý B, Moore I, Kristoffersen P, Bako L, Campos N, et al. Release of active cytokinin by a beta-glucosidase localized to the maize root meristem. Science. 1993;12:1051–1054. PubMed

Cairns JRK, Esen A. β-Glucosidases. Cell Mol Life Sci. 2010;67:3389–3405. PubMed PMC

Filipi T, Mazura P, Janda L, Kiran NS, Brzobohatý B. Engineering the cytokinin-glucoside specificity of the maize β-D-glucosidase Zm-p60.1 using site-directed random mutagenesis. Phytochemistry. 2012;74:40–48. PubMed

Faiss M, Zalubilová J, Strnad M, Schmülling T. Conditional transgenic expression of the ipt gene indicates a function of cytokinins in paracrine signaling in whole tobacco plants. Plant J. 1997;12:401–415. PubMed

Novák O, Hauserová E, Amakorová P, Doležal K, Strnad M. Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry. Phytochemistry. 2008;69:2214–2224. PubMed

Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, et al. The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol. 2001;42:1017–1023. PubMed

Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B Met. 1995;57:289–300.

Kurakawa T, Ueda N, Maekawa M, Kobayashi K, Kojima M, et al. Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature. 2007;445:652–655. PubMed

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