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The Pea Nodulation Mutant R50 (sym16) Displays Altered Activity and Expression Profiles for Cytokinin Dehydrogenase

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Abstract

R50 (sym16) is a pleiotropic mutant of pea (Pisum sativum L.) which develops few, pale nodules and has pale young leaves. This phenotype coincides with elevated cytokinin content in vegetative organs, especially mature shoots. Because cytokinin content is known to be tightly regulated by the catabolic action of cytokinin dehydrogenase (CKX), this study focuses on whether CKX-mediated regulation of cytokinin content is involved in the R50 phenotype. Thus, we analyzed the biochemical activity of this enzyme in vitro and found that R50 displayed an aberrant activity profile. During development, PsCKX activity was significantly reduced when compared to wild-type (WT); this was observed in many tissues, specifically in mature shoots and nodules where decrease in activity correlated with elevated cytokinin content. To further address this issue, a full-length cDNA corresponding to CKX1 from pea (PsCKX1) was obtained via RACE-PCR. Although sequencing the entire PsCKX1 cDNA from R50 did not reveal any significant mutations that could have linked PsCKX1 to the sym16 mutation, relative transcript levels of PsCKX1 and of another PsCKX homolog (PsCKX2) were compared between R50 and WT using semiquantitative reverse transcriptase PCR. Interestingly, transcription of these homologs was upregulated in the tissues of R50 displaying the most aberrant phenotype, namely, the mature shoots and nodules. We propose that the R50 phenotype is linked to elevated cytokinin content as a result of deficient PsCKX activity and that transcription of two PsCKX homologs is upregulated as a means to compensate for the biochemical deficiency of this enzyme in R50 mutants.

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References

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M (2005) Cytokinin oxidase regulates rice grain production. Science 309:741–745

    Article  PubMed  CAS  Google Scholar 

  • Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18:298–305

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • 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 950:21–29

    Article  Google Scholar 

  • Ellis THN, Poyser SJ (2002) An integrated and comparative view of pea genetic and cytogenetic maps. New Phytol 153:17–25

    Article  CAS  Google Scholar 

  • Emery RJN, Leport L, Barton JE, Turner NC, Atkins CA (1998) cis-isomers of cytokinins predominate Cicer arietinum throughout their development. Plant Physiol 117:1515–1523

    Article  PubMed  CAS  Google Scholar 

  • Emery RJN, Ma Q, Atkins CA (2000) The forms and sources of cytokinins in developing Lupinus albus seeds and fruits. Plant Physiol 123:1593–1604

    Article  PubMed  CAS  Google Scholar 

  • Fang Y, Hirsch AM (1998) Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in transgenic alfalfa. Plant Physiol 116:53–68

    Article  PubMed  CAS  Google Scholar 

  • Ferguson BJ, Wiebe EM, Emery RJN, Guinel FC (2005) Cytokinin accumulation and an altered ethylene response mediate the pleiotropic phenotype of the pea nodulation mutant R50 (sym 16). Can J Bot 83:989–1000

    Article  Google Scholar 

  • Frébort I, Šebela M, Galuszka P, Werner T (2002) Cytokinin oxidase/dehydrogenase assay: Optimized procedures and applications. Anal Biochem 306:1–7

    Article  PubMed  CAS  Google Scholar 

  • 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 271:3990–4002

    Article  PubMed  CAS  Google Scholar 

  • Galuszka P, Frébortová J, Luhová L, Bilyeu KD, English JT, Frébort I (2005) Tissue localization of cytokinin dehydrogenase in maize: Possible involvement of quinone species generated from plant phenolics by other enzymatic systems in the catalytic reaction. Plant Cell Physiol 46:716–728

    Article  PubMed  CAS  Google Scholar 

  • 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 26:255–267

    Article  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  CAS  Google Scholar 

  • Gonzalez-Rizzo S, Crespi M, Frugier F (2006) The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti. Plant Cell 18:2680–2693

    Article  PubMed  CAS  Google Scholar 

  • Guinel FC, Sloetjes LL (2000) Ethylene is involved in the nodulation phenotype of Pisum sativum R50 (sym16), a pleiotropic mutant that nodulates poorly and has pale green leaves. J Exp Bot 51:885–894

    Article  PubMed  CAS  Google Scholar 

  • Higgins DG, Thompson JD, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  Google Scholar 

  • Houba-Hérin N, Pethe C, d’Alayer J, Laloue M (1999) Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts. Plant J 17:615–626

    Article  PubMed  Google Scholar 

  • Kamínek M, Motyka V, Vaňkova R (1997) Regulation of cytokinin content in plant cells. Physiol Plant 101:689–700

    Article  Google Scholar 

  • Kneen BE, Weeden NF, LaRue TA (1994) Non-nodulating mutants of Pisum sativum (L.) cv. Sparkle. J Hered 85:129–133

    Google Scholar 

  • Liberos-Minotta CA, Tipton PA (1995) A colorimetric assay for cytokinin oxidase. Anal Biochem 231:339–341

    Article  Google Scholar 

  • Lohar DP, Schaff JE, Laskey JG, Kieber JJ, Bilyeu KD, Bird DM (2004) Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses. Plant J 38:203–214

    Article  PubMed  CAS  Google Scholar 

  • Lorteau MA, Ferguson BJ, Guinel FC (2001) Effects of cytokinin on ethylene production and nodulation in pea (Pisum sativum) cv. Sparkle. Physiol Plant 112:421–428

    Article  PubMed  CAS  Google Scholar 

  • Malito E, Coda A, Bilyeu KD, Fraaije MW, Mattevi A (2004) Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis. J Mol Biol 341:1237–1249

    Article  PubMed  CAS  Google Scholar 

  • Massonneau A, Houba-Hérin N, Pethe C, Madzak C, Falque M, Mercy M, Kopecny D, Majira A, Rogowsky P, Laloue M (2004) Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs. J Exp Bot 55:2549–2557

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Morris RO, Bilyeu KD, Laskey JG, Cheikh NN (1999) Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun 255:328–333

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Murray JD, Karas BJ, Sato S, Tabata S, Amyot L, Szczyglowski K (2007) A cytokinin perception mutant colonized by Rhizobium in the absence of nodule organogenesis. Science 315:101–104

    Article  PubMed  CAS  Google Scholar 

  • Pepper AN, Morse AP, Guinel FC (2007) Abnormal root and nodule vasculature in R50 (sym16), a pea nodulation mutant which accumulates cytokinins. Ann Bot 99:765–776

    Article  PubMed  CAS  Google Scholar 

  • Prinsen E, Redig P, Van Dongen W, Esmans EL, Van Onckelen HA (1995) Quantitative analysis of cytokinins by electrospray tandem mass spectrometry. Rapid Commun Mass Spectr 9:948–953

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Schmülling T, Werner T, Riefler M, Krupkova E, Manns YB (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis, and other species. J Plant Res 116:241–252

    Article  PubMed  CAS  Google Scholar 

  • Syõno K, Torrey JG (1976) Identification of cytokinins of root nodules of the garden pea, Pisum sativum L. Plant Physiol 57:602–606

    PubMed  Google Scholar 

  • Syõno K, Newcomb W, Torrey JG (1976) Cytokinin production in relation to the development of pea root nodules. Can J Bot 54:2155–2162

    Article  Google Scholar 

  • Tirichine L, Sandal N, Madsen LH, Radutoiu S, Albrektsen AS, Sato S, Asamizu E, Tabata S, Stougaard J (2007) A gain-of-function mutation in a cytokinin receptor triggers spontaneous root nodule organogenesis. Science 315:104–107

    Article  PubMed  CAS  Google Scholar 

  • Todorova D, Vaseva-Gemisheva I, Petrov P, Stoynova-Bakalova E, Alexieva V, Karanov E, Smith A, Hall M (2006) Cytokinin oxidase/dehydrogenase (CKX) activity in wild and ethylene-insensitive mutant eti5 type of Arabidopsis thaliana (L.) Heynh plants and the effect of cytokinin N1-(2-chloro-4-pyridyl)-N2-phenylurea on enzymatic activity and leaf morphology. Acta Physiol Plant 28:613–617

    Google Scholar 

  • Vaseva-Gemisheva IV, Lee D, Alexieva V, Karanov E (2004) Cytokinin oxidase/dehydrogenase in Pisum sativum plants during vegetative development. Influence of UV-B irradiation and high temperature on enzymatic activity. Plant Growth Regul 42:1–5

    Article  CAS  Google Scholar 

  • Vaseva-Gemisheva I, Lee D, Karanov E (2005a) Response of Pisum sativum cytokinin oxidase/dehydrogenase expression and specific activity to drought stress and herbicide treatments. Plant Growth Regul 46:199–208

    Article  CAS  Google Scholar 

  • Vaseva-Gemisheva I, Lee D, Karanov E (2005b) Antagonistic effects of triazolo[4,5d]pyrimidine and pyridylurea derivatives on cytokinin-induced cytokinin oxidase/dehydrogenase activity in young pea plants. Plant Growth Regul 46:193–197

    Article  CAS  Google Scholar 

  • Vogel JP, Woeste KE, Theologis A, Kieber JJ (1998) Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively. Proc Natl Acad Sci U S A 95:4766–4771

    Article  PubMed  CAS  Google Scholar 

  • Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmülling T (2003) 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 15:2532–2550

    Article  PubMed  CAS  Google Scholar 

  • Werner T, Köllmer I, Bartrina I, Holst K, Schmülling T (2006) New insights into the biology of cytokinin degradation. Plant Biol 8:371–381

    Article  PubMed  CAS  Google Scholar 

  • Wilkins MR, Lindskog I, Gasteiger E, Bairoch A, Sanchez JC, Hochstrasser DF, Appel RD (1997) Detailed peptide characterisation using PEPTIDEMASS - a World-Wide Web accessible tool. Electrophoresis 18:403–408

    Article  PubMed  CAS  Google Scholar 

  • Yang S, Yu H, Goh CJ (2003) Functional characterization of a cytokinin oxidase gene DsCKX1 in Dendrobium orchid. Plant Mol Biol 51:237–248

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank Dr. T.A. LaRue for his gift of R50 seeds, and Dr. B.A. Moffatt (University of Waterloo) for her helpful suggestions and advice. M. Held acknowledges the seminal work of Dr. Vaseva-Gemisheva regarding the isolation of PsCKX sequence data and her aid in analyzing the PsCKX activity data. The research was supported by Natural Sciences and Engineering Research Council of Canada operating grants to MDS, RJNE, and FCG, and one Natural Sciences and Engineering Research Council of Canada Undergraduate Student Research Assistantship to ANP.

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  1. Department of Biology, Wilfrid Laurier University, 75 University Avenue West, Waterloo, Ontario, Canada, N2L 3C5

    Mark Held, Alicia N. Pepper, Johny Bozdarov, Matthew D. Smith & Frédérique C. Guinel

  2. Department of Biology, Trent University, 1600 West Bank Drive, Peterborough, Ontario, K9J 7B8, Canada

    R. J. Neil Emery

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  1. Mark Held
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Correspondence to Frédérique C. Guinel.

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Held, M., Pepper, A.N., Bozdarov, J. et al. The Pea Nodulation Mutant R50 (sym16) Displays Altered Activity and Expression Profiles for Cytokinin Dehydrogenase. J Plant Growth Regul 27, 170–180 (2008). https://doi.org/10.1007/s00344-008-9043-x

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  • DOI: https://doi.org/10.1007/s00344-008-9043-x

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