Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 120, Issue 2, pp 767–777 | Cite as

Changes in cytokinin content and altered cytokinin homeostasis in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro

  • Milana Trifunović
  • Václav Motyka
  • Aleksandar Cingel
  • Angelina Subotić
  • Slađana Jevremović
  • Marija Petrić
  • Josef Holík
  • Jiří Malbeck
  • Petre I. Dobrev
  • Ivana Č. Dragićević
Original Paper


The plant hormones cytokinins (CKs) regulate a number of physiological processes. Their homeostasis is controlled by the rate of de novo synthesis and the rate of catabolism. The aim of this work was to analyze the content of total as well as individual groups of endogenous CKs in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro. Transgenic CKX plants represent a suitable model system for studying physiological and morphological processes controlled by CKs. In this work we clearly demonstrate a significant effect of AtCKX transgenes on CK metabolism in transgenic centaury plants. However, shoots and roots of only one AtCKX1 line and three AtCKX2 lines with a significant reduction of bioactive CKs were obtained. We also show that changes in the CKs metabolism considerably affected endogenous indole-3-acetic acid (IAA) levels in plant tissues. All analyzed transgenic AtCKX centaury lines exhibited decreased amount of endogenous IAA in shoots as well as in roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all analyzed AtCKX centaury transformants.


Centaurium erythraea Rafn. Cytokinin Cytokinin oxidase/dehydrogenase AtCKX genes Hormone metabolism Plant development 





Cytokinin oxidase/dehydrogenase


Indole-3-acetic acid




Murashige and Skoog medium



This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. ON173015) and the Czech Science Foundation (P506/11/0774).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2014_646_MOESM1_ESM.doc (140 kb)
Supplementary material 1 (DOC 140 kb)


  1. Aremu AO, Plačková L, Bairu MW, Novák O, Plíhalová L, Doležal K, Finnie JF, Van Staden J (2014) How does exogenously applied cytokinin type affect growth and endogenous cytokinins in micropropagated Merwilla plumbea? Plant Cell, Tissue Organ Cult 118:245–256CrossRefGoogle Scholar
  2. Armstrong DJ (1994) Cytokinin oxidase and the regulation of cytokinin degradation. In: Mok DWS, Mok MC (eds) Cytokinins: chemistry, activity and function. CRC Press, Boca Raton, pp 139–154Google Scholar
  3. Behr M, Motyka V, Weihmann F, Malbeck J, Deising HB, Wirsel SG (2012) Remodeling of cytokinin metabolism at infection sites of Colletotrichum graminicola on maize leaves. Mol Plant Microbe Interact 25:1073–1082PubMedCrossRefGoogle Scholar
  4. Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice, a, Revised edn. Elsevier, New YorkGoogle Scholar
  5. Bilyeu KD, Cole JL, Laskey JG, Riekhof WR, Esparza TJ (2001) Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol 125:378–386PubMedCentralPubMedCrossRefGoogle Scholar
  6. Che P, Gingerich D, Lall S, Howell S (2002) Global and hormone-induced gene expression changes during shoot development in Arabidopsis. Plant Cell 14:2271–2279CrossRefGoogle Scholar
  7. Cheng ZJ, Wang L, Sun W, Zhang Y, Zhou C, Su YH, Li W, Sun TT, Zhao XY, Li XG, Cheng Y, Zhao Y, Xie Q, Zhang XS (2013) Pattern of auxin and cytokinin responses for shoot meristem induction results from the regulation of cytokinin biosynthesis by auxin response factor3. Plant Physiol 161:240–251PubMedCentralPubMedCrossRefGoogle Scholar
  8. Dobrev PI, 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 950:21–29PubMedCrossRefGoogle Scholar
  9. Dobrev PI, Havlíček L, Vágner M, Malbeck J, Kamínek M (2005) Purification and determination of plant hormones auxin and abscisic acid using solid phase extraction and two-dimensional high performance liquid chromatography. J Chromatogr A 1075:159–166PubMedCrossRefGoogle Scholar
  10. Dwivedi S, Vaňková R, Motyka V, Herrera C, Žižková E, Auer C (2010) Characterization of Arabidopsis thaliana mutant ror-1 (roscovitine-resistant) and its utilization in understanding of the role of cytokinin N-glucosylation pathway in plants. Plant Growth Regul 61:231–242CrossRefGoogle Scholar
  11. Eklöff S, Astot C, Blackwell J, Mortiz T, Olsson O, Sandberg G (1997) Auxin–cytokinin interactions in wild-type and transgenic tobacco. Plant Cell Physiol 38:225–235CrossRefGoogle Scholar
  12. Feng DS, Wang HG, Zhang XS, Kong LR, Tian JC, Li XF (2008) Using an inverse PCR method to clone the wheat cytokinin oxidase/dehydrogenase gene TaCKX1. Plant Mol Biol Rep 26:143–155CrossRefGoogle Scholar
  13. Gajdošová S, Spíchal L, Kamínek M, Hoyerová K, Novák O, Dobrev PI, Galuszka P, Klíma P, Gaudinová A, Žižková E, Hanuš J, Dančák M, Trávníček B, Pešek B, Krupička M, Vaňková R, Strnad M, Motyka V (2011) Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants. J Exp Bot 62:2827–2840PubMedCrossRefGoogle Scholar
  14. Galuszka P, Frébortová J, Werner T, Yamada M, Strnad M, Schmülling T, Frébort I (2004) Cytokinin oxidase/dehydrogenase genes in barley and wheat cloning and heterologous expression. Eur J Biochem 271:3990–4002PubMedCrossRefGoogle Scholar
  15. 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 Arabidopsis thaliana expressed in Nicotiana tabacum L. J Plant Growth Regul 26:255–267CrossRefGoogle Scholar
  16. Hartmann A, Senning M, Hedden P, Sonnewald U, Sonnewald S (2011) Reactivation of meristem activity and sprout growth in potato tuber require both cytokinin and gibberellin. Plant Physiol 155:776–796PubMedCentralPubMedCrossRefGoogle Scholar
  17. Houba-Hérin N, Pethe C, d’Alayer J, Laloue M (1999) Cytokinin oxidase from Zea mays: purufication, cDNA cloning and expression in moss protoplasts. Plant J 17:615–626PubMedCrossRefGoogle Scholar
  18. Janković T, Krstić D, Šavikin-Fodulović K, Menković N, Grubišić D (1997) Comparative investigation of secoiridoid compounds of Centaurium erythraea grown in nature and cultured in vitro. Pharm Pharmacol Lett 7:30–32Google Scholar
  19. Jones RJ, Schreiber BMN (1997) Role and function of cytokinin oxidase in plants. Plant Growth Regul 23:123–134CrossRefGoogle Scholar
  20. Jones B, Andersson Gunnears S, Petrsson SV, Tarkowski P, Graham N, May S, Doležal K, Sandberg G, Ljung K (2010) Cytokinin regulation of auxin synthesis in Arabidopsis involves a homeostatic feedback loop regulated via auxin and cytokinin signal transduction. Plant Cell 22:2956–2969PubMedCentralPubMedCrossRefGoogle Scholar
  21. Kamínek M, Motyka V, Vaňková R (1997) Regulation of cytokinin content in plant cells. Physiol Plant 101:689–700CrossRefGoogle Scholar
  22. Kamínek M, Březinová A, Gaudinová A, Motyka V, Vaňková R, Zažímalová E (2000) Purine cytokinins: a proposal of abbreviations. Plant Growth Regul 32:253–256CrossRefGoogle Scholar
  23. Letham DS (1994) Cytokinins as phytohormones-sites of biosynthesis, translocation and function of translocated cytokinin. In: Mok DWS, Mok MC (eds) Cytokinins-chemistry, activity and function. CRC Press, Boca Raton, pp 57–80Google Scholar
  24. Liu J, Mehdi S, Topping J, Tarkowski P, Lindsey K (2010) Modelling and experimental analysis of hormonal crosstalk in Arabidopsis. Mol Systems Biol 6:1–13CrossRefGoogle Scholar
  25. Macková H, Hronková M, Dobrá J, Turečková V, Novák O, Lubovská Z, Motyka V, Haisel D, Hájek T, Prášil IT, Gaudinová A, Štorchová H, Ge E, Werner T, Schmülling T, Vaňková R (2013) Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression. J Exp Bot 64:2805–2815PubMedCentralPubMedCrossRefGoogle Scholar
  26. Massonneau A, Houba-Hérin N, Pethe C, Madzak C, Falque M, Mercy M, Kopečný D, Majira A, Rogowsky P, Laloue M (2004) Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs. J Exp Bot 55:2549–2557PubMedCrossRefGoogle Scholar
  27. McGaw BA, Horgan R (1983) Cytokinin catabolism and cytokinin oxidase. Phytochemistry 22:1103–1105CrossRefGoogle Scholar
  28. Mok DW, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Phys 52:89–118CrossRefGoogle Scholar
  29. Morris RO, Bilyeu KD, Laskey JG, Cheikh NN (1999) Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Bioph Res Commun 255:328–333CrossRefGoogle Scholar
  30. Motyka V, Kamínek M (1994) Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.). J Plant Growth Regul 13:1–9CrossRefGoogle Scholar
  31. Motyka V, Faiss M, Strnad M, Kamínek M, Schmülling T (1996) Changes in cytokinin content and cytokinin oxidase activity in response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol 112:1035–1043PubMedCentralPubMedGoogle Scholar
  32. Motyka V, Vaňková R, Čapková V, Petrášek J, Kamínek M, Schmülling T (2003) Cytokinin induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion. Physiol Plant 117:11–21CrossRefGoogle Scholar
  33. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479CrossRefGoogle Scholar
  34. Mýtinová Z, Motyka V, Haisel D, Lubovská Z, Trávníčková A, Dobrev P, Holík J, Wilhelmová N (2011) Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion. Plant Growth Regul 65:23–34CrossRefGoogle Scholar
  35. Nordström A, Tarkowski P, Tarkowska D, Norbaek R, Astot C, Doležal K, Sandberg G (2004) Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana: a factor of potential importance for auxin–cytokinin-regulated development. Proc Natl Acad Sci USA 101:8039–8044PubMedCentralPubMedCrossRefGoogle Scholar
  36. Podwyszynska M, Novák O, Doležal K, Strnad M (2014) Endogenous cytokinin dynamics in micropropagated tulips during bulb formation process influenced by TDZ and iP pretreatment. Plant Cell Tissue Organ Cult 1–16. doi: 10.1007/s11240-014-0537-x
  37. Raspor M, Motyka M, Žižková E, Dobrev P, Trávníčková A, Zdravković-Korać S, Simonović A, Ninković S, Dragićević I (2012) Cytokinin profiles of AtCKX2-overexpressing potato plants and the impact of altered cytokinin homeostasis on tuberization in vitro. J Plant Growth Regul 31:460–470CrossRefGoogle Scholar
  38. Redig P, Motyka V, Van Onckelen HA, Kamínek M (1997) Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene. Physiol Plant 99:89–96CrossRefGoogle Scholar
  39. Schmülling T, Werner T, Riefler M, Krupková E, Bartrina y Manns I (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116:241–252PubMedCrossRefGoogle Scholar
  40. Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissue cultured in vitro. Symp Soc Exp Biol 11:118–131PubMedGoogle Scholar
  41. Šmehilová M, Galuszka P, Bilyeu KD, Jaworek P, Kowalska M, Šebela M, Sedlářová M, English JT, Frébort I (2009) Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehydrogenase enzymes from maize. J Exp Bot 60:2701–2712PubMedCrossRefGoogle Scholar
  42. Spíchal L (2012) Cytokinins—recent news and views of evolutionally old molecules. Funct Plant Biol 39:267–284CrossRefGoogle Scholar
  43. Subotić A, Budimir S, Grubišić D, Momčilović I (2003/2004) Direct regeneration of shoots from hairy root cultures of Centaurium erythraea inoculated with Agrobacterium rhizogenes. Biol Plant 47:617–619Google Scholar
  44. Subotić A, Janković T, Jevremović S, Grubišić D (2006) Plant tissue culture and secondary metabolite production of Centaurium erythraea Rafn., a medicinal plant. In: Bajaj YPS (ed) Floriculture, ornamental and plant biotechnology, vol 2. Global Science Books, UK, pp 564–570Google Scholar
  45. Subotić A, Jevremović S, Grubišić D, Janković T (2009) Spontaneous plant regeneration and production of secondary metabolites from hairy root cultures of Centaurium erythraea Rafn. In: Jain SM, Saxena PK (eds) Protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, series: methods in molecular biology, vol 547. Springer, Berlin, pp 205–217Google Scholar
  46. Suttle JC, Huckle LL, Lu S, Knauber DC (2014) Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression. J Plant Physiol 171:448–457PubMedCrossRefGoogle Scholar
  47. Trifunović M, Cingel A, Simonović A, Jevremović S, Petrić M, Dragićević IČ, Motyka V, Dobrev PI, Zahajská L, Subotić A (2013) Overexpression of Arabidopsis cytokinin oxidase/dehydrogenase genes AtCKX1 and AtCKX2 in transgenic Centaurium erythraea Rafn. Plant Cell, Tissue Organ Cult 115:139–150CrossRefGoogle Scholar
  48. Van Staden J, Crouch NR (1996) Benzyladenine and derivates—their significance and interconversion in plants. Plant Growth Regul 19:153–175CrossRefGoogle Scholar
  49. Vaseva-Gemisheva I, Lee D, Karanov E (2005) Response of Pisum sativum cytokinin oxidase/dehydrogenase expression and specific activity to drought stress and herbicide treatments. Plant Growth Regul 46:199–208CrossRefGoogle Scholar
  50. Von Schwartzenberg K, Nunez MF, Blaschke H, Dobrev PI, Novák O, Motyka V, Strnad M (2007) Cytokinins in the bryophyte Physcomitrella patens: analyses of activity, distribution and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins. Plant Physiol 145:786–800CrossRefGoogle Scholar
  51. Wang Y, Luo JP, Wei ZJ, Zhang JC (2009) Molecular cloning and expression analysis of a cytokinin oxidase (DhCKX) gene in Dendrobium huoshanense. Mol Biol Rep 36:1331–1338PubMedCrossRefGoogle Scholar
  52. Wang Y, Liu H, Xin Q (2014) Genome-wide analysis and identification of cytokinin oxidase/dehydrogenase (CKX) gene family in foxtail millet (Setaria italica). Crop J 2:244–254CrossRefGoogle Scholar
  53. Werner T, Motyka V, Strnad M, Schmülling T (2001) Regulation of plant growth by cytokinin. Proc Natl Acad Sci USA 98:10487–10492PubMedCentralPubMedCrossRefGoogle Scholar
  54. Werner T, Motyka V, Laucou V, Stems R, Van Onckelen H, Schmülling T (2003) Cytokinin deficient transgenic Arabidopsis plant show multiple developmental alterations indicating opposite function of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550PubMedCentralPubMedCrossRefGoogle Scholar
  55. Werner T, Köllmer I, Bartrina K, Holst K, Schmülling T (2006) New insights into the biology of cytokinin degradation. Plant Biol 8:371–381PubMedCrossRefGoogle Scholar
  56. Yang SH, Yu H, Goh YC (2002) Isolation and characterization of the orchid cytokinin oxidase DSCKX1 promoter. J Exp Biol 53:1899–1907Google Scholar
  57. Yang SH, Yu H, Xu Y, Goh YC (2003a) Functional characterization of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid. Plant Mol Biol 51:237–248PubMedCrossRefGoogle Scholar
  58. Yang SH, Yu H, Xu Y, Goh YC (2003b) Investigation of cytokinin-deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1. FEBS Lett 555:291–296PubMedCrossRefGoogle Scholar
  59. Zhang R, Zhang X, Wang J, Letham DS, McKinney SA, Higgins TJV (1995) The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene. Planta 196:84–94CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Milana Trifunović
    • 1
  • Václav Motyka
    • 2
  • Aleksandar Cingel
    • 1
  • Angelina Subotić
    • 1
  • Slađana Jevremović
    • 1
  • Marija Petrić
    • 1
  • Josef Holík
    • 3
  • Jiří Malbeck
    • 2
  • Petre I. Dobrev
    • 2
  • Ivana Č. Dragićević
    • 4
  1. 1.Institute for Biological Research “Siniša Stanković”University of BelgradeBelgradeSerbia
  2. 2.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPrague 6Czech Republic
  3. 3.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPrague 4Czech Republic
  4. 4.Faculty of BiologyUniversity of BelgradeBelgradeSerbia

Personalised recommendations