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Changes in cytokinin levels and metabolism in tobacco (Nicotiana tabacum L.) explants during in vitro shoot organogenesis induced by trans-zeatin and dihydrozeatin

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Abstract

The uptake and metabolism of trans-zeatin and/or dihydrozeatin, in correlation with cytokinin oxidase/dehydrogenase (CKX) and β-glucosidase activity, were studied in leaf segments derived from wild-type (WT) and transgenic (T) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) during in vitro induction of shoot organogenesis. T explants harbored the maize gene Zm-p60.1β-glucosidase. Higher levels of shoot regeneration were observed on T explants in the early stages of cultivation. In WT explants, the content of cytokinin (CK)-O- and N-glucosides increased. In T explants, a higher content of Z-9-riboside and Z-9-riboside-5′-monophosphate and higher CKX activity during the early stage of cultures were found. A positive correlation was obtained for bioactive CK content and the organogenic response in T explants. Our results indicate a connection between the organogenic capacity of tobacco explants, metabolism of endogenous CKs and uptake of exogenous CKs from the cultivation medium.

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Abbreviations

BA:

N 6-benzyladenine

CK:

Cytokinin

CKX:

Cytokinin oxidase/dehydrogenase

2,4-D:

2,4-dichlorophenoxyacetic acid

DHZ:

Dihydrozeatin

DHZR:

Dihydrozeatin-9-riboside

DHZRMP:

Dihydrozeatin-9-riboside-5′-monophosphate

DHZOG:

Dihydrozeatin-O-glucoside

DHZROG:

Dihydrozeatin-9-riboside-O-glucoside

DHZ7G:

Dihydrozeatin-7-glucoside

DHZ9G:

Dihydrozeatin-9-glucoside

GRZ+:

Induction medium containing trans-zeatin

GRDHZ+:

Induction medium containing dihydrozeatin

GR−:

Growth regulator-free medium

GRZ+/GR−:

Subcultivation from zeatin induction to growth regulator-free medium

GRDHZ+/GR−:

Subcultivation from dihydrozeatin induction to growth regulator-free medium

IBA:

Indole-3-butyric acid

iP:

N 6-(Δ2-isopentenyl)adenine

ipt :

Gene coding isopentenyltransferase

iP7G:

N 6-(Δ2-isopentenyl)adenine-7-glucoside

T:

Transgenic tobacco

WT:

Wild-type tobacco

Z:

trans-zeatin

ZR:

trans-zeatin-9-riboside

ZRMP:

trans-zeatin-9-riboside-5′-monophosphate

ZOG:

trans-zeatin-O-glucoside

Z7G:

trans-zeatin-7-glucoside (abbreviations for cytokinins according to Kamínek et al. 2000)

References

  • Allen M, Qin WS, Moreau F, Moffatt B (2002) Adenine phosphoribosyltransferase isoforms of Arabidopsis and their potential contributions to adenine and cytokinin metabolism. Physiol Plant 115:56–68

    Article  PubMed  CAS  Google Scholar 

  • 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, FL, pp 139–154

    Google Scholar 

  • Auer CA, Motyka V, Březinová A, Kamínek M (1999) Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida. Physiol Plant 105:141–147

    Article  CAS  Google Scholar 

  • Blagoeva E, Dobrev PI, Malbeck J, Motyka V, Gaudinová A, Vaňková R (2004) Effect of exogenous cytokinins, auxins and adenine on cytokinin N-glucosylation and cytokinin oxidase/dehydrogenase activity in de-rooted radish seedlings. Plant Growth Regul 44:15–23

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Brzobohatý B, Moore I, Kristoffersen P, Bako L, Campos N, Schell J, Palme K (1993) Release of active cytokinin by β-glucosidase localized to the maize root meristem. Science 262:051–1054

    Article  Google Scholar 

  • Brzobohatý B, Moore I, Palme K (1994) Cytokinin metabolism: implication for regulation of plant growth and development. Plant Mol Biol 26:1483–1497

    Article  PubMed  Google Scholar 

  • Casanova E, Valdés AE, Fernandés B, Moysset L, Trillas MI (2004) Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation. J Plant Physiol 161:95–104

    Article  PubMed  CAS  Google Scholar 

  • Centeno ML, Rodríguez A, Feito I, Sánches-Tamés R, Fernandés B (2003) Uptake and metabolism of N6-benzyladenine and 1-naphthaleneacetic acid and dynamics of indole-3-acetic acid and cytokinins in two callus lines of Actinidia deliciosa differing in growth and shoot organogenesis. Physiol Plant 118(4):579–588

    Article  CAS  Google Scholar 

  • Chatfield JM, Armstrong DJ (1986) Regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv. Great Northern. Plant Physiol 80:493–499

    Article  PubMed  CAS  Google Scholar 

  • Christianson ML, Warnick DA (1984) Phenocritical times in the process of in vitro shoot organogenesis. Develop Biol 101:382–390

    Article  PubMed  CAS  Google Scholar 

  • Collier MD, Sheppard LJ, Crossley A, Hanke DE (2003) Needle cytokinin content as a sensitive bioindicator of N pollution in Sitka spruce. Plant Cell Environ 26:1929–1939

    Article  CAS  Google Scholar 

  • Crouch NR, van Staden J (1995) Promotion by 2, 4-D of 7-glucosylation of benzyladenine in seed-derived and shoot apex-derived cell culture of Dianthus zeyheri. Physiol Plant 93:645–650

    Article  CAS  Google Scholar 

  • Dhaliwal HS, Ramesar-Fortner NS, Yeung EC, Thorpe TA (2003) Competence, determination, and meristemoid plasticity in tobacco organogenesis in vitro. Canad J Bot 81:611–621

    Article  Google Scholar 

  • 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 Chromatography A 950:21–29

    Article  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  PubMed  CAS  Google Scholar 

  • Gaudinová A, Dobrev PI, Šolcová B, Novák O, Strnad M, Friedecký D, Motyka V (2005) The involvement of cytokinin oxidase/dehydrogenase and zeatin reductase in regulation of cytokinin levels in pea (Pisum sativum L.) leaves. J Plant Growth Regul 24:188–200

    Article  Google Scholar 

  • Haberer G, Kieber JJ (2002) Cytokinins. New insights into a classic phytohormone. Plant Physiol 128:354–362

    Article  PubMed  CAS  Google Scholar 

  • Hou B, Lim E-K, Higgins GS, Bowles DJ (2004) N-Glucosylation of cytokinins by glycosyltransferases of Arabidopsis thaliana. J Biol Chem 279:47822–47832

    Article  PubMed  CAS  Google Scholar 

  • Jelic G, Bogdanovic M (1990) The relationship between chlorophyll accumulation and endogenous cytokinin in the greening cotyledons of Pinus nigra. Plant Sci 71(2):53–157

    Article  Google Scholar 

  • Kakimoto T (2003) Perception and signal transduction of cytokinins. Ann Rev Plant Biol 54:605–627

    Article  CAS  Google Scholar 

  • Kamínek M, Armstrong DJ (1990) Genotypic variation in cytokinin oxidase from Phaseolus callus-cultures. Plant Physiol 93:1530–1538

    Article  PubMed  Google Scholar 

  • Kamínek M, Březinová A, Gaudinová A, Motyka V, Vaňková R, Zažímalová E (2000) Purine cytokinins: a proposal of abbreviations. Plant Growth Regul 32:253–256

    Article  Google Scholar 

  • Kiran NS, Polanská L, Fohlerová R, Mazura P, Válková M, Šmeral M, Zouhar J, Malbeck J, Dobrev PI, Macháčková I, Brzobohatý B (2006) Ectopic over-expression of the maize β-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco. J Exp Bot 57:985–996

    Article  PubMed  CAS  Google Scholar 

  • Letham DS (1963) Zeatin, a factor inducing cell division from Zea mays. Life Sci 8:596–599

    Google Scholar 

  • Lexa M, Genkov T, Malbeck J, Macháčková I, Brzobohatý B (2003) Dynamic of endogenous cytokinin pools in tobacco seedlings: a modeling approach. Ann Bot 91:585–597

    Article  PubMed  CAS  Google Scholar 

  • Ma QH, Lin ZB, Fu DZ (2002) Increased seed cytokinin levels in transgenic tobacco influence embryo and seedling development. Func Plant Biol 29:1107–1113

    Article  CAS  Google Scholar 

  • Malá J, Gaudinová A, Dobrev P, Eder J, Cvikrová M (2005) Role of phytohormones inorganogenic ability of elm multiplicated shoots. Biol Plant 50(1):8–14

    Article  Google Scholar 

  • Martin RC, Mok MC, Shaw G, Mok DW (1989) An enzyme mediating the conversion of zeatin to dihydrozeatin in Phaseolus embryo. Plant Physiol 90:1630–1635

    Article  PubMed  CAS  Google Scholar 

  • McGaw BA, Burch LR (1995) Cytokinin biosynthesis and metabolism. In: Davies PJ (ed) Plant hormones: physiology, biochemistry and molecular biology. Kluwer Academic Publisher, Dortrecht

    Google Scholar 

  • McGaw BA, Scott IM, Horgan R (1984) Cytokinin biosynthesis and metabolism. In: Crosier A, Hilmann JP (eds) Biosynthesis and metabolism of plant hormones. Cambridge University Press, Cambridge, pp 105–133

    Google Scholar 

  • Moffatt B, Pethe C, Laloue M (1991) Metabolism of benzyladenine is impaired in a mutant of Arabidopsis thaliana lacking adenine phosphoribosyltransferase activity. Plant Physiol 95:900–908

    Article  PubMed  CAS  Google Scholar 

  • Mok DWS, Mok MC (2001) Cytokinin metabolism and action. Ann Rev Plant Physiol Plant Mol Biol 52:89–118

    Article  CAS  Google Scholar 

  • Mok MC, Martin RC, Dobrev PI, Vaňková R, Ho PS, Yonekura-Sakakibara K, Sakakibara H, Mok DWS (2005) Topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin thidiazuron derivatives O-glucosyltransferase with position specificity related to receptor recognition Plant Physiol 137:1057–1066

    CAS  Google Scholar 

  • Moncaleán P, Alonso P, Centeno ML, Cortizo M, Rodríguez A, Fernández B, Ordás RJ (2005) Organogenic responses of Pinus pinea cotyledons to hormonal treatments: BA metabolism and cytokinin content. Tree Physiol 25:1–9

    PubMed  Google Scholar 

  • Motyka V, Kamínek M (1992) Characterization of cytokinin oxidase from tobacco and poplar casus cultures. In: Kamínek M, Mok DWS, Zažímalová E (eds) Physiology and biochemistry of cytokinins in plants. SPB Academic Publishing, The Hague, pp 33–39

    Google Scholar 

  • Motyka V, Faiss M, Strnad M, Kaamínek M, Schműlling 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 112:1035–1043

    PubMed  CAS  Google Scholar 

  • Motyka V, Vaňková R, Čapková V, Petrášek J, Kamínek M, Schműlling T (2003) Cytokinin-induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion. Physiol Plant 117:11–21

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Rao SM, Ravishankar GA (2002) Plant cell cultures: Chemical factories of secondary metabolites. Biotechnol Advances 20:101–153

    Article  CAS  Google Scholar 

  • Rotrekl V, Nejedlá E, Kučera I, Abdallah F, Palme K, Brzobohatý B (1999) The role of cysteine residues in structure and enzyme activity of a maize β-glucosidase. Europ J Biochem 266:1056–1065

    Article  PubMed  CAS  Google Scholar 

  • Sakakibara H (2006) Cytokinins: activity, biosynthesis, and translocation. Ann Rev Plant Biol 57:431–449

    Article  CAS  Google Scholar 

  • Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. In: Porter HK (ed). The biological action of growth substances. Acadamic Press, New York, pp 118−131

  • Sondheimer E, Tzou DS (1971) The metabolism of hormones during seed germination and dormancy II. The metabolism of 8–14C-zeatin in bean axes. Plant Physiol 47:516–520

    Article  PubMed  CAS  Google Scholar 

  • Souza BM, Kraus JE, Enders L, Mercier H (2003) Relationships between endogenous hormonal levels and axillary bud development of Ananas comosus nodal segments. Plant Physiol Biochem 41:733–739

    Article  CAS  Google Scholar 

  • Sugiyama M (1999) Organogenesis in vitro. Curr Opin Plant Biol 2(1):61–64

    Article  PubMed  CAS  Google Scholar 

  • van der Krieken WM, Croes AF, Smulders MJ, Wullems GJ (1990) Cytokinin and flower bud formation in vitro in tobacco. Plant Physiol 92:565–569

    Article  PubMed  Google Scholar 

  • van Staden J, Crouch NR (1996) Benzyladenine and derivates—their significance and interconversion in plants. Plant Growth Regul (19):153–175

  • Vaňková R (1999) Cytokinin glucoconjugates–distribution, metabolism and function. In: Strnad M, Peč P, Beck E (eds) Advances in regulation of plant growth and development. Peres Publishers, Prague, pp 67–78

    Google Scholar 

  • Vaňková R, Gaudinová A, Kamínek M, Eder J (1992) The effect of interaction of syntetic cytokinin and auxin on production of natural cytokinin by immobilised of tobacco cells. In: Kamínek M, Mok DWS, Zažímalová E (eds) Physiology and biochemistry of cytokinins in plants. SPB Academic Publishings, The Hague

    Google Scholar 

  • Werbrouck SPO, Strnad M, Van Onckelen HA, Deberght PC (1996) Meta-topolin, an alternative to benzyladenine in tissue culture. Physiol Plant 98:291–297

    Article  CAS  Google Scholar 

  • Yamaguchi M, Kato H, Yoshida S, Yamamura S, Uchimiya H, Umeda M (2003) Control of in vitro organogenesis by cyclin-dependent kinase activities in plants. PNAS 100(13):8019–8023

    Article  PubMed  CAS  Google Scholar 

  • 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 196:84–94

    Article  CAS  Google Scholar 

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Acknowledgments

This study was funded by the Ministry of Education for Youth and Sports of the Czech Republic (1M0603 and LC06034), the Czech Science Foundation (P506/11/0774), the Grant Agency of the Academy of Sciences of the Czech Republic (IAA 600380701) and the Grant Agency of MUAF (IGA 4/4).

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Authors and Affiliations

  1. Department of Plant Biology, Mendel University, Zemědělská 1, Brno, 613 00, Czech Republic

    Marek Klemš, Zdeňka Slámová & Stanislav Procházka

  2. Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, Lysolaje, Prague 6, 165 02, Czech Republic

    Václav Motyka, Jiří Malbeck, Alena Trávníčková & Ivana Macháčková

  3. Isotope Laboratory, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4, 142 20, Czech Republic

    Josef Holík

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  1. Marek Klemš
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Klemš, M., Slámová, Z., Motyka, V. et al. Changes in cytokinin levels and metabolism in tobacco (Nicotiana tabacum L.) explants during in vitro shoot organogenesis induced by trans-zeatin and dihydrozeatin. Plant Growth Regul 65, 427–437 (2011). https://doi.org/10.1007/s10725-011-9612-z

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