Evidence for Importance of tRNA-Dependent Cytokinin Biosynthetic Pathway in the Moss Physcomitrella patens

  • Natalya A. Yevdakova
  • Václav Motyka
  • Jiri Malbeck
  • Alena Trávníčková
  • Ondrej Novák
  • Miroslav Strnad
  • Klaus von Schwartzenberg


To study cytokinin biosynthesis, we characterized a temperature-sensitive cytokinin-overproducing mutant, oveST25, of the moss Physcomitrella patens with respect to changes in cytokinin content during thermal induction in comparison to wild type. Our findings, based on combined liquid chromatography-mass spectrometry (LC-MS) analyses, show that thermoinduction caused a strong increase of extracellular N 6-(Δ2-isopentenyl)adenine (iP), N 6-(Δ2-isopentenyl)adenosine (iPR), cis-zeatin (cZ), cis-zeatin riboside (cZR) and its O-glucoside cZROG in oveST25. In contrast, no significant changes were measured in the wild type. To investigate the relevance of tRNA for cytokinin production in Physcomitrella, we determined cytokinins in tissue and culture medium as well as in tRNA hydrolysates. The analysis of cytokinins from whole-culture extracts of wild type revealed 56% of iP-type, 32% of cZ-type, and 11% of trans-zeatin (tZ)-type forms. In tRNA, 90% of cytokinins were represented by cZ-type and 8% by iP-type forms; tZ-type cytokinins were found only in trace amounts. The finding that the major free cytokinins are, albeit with altered proportions, also major forms in tRNA is compatible with the hypothesis of a strong tRNA-mediated biogenesis of cytokinins in this plant. Our RT-PCR-based studies on the expression of the tRNA-IPT gene, PpIPT1, revealed enhanced transcription levels in the cytokinin-overproducing oveST25 mutant at the inducing temperature of 25°C, but not at noninducing conditions (15°C). A wild-type transgenic line with cytokinin deficiency due to heterologous cytokinin oxidase/dehydrogenase overexpression (AtCKX2) also exhibited enhanced PpIPT1 expression levels, indicating that cytokinin deficiency might upregulate tRNA-mediated cytokinin biosynthesis. The evidence that the tRNA-mediated pathway might be mainly responsible for biosynthesis of isoprenoid cytokinins in Physcomitrella is strongly supported by the recent release of the Physcomitrella genomic sequence where only tRNA-IPTs but no adenylate-IPTs are present.


Physcomitrella ove mutant Cytokinin biosynthesis tRNA-isopentenyl transferase tRNA-bound cytokinins 



The authors thank Petra Amakorová, Hana Martínková (both University of Olomouc), Marie Korecká (Prague), and Susanne Bringe (University of Hamburg) for skillful technical assistance, and Douglas Dunlop (University of Waterloo, Canada) for help with manuscript preparation. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Schw687/4), the Ministry of Education, Youth and Sports of the Czech Republic (MSM6198959216 and LC 06034), the Grant Agency of the Czech Republic (206/05/0894), and the Grant Agency of the Academy of Sciences of the Czech Republic (IAA600380701).


  1. Ashton NW, Cove DJ, Featherstone DR (1979) The isolation and physiological analysis of mutants of the moss Physcomitrella patens, which over-produce gametophores. Planta 144:437–442CrossRefGoogle Scholar
  2. Brandes H, Kende H (1968) Studies on cytokinin-controlled bud formation in moss protonema. Plant Physiol 43:827–837PubMedCrossRefGoogle Scholar
  3. Buck M, Connick M, Ames BN (1983) Complete analysis of tRNA-modified nucleosides by high-performance liquid chromatography: the 29 modified nucleosides of Salmonella typhimurium and Escherichia coli tRNA. Anal Biochem 129:1–13PubMedCrossRefGoogle Scholar
  4. Cove DJ (2005) The moss Physcomitrella patens. Annu Rev Genet 39:339–358PubMedCrossRefGoogle Scholar
  5. Chaudhury AM, Letham S, Craig, Dennis ES (1993) amp1—a mutant with high cytokinin levels and altered embryonic pattern, faster vegetative growth, constitutive photomorphogenesis and precocious flowering. Plant J 4:907–916CrossRefGoogle Scholar
  6. Decker EL, Frank W, Sarnighausen E, Reski R (2006) Moss systems biology en route: phytohormones in Physcomitrella development. Plant Biol 8:397–405PubMedCrossRefGoogle Scholar
  7. Dihanich ME, Najarian D, Clark R, Gillman EC, Martin NC, Hopper AK (1987) Isolation and characterization of mod5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae. Mol Cell Biol 7:177–184PubMedGoogle Scholar
  8. Dobrev PI, Kaminek 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. Edmonds CG, Crain PF, Gupta R, Hashizume T, Hocart CH, Kowalak JA, Pomerantz SC, Stetter KO, McCloskey JA (1991) Posttranscriptional modification of tRNA in thermophilic Archea (Archaebacteria). J Bacteriol 173:3138–3148PubMedGoogle Scholar
  10. Faiss M, Zalubilova J, Strnad M, Schmülling T (1997) Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants. Plant J 12:401–415PubMedCrossRefGoogle Scholar
  11. Featherstone DR, Cove DJ, Ashton NW (1990) Genetic analysis by somatic hybridization of cytokinin overproducing developmental mutants of the moss, Physcomitrella patens. Mol Gen Genet 222:217–224PubMedCrossRefGoogle Scholar
  12. Futers TS, Wang TL, Cove DJ (1986) Characterisation of a temperature-sensitive gametophore over-producing mutant of the moss, Physcomitrella patens. Mol Gen Genet 203:529–532CrossRefGoogle Scholar
  13. Gray J, Wang J, Gelvin SB (1992) Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression. J Bacteriol 174:1086–1098PubMedGoogle Scholar
  14. Gray J, Gelvin SB, Meilan R, Morris RO (1996) Transfer RNA is the source of extracellular isopentenyladenine in a Ti-plasmidless strain of Agrobacterium tumefaciens. Plant Physiol 110:431–438PubMedGoogle Scholar
  15. Haberer G, Kieber JJ (2002) Cytokinins. New insights into a classic phytohormone. Plant Physiol 128:354–362PubMedCrossRefGoogle Scholar
  16. Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases. Plant Cell Physiol 42:677–685PubMedCrossRefGoogle Scholar
  17. Kakimoto T (2003) Biosynthesis of cytokinins. J Plant Res 116:233–239PubMedCrossRefGoogle Scholar
  18. Kaminek M (1974) Evolution of tRNA and origin of the two positional isomers of zeatin. J Theor Biol 48:489–492PubMedCrossRefGoogle Scholar
  19. Kasahara H, Takei K, Ueda N, Hishiyama S, Yamaya T, Kamiya Y, Yamaguchi S, Sakakibara H (2004) Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis. J Biol Chem 279:14049–14054PubMedCrossRefGoogle Scholar
  20. Koenig RL, Morris RO, Polacco JC (2002) tRNA is the source of low-level trans-zeatin production in Methylobacterium spp. J Bacteriol 184:1832–1842PubMedCrossRefGoogle Scholar
  21. Konevega AL, Soboleva NG, Makhno VI, Peshekhonov AV, Katunin VI (2006) Effect of modification of tRNA nucleotide 37 on the tRNA interaction with the A and P sites of the Escherichia coli 70S ribosome. Mol Biol 40:597–610CrossRefGoogle Scholar
  22. Kurakawa T, Ueda N, Maekawa M, Kobayashi K, Kojima M, Nagato Y, Sakakibara H, Kyozuka J (2007) Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445:652–655PubMedCrossRefGoogle Scholar
  23. Lexa M, Genkov T, Malbeck J, Machackova I, Brzohohaty B (2003) Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach. Ann Bot 91:585–597PubMedCrossRefGoogle Scholar
  24. Maaß H, Klämbt D (1981) On the biogenesis of cytokinins in roots of Phaseolus vulgaris. Planta 151:353–358CrossRefGoogle Scholar
  25. Martin NC, Hopper AK (1982) Isopentenylation of both cytoplasmic and mitochondrial tRNA is affected by a single nuclear mutation. J Biol Chem 257:10562–10565PubMedGoogle Scholar
  26. Miyawaki K, Matsumoto-Kitano M, Kakimoto T (2004) Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin and nitrate. Plant J 37:128–138PubMedCrossRefGoogle Scholar
  27. Miyawaki K, Tarkowski P, Matsumoto-Kitano M, Kato T, Sato S, Tarkowska D, Tabata S, Sandberg G, Kakimoto T (2006) Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis. Proc Natl Acad Sci U S A 103:16598–16603PubMedCrossRefGoogle Scholar
  28. Mok DWS, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Mol Biol 52:89–118CrossRefGoogle Scholar
  29. 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
  30. Nakamura T, Sugiura C, Kobayashi Y, Sugita M (2005) Transcript profiling in plastid arginine tRNA-CCG gene knockout moss: construction of Physcomitrella patens plastid DNA microarray. Plant Biol 7:258–265PubMedCrossRefGoogle Scholar
  31. Perry KC, Cove DJ (1986) Transfer RNA pool sizes and half lives in wild-type and cytokinin over-producing strains of the moss Physcomitrella patens. Physiol Plant 67:680–684CrossRefGoogle Scholar
  32. Petrášek J, Březinová A, Holík J, Zažimalová E (2002) Excretion of cytokinins into the cultivation medium by suspension-cultured tobacco cells. Plant Cell Rep 21:97–104CrossRefGoogle Scholar
  33. Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H, Nishiyama T et al (2008) The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 319:64–69Google Scholar
  34. Reutter K, Atzorn R, Hadeler B, Schmülling T, Reski R (1998) Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division. Planta 206:196–203CrossRefGoogle Scholar
  35. Rosenbaum N, Gefter ML (1972) Δ2-Isopentenylpyrophosphate: transfer ribonucleic acid. Δ2-isopentenyltransferase from Escherichia coli. Purification and properties of the enzyme. J Biol Chem 247:5675–5680PubMedGoogle Scholar
  36. Sakakibara K, Nishiyama T, Deguchi H, Hasebe M (2006) Evolution of cytokinin biosynthesis in land plants. Abstract available at
  37. Schulz P, Reski R, Maldiney R, Laloue M, von Schwartzenberg K (2000) Kinetics of cytokinin production and bud formation in Physcomitrella: analysis of wild type, a developmental mutant and two of its ipt transgenics. J Plant Physiol 156:768–774Google Scholar
  38. Schulz PA, Hofmann AH, Russo VEA, Hartmann E, Laloue M, von Schwartzenberg K (2001) Cytokinin overproducing ove mutants of Physcomitrella patens show increased riboside to base conversion. Plant Physiol 126:1224–1231PubMedCrossRefGoogle Scholar
  39. Stirk WA, Ördög V, van Staden J (1999) Identification of the cytokinin isopentenyladenine in a strain of Arthronema africanum (Cyanobacteria) J. Phycol 35:89–92CrossRefGoogle Scholar
  40. Stirk WA, Novák O, Strnad M, van Staden J (2003) Cytokinins in macroalgae. Plant Growth Regul 41:13–24CrossRefGoogle Scholar
  41. Taller BJ (1994) Distribution, biosynthesis and function of cytokinins in tRNA. In: Mok DWS, Mok MC (eds), Cytokinins: chemistry, activity and function. CRC Press, Boca Raton, FL, pp 101–112Google Scholar
  42. Takei K, Sakakibara H, Sugiyama T (2001) Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthasis enzyme, in Arabidopsis thaliana. J Biol Chem 276:26405–26410PubMedCrossRefGoogle Scholar
  43. von Schwartzenberg K (2006) Moss biology and phytohormones—cytokinins in Physcomitrella. Plant Biol 8:382–388CrossRefGoogle Scholar
  44. von Schwartzenberg K, Pethe C, Laloue M (2003) Cytokinin metabolism in Physcomitrella patens differences and similarities to higher plants. Plant Growth Regul 39:99–106CrossRefGoogle Scholar
  45. von Schwartzenberg K, Fernández Núñez M, 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 role of extracellular cytokinins. Plant Physiol 145:786-800CrossRefGoogle Scholar
  46. Vreman HJ, Thomas R, Corse J, Swaminathan S, Murai N (1978) Cytokinins in tRNA obtained from Spinacia oleracea L. leaves and isolated chloroplasts. Plant Physiol 61:296–306PubMedGoogle Scholar
  47. Wang TL, Horgan R, Cove D (1981) Cytokinins from the moss Physcomitrella patens. Plant Physiol 68:735–738PubMedGoogle Scholar
  48. Yevdakova NA, von Schwartzenberg K (2007) Characterisation of a prokaryote-type tRNA-isopentenyltransferase gene from the moss Physcomitrella patens. Planta 226:683–695PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Natalya A. Yevdakova
    • 1
    • 2
  • Václav Motyka
    • 3
  • Jiri Malbeck
    • 3
  • Alena Trávníčková
    • 3
  • Ondrej Novák
    • 4
  • Miroslav Strnad
    • 4
  • Klaus von Schwartzenberg
    • 1
  1. 1.Biocenter Klein Flottbek and Botanical GardenUniversity of HamburgHamburgGermany
  2. 2.Institute of Plant Biology and BiotechnologyNational Centre of BiotechnolyAlmatyKazakhstan
  3. 3.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPragueCzech Republic
  4. 4.Laboratory of Growth RegulatorsPalacký University and Institute of Experimental Botany, Academy of Sciences of the Czech RepublicOlomoucCzech Republic

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