Cytokinin is an indispensable phytohormone responsible for physiological processes ranging from root development to leaf senescence. The term "cytokinin" refers to several dozen adenine-derived compounds occurring naturally in plants. Cytokinins (CKs) can be divided into various classes and forms; base forms are generally considered to be active while highly abundant cytokinin-N-glucosides (CKNGs), composed of a CK base irreversibly conjugated to a glucose molecule, are considered inactive. However, results from early CK studies suggest CKNGs do not always lack activity despite the perpetuation over several decades in the literature that they are inactive. Here we show that exogenous application of trans-Zeatin-N-glucosides (tZNGs, a specific class of CKNGs) to Arabidopsis results in CK response comparable to the application of an active CK base. These results are most apparent in senescence assays where both a CK base (tZ) and tZNGs (tZ7G, tZ9G) delay senescence in cotyledons. Further experiments involving root growth and shoot regeneration revealed tZNGs do not always have the same effects as tZ, and have largely distinct effects on the transcriptome and proteome. These data are in contrast to previous reports of CKNGs being inactive and raise questions about the function of these compounds as well as their mechanism of action.

Department of Biological Sciences Auburn University Auburn AL United States of America

Department of Molecular Biology and Radiobiology Faculty of AgriSciences Mendel University in Brno Brno Czech Republic

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Kieber JJ, Schaller GE. Cytokinins. Arab Book Am Soc Plant Biol. 2014. January 2;12 Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894907/ PubMed PMC

Sakakibara H. CYTOKININS: Activity, Biosynthesis, and Translocation. Annu Rev Plant Biol. 2006;57(1):431–49. PubMed

Schmitz RY, Skoog F, Hecht SM, Bock RM, Leonard NJ. Comparison of cytokinin activities of naturally occurring ribonucleosides and corresponding bases. Phytochemistry. 1972. May 1;11(5):1603–10.

Spiess LD. Comparative Activity of Isomers of Zeatin and Ribosyl-Zeatin on Funaria hygrometrica. Plant Physiol. 1975. March 1;55(3):583–5. 10.1104/pp.55.3.583 PubMed DOI PMC

Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, Miwa K, et al. The Arabidopsis AHK4 Histidine Kinase is a Cytokinin-Binding Receptor that Transduces Cytokinin Signals Across the Membrane. Plant Cell Physiol. 2001. September 15;42(9):1017–23. 10.1093/pcp/pce127 PubMed DOI

Brzobohaty B, Moore I, Kristoffersen P, Bako L, Campos N, Schell J, et al. Release of active cytokinin by a beta-glucosidase localized to the maize root meristem. Science. 1993. November 12;262(5136):1051–4. 10.1126/science.8235622 PubMed DOI

Entsch B, Letham DS. Enzymic glucosylation of the cytokinin, 6-benzylaminopurine. Plant Sci Lett. 1979. February 1;14(2):205–12.

Falk A, Rask L. Expression of a Zeatin-O-Glucoside-Degrading [beta]-Glucosidase in Brassica napus. Plant Physiol. 1995. August 1;108(4):1369–77. 10.1104/pp.108.4.1369 PubMed DOI PMC

Yoshida R, Oritani T. Cytokinin glucoside in roots of the rice plant. Plant Cell Physiol. 1972. April 1;13(2):337–43.

Bairu MW, Novák O, Doležal K, Van Staden J. Changes in endogenous cytokinin profiles in micropropagated Harpagophytum procumbens in relation to shoot-tip necrosis and cytokinin treatments. Plant Growth Regul. 2011. March 1;63(2):105–14.

Fox JE, Cornette J, Deleuze G, Dyson W, Giersak C, Niu P, et al. The Formation, Isolation, and Biological Activity of a Cytokinin 7- Glucoside. Plant Physiol. 1973. December 1;52(6):627–32. 10.1104/pp.52.6.627 PubMed DOI PMC

Spíchal L. Cytokinins–recent news and views of evolutionally old molecules. Funct Plant Biol. 2012. May 15;39(4):267–84. PubMed

Kiba T, Takebayashi Y, Kojima M, Sakakibara H. Sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated CO 2. Sci Rep. 2019. May 23;9(1):1–15. 10.1038/s41598-018-37186-2 PubMed DOI PMC

Tokunaga H, Kojima M, Kuroha T, Ishida T, Sugimoto K, Kiba T, et al. Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation. Plant J. 2012;69(2):355–65. 10.1111/j.1365-313X.2011.04795.x PubMed DOI

Ko D, Kang J, Kiba T, Park J, Kojima M, Do J, et al. Arabidopsis ABCG14 is essential for the root-to-shoot translocation of cytokinin. Proc Natl Acad Sci. 2014. May 13;111(19):7150–5. 10.1073/pnas.1321519111 PubMed DOI PMC

Kiba T, Takei K, Kojima M, Sakakibara H. Side-Chain Modification of Cytokinins Controls Shoot Growth in Arabidopsis. Dev Cell. 2013. November 25;27(4):452–61. 10.1016/j.devcel.2013.10.004 PubMed DOI

Nishiyama R, Watanabe Y, Fujita Y, Le DT, Kojima M, Werner T, et al. Analysis of Cytokinin Mutants and Regulation of Cytokinin Metabolic Genes Reveals Important Regulatory Roles of Cytokinins in Drought, Salt and Abscisic Acid Responses, and Abscisic Acid Biosynthesis. Plant Cell. 2011. June 1;23(6):2169–83. 10.1105/tpc.111.087395 PubMed DOI PMC

Sakakibara H, Kasahara H, Ueda N, Kojima M, Takei K, Hishiyama S, et al. Agrobacterium tumefaciens increases cytokinin production in plastids by modifying the biosynthetic pathway in the host plant. Proc Natl Acad Sci. 2005. July 12;102(28):9972–7. 10.1073/pnas.0500793102 PubMed DOI PMC

Zhang K, Novak O, Wei Z, Gou M, Zhang X, Yu Y, et al. Arabidopsis ABCG14 protein controls the acropetal translocation of root-synthesized cytokinins. Nat Commun. 2014. December;5(1). PubMed

Letham DS, Palni LMS. The Biosynthesis and Metabolism of Cytokinins. Annu Rev Plant Physiol. 1983;34(1):163–97.

Letham DS, Palni LMS, Tao G-Q, Gollnow BI, Bates CM. Regulators of cell division in plant tissues XXIX. The activities of cytokinin glucosides and alanine conjugates in cytokinin bioassays. J Plant Growth Regul. 1983. August 1;2(1):103–15.

Palni LMS, Palmer MV, Letham DS. The stability and biological activity of cytokinin metabolites in soybean callus tissue. Planta. 1984. March 1;160(3):242–9. 10.1007/BF00402861 PubMed DOI

Deleuze GG, McChesney JD, Fox JE. Identification of a stable cytokinin metabolite. Biochem Biophys Res Commun. 1972. September 26;48(6):1426–32. 10.1016/0006-291x(72)90872-8 PubMed DOI

Hou B, Lim E-K, Higgins GS, Bowles DJ. N-Glucosylation of Cytokinins by Glycosyltransferases of Arabidopsis thaliana. J Biol Chem. 2004. November 12;279(46):47822–32. 10.1074/jbc.M409569200 PubMed DOI

Li Y, Wang B, Dong R, Hou B. AtUGT76C2, an Arabidopsis cytokinin glycosyltransferase is involved in drought stress adaptation. Plant Sci. 2015. July 1;236(Supplement C):157–67. PubMed

Wang J, Ma X-M, Kojima M, Sakakibara H, Hou B-K. N-Glucosyltransferase UGT76C2 is Involved in Cytokinin Homeostasis and Cytokinin Response in Arabidopsis thaliana. Plant Cell Physiol. 2011. December 1;52(12):2200–13. 10.1093/pcp/pcr152 PubMed DOI

Hošek P, Hoyerová K, Kiran NS, Dobrev PI, Zahajská L, Filepová R, et al. Distinct metabolism of N-glucosides of isopentenyladenine and trans-zeatin determines cytokinin metabolic spectrum in Arabidopsis. New Phytol. 2019; Available from: https://nph.onlinelibrary.wiley.com/doi/abs/10.1111/nph.16310 PubMed DOI

Jiskrová E, Novák O, Pospíšilová H, Holubová K, Karády M, Galuszka P, et al. Extra- and intracellular distribution of cytokinins in the leaves of monocots and dicots. New Biotechnol. 2016. September;33(5):735–42. PubMed

Fletcher RA, McCullagh D. Cytokinin-induced chlorophyll formation in cucumber cotyledons. Planta. 1971. March 1;101(1):88–90. 10.1007/BF00387693 PubMed DOI

Letham DS. Chemistry and Physiology of Kinetin-Like Compounds. Annu Rev Plant Physiol. 1967;18(1):349–64.

Gajdošová S. Biological effects and metabolism of cis-Zeatin-type cytokinins in plants. Prague: Charles University; 2011. Available from: https://dspace.cuni.cz/handle/20.500.11956/47271 PubMed

Sumanta N, Haque CI, Nishika J, Suprakash R. Spectrophotometric Analysis of Chlorophylls and Carotenoids from Commonly Grown Fern Species by Using Various Extracting Solvents. Res J Chem Sci. 2014;9(4).

Rashotte AM, Mason MG, Hutchison CE, Ferreira FJ, Schaller GE, Kieber JJ. A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway. Proc Natl Acad Sci. 2006. July 18;103(29):11081–5. 10.1073/pnas.0602038103 PubMed DOI PMC

Andrews S. FastQC: A quality control tool for high throughput sequence data. 2010. Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014. August 1;30(15):2114–20. 10.1093/bioinformatics/btu170 PubMed DOI PMC

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014. December 5;15(12):550 10.1186/s13059-014-0550-8 PubMed DOI PMC

Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc. 2016. September;11(9):1650–67. 10.1038/nprot.2016.095 PubMed DOI PMC

Tian T, Liu Y, Yan H, You Q, Yi X, Du Z, et al. agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Res. 2017. July 3;45:W122–9. 10.1093/nar/gkx382 PubMed DOI PMC

Zwack PJ, Robinson BR, Risley MG, Rashotte AM. Cytokinin Response Factor 6 Negatively Regulates Leaf Senescence and is Induced in Response to Cytokinin and Numerous Abiotic Stresses. Plant Cell Physiol. 2013. June 1;54(6):971–81. 10.1093/pcp/pct049 PubMed DOI

Hooper CM, Castleden IR, Tanz SK, Aryamanesh N, Millar AH. SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations. Nucleic Acids Res. 2017. January 4;45(D1):D1064–74. 10.1093/nar/gkw1041 PubMed DOI PMC

Cerna H, Černý M, Habánová H, Šafářová D, Abushamsiya K, Navrátil M, et al. Proteomics offers insight to the mechanism behind Pisum sativum L. response to pea seed-borne mosaic virus (PSbMV). J Proteomics. 2017. February 5;153:78–88. 10.1016/j.jprot.2016.05.018 PubMed DOI

Letham DS. Regulators of Cell Division in Plant Tissues XII. A Cytokinin Bioassay Using Excised Radish Cotyledons. Physiol Plant. 1971. December 1;25(3):391–6.

Zwack PJ, Rashotte AM. Cytokinin inhibition of leaf senescence. Plant Signal Behav. 2013. July 1;8(7). Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908980/ PubMed PMC

Hörtensteiner S. Chlorophyll Degradation During Senescence. Annu Rev Plant Biol. 2006;57(1):55–77. PubMed

Stenlid G. Cytokinins as inhibitors of root growth. Physiol Plant. 1982;56(4):500–6.

Werner T, Motyka V, Strnad M, Schmülling T. Regulation of plant growth by cytokinin. Proc Natl Acad Sci. 2001. August 28;98(18):10487–92. 10.1073/pnas.171304098 PubMed DOI PMC

Skoog F, Miller CO. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol. 1957;54:118–30. PubMed

Kubo M, Kakimoto T. The CYTOKININ-HYPERSENSITIVE genes of Arabidopsis negatively regulate the cytokinin-signaling pathway for cell division and chloroplast development. Plant J. 2000;23(3):385–94. 10.1046/j.1365-313x.2000.00796.x PubMed DOI

Suzuki T, Ishikawa K, Yamashino T, Mizuno T. An Arabidopsis Histidine-Containing Phosphotransfer (HPt) Factor Implicated in Phosphorelay Signal Transduction: Overexpression of AHP2 in Plants Results in Hypersensitiveness to Cytokinin. Plant Cell Physiol. 2002. January 15;43(1):123–9. 10.1093/pcp/pcf007 PubMed DOI

Jones RJ, Schreiber BMN. Role and function of cytokinin oxidase in plants. Plant Growth Regul. 1997. October 1;23(1):123–34.

McGaw BA, Horgan R. Cytokinin catabolism and cytokinin oxidase. Phytochemistry. 1983. January 1;22(5):1103–5.

Gepstein S, Sabehi G, Carp M-J, Hajouj T, Nesher MFO, Yariv I, et al. Large-scale identification of leaf senescence-associated genes. Plant J. 2003;36(5):629–42. 10.1046/j.1365-313x.2003.01908.x PubMed DOI

Kliebenstein DJ, Monde R-A, Last RL. Superoxide Dismutase in Arabidopsis: An Eclectic Enzyme Family with Disparate Regulation and Protein Localization. Plant Physiol. 1998. October 1;118(2):637–50. 10.1104/pp.118.2.637 PubMed DOI PMC

Reiter RS, Coomber SA, Bourett TM, Bartley GE, Scolnik PA. Control of leaf and chloroplast development by the Arabidopsis gene pale cress. Plant Cell. 1994. September 1;6(9):1253–64. 10.1105/tpc.6.9.1253 PubMed DOI PMC

Černý M, Dyčka F, Bobál’ová J, Brzobohatý B. Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling. J Exp Bot. 2011. January 1;62(3):921–37. 10.1093/jxb/erq322 PubMed DOI PMC

Černý M, Kuklová A, Hoehenwarter W, Fragner L, Novák O, Rotková G, et al. Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation. J Exp Bot. 2013. November 1;64(14):4193–206. 10.1093/jxb/ert227 PubMed DOI PMC

Černý M, Jedelský PL, Novák J, Schlosser A, Brzobohatý B. Cytokinin modulates proteomic, transcriptomic and growth responses to temperature shocks in Arabidopsis. Plant Cell Environ. 2014. July 1;37(7):1641–55. 10.1111/pce.12270 PubMed DOI

Lochmanová G, Zdráhal Z, Konečná H, Koukalová Š, Malbeck J, Souček P, et al. Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis. J Exp Bot. 2008. October 1;59(13):3705–19. 10.1093/jxb/ern220 PubMed DOI

Žd’árská M, Zatloukalová P, Benítez M, Šedo O, Potěšil D, Novák O, et al. Proteome Analysis in Arabidopsis Reveals Shoot- and Root-Specific Targets of Cytokinin Action and Differential Regulation of Hormonal Homeostasis. Plant Physiol. 2013. February 1;161(2):918–30. 10.1104/pp.112.202853 PubMed DOI PMC

Zhang Y, Liu S, Dai SY, Yuan JS. Integration of shot-gun proteomics and bioinformatics analysis to explore plant hormone responses. BMC Bioinformatics. 2012. September 11;13(15):S8. PubMed PMC

Bhargava A, Clabaugh I, To JP, Maxwell BB, Chiang Y-H, Schaller EG, et al. Identification of Cytokinin Responsive Genes Using Microarray Meta-analysis and RNA-seq in Arabidopsis thaliana. Plant Physiol. 2013. January 1;pp.113.217026. PubMed PMC

Rashotte AM, Carson SDB, To JPC, Kieber JJ. Expression Profiling of Cytokinin Action in Arabidopsis. Plant Physiol. 2003. August 1;132(4):1998–2011. 10.1104/pp.103.021436 PubMed DOI PMC

Galuszka P, Popelková H, Werner T, Frébortová J, Pospíšilová H, Mik V, et al. Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from Arabidopsis thaliana Expressed in Nicotiana tabacum L. J Plant Growth Regul. 2007. September 1;26(3):255–67.

Bürkle L, Cedzich A, Döpke C, Stransky H, Okumoto S, Gillissen B, et al. Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis. Plant J. 2003;34(1):13–26. 10.1046/j.1365-313x.2003.01700.x PubMed DOI

Hirose N, Makita N, Yamaya T, Sakakibara H. Functional Characterization and Expression Analysis of a Gene, OsENT2, Encoding an Equilibrative Nucleoside Transporter in Rice Suggest a Function in Cytokinin Transport. Plant Physiol. 2005. May 1;138(1):196–206. 10.1104/pp.105.060137 PubMed DOI PMC

Sun J, Hirose N, Wang X, Wen P, Xue L, Sakakibara H, et al. Arabidopsis SOI33/AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta. J Integr Plant Biol. 2005;47(5):588–603.

Graf A, Coman D, Uhrig RG, Walsh S, Flis A, Stitt M, et al. Parallel analysis of Arabidopsis circadian clock mutants reveals different scales of transcriptome and proteome regulation. Open Biol. 7(3):160333 10.1098/rsob.160333 PubMed DOI PMC

Ponnala L, Wang Y, Sun Q, Wijk KJ van. Correlation of mRNA and protein abundance in the developing maize leaf. Plant J. 2014;78(3):424–40. 10.1111/tpj.12482 PubMed DOI

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. December 1;57(15):4051–8. 10.1093/jxb/erl179 PubMed DOI

Spíchal L, Rakova NY, Riefler M, Mizuno T, Romanov GA, Strnad M, 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. September 15;45(9):1299–305. 10.1093/pcp/pch132 PubMed DOI

Deng Y, Dong H, Mu J, Ren B, Zheng B, Ji Z, et al. Arabidopsis Histidine Kinase CKI1 Acts Upstream of HISTIDINE PHOSPHOTRANSFER PROTEINS to Regulate Female Gametophyte Development and Vegetative Growth. Plant Cell. 2010. April 1;22(4):1232–48. 10.1105/tpc.108.065128 PubMed DOI PMC

Kakimoto T. CKI1, a Histidine Kinase Homolog Implicated in Cytokinin Signal Transduction. Science. 1996. November 8;274(5289):982–5. 10.1126/science.274.5289.982 PubMed DOI

Žižková E, Kubeš M, Dobrev PI, Přibyl P, Šimura J, Zahajská L, et al. Control of cytokinin and auxin homeostasis in cyanobacteria and algae. Ann Bot. 2017. January 1;119(1):151–66. 10.1093/aob/mcw194 PubMed DOI PMC

Drábková LZ, Dobrev PI, Motyka V. Phytohormone Profiling across the Bryophytes. PLOS ONE. 2015. May 14;10(5):e0125411 10.1371/journal.pone.0125411 PubMed DOI PMC

Auer CA. Cytokinin conjugation: recent advances and patterns in plant evolution. Plant Growth Regul. 1997;23 10.1023/a:1005853128971 DOI

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