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Quantification of Cytokinin Levels and Responses in Abiotic Stresses

  • Alfonso AlbaceteEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1569)

Abstract

Since their discovery in the 1950s, it has been established that cytokinins (CKs) play important regulatory roles in various physiological processes in plants. Only recently have CKs been also implicated in the response of plants to biotic and abiotic stresses. During the last years, several analytical methods have been developed to determine CK concentrations in plant tissues. Here we present a simple and robust method for CK extraction, purification and analysis in plant tissues, using ultrahigh-performance liquid chromatography coupled to high resolution mass spectrometry (U-HPLC-HRMS). The main advantage of this methodology is the simplicity of the purification protocol and the possibility to extend it to the analysis of other plant hormones and derivatives.

Key words

Plant hormones Cytokinins Abiotic stress Quantification Internal standards High-resolution mass spectrometry 

Notes

Acknowledgments

I would like to thank CSIC (Spain) for a postdoc research grant (JAE program), Prof. Francisco Pérez-Alfocea for supporting my research, and Ms. Cristina Soriano-Carpena for her help in editing the English version of the manuscript. Research funded by the Spanish MINECO-FEDER (project AGL2014-59197-JIN).

References

  1. 1.
    Sakakibara H (2006) Cytokinins: activity, biosynthesis, and translocation. Annu Rev Plant Biol 57:431–449CrossRefPubMedGoogle Scholar
  2. 2.
    Kieber JJ, Schaller GE (2014) Cytokinins. The Arabidopsis Book 12:e0168CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Mok DWS, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Biol 52:89–118CrossRefGoogle Scholar
  4. 4.
    Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases. Plant Cell Physiol 42:677–685CrossRefPubMedGoogle Scholar
  5. 5.
    Albacete A, Martínez-Andújar C, Pérez-Alfocea F (2014) Hormonal and metabolic regulation of source–sink relations under salinity and drought: from plant survival to crop yield stability. Biotechnol Adv 32:12–30CrossRefPubMedGoogle Scholar
  6. 6.
    Albacete A, Cantero-Navarro E, Großkinsky DK, Arias CL, Balibrea ME, Bru R, Fragner L, Ghanem ME, González MC, Hernández JA, Martínez-Andújar C, van der Graaff E, Weckwerth W, Zellnig G, Pérez-Alfocea F, Roitsch T (2015) Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato. J Exp Bot 66:863–878CrossRefPubMedGoogle Scholar
  7. 7.
    Albacete A, Cantero-Navarro E, Balibrea ME, Großkinsky DK, de la Cruz González M, Martínez-Andújar C, Smigocki AC, Roitsch T, Pérez-Alfocea F (2014) Hormonal and metabolic regulation of tomato fruit sink activity and yield under salinity. J Exp Bot 65:6081–6095CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Dodd IC, Davies WJ (1996) The relationship between leaf growth and ABA accumulation in the grass leaf elongation zone. Plant Cell Environ 19:1047–1056CrossRefGoogle Scholar
  9. 9.
    Albacete A, Ghanem ME, Martínez-Andújar C, Acosta M, Sánchez-Bravo J, Martínez V, Lutts S, Dodd IC, Pérez-Alfocea F (2008) Hormonal changes in relation to biomass partitioning and shoot growth impairment in salinized tomato (Solanum lycopersicum L.) plants. J Exp Bot 59:4119–4131CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ghanem ME, Albacete A, Martínez-Andújar C, Acosta M, Romero-Aranda R, Dodd IC, Lutts S, Pérez-Alfocea F (2008) Hormonal changes during salinity-induced leaf senescence in tomato (Solanum lycopersicum L.). J Exp Bot 59:3039–3050CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Magome H, Yamaguchi S, Hanada A, Kamiya Y, Oda K (2004) Dwarf and delayed-flowering 1, a novel Arabidopsis mutant deficient in gibberellin biosynthesis because of overexpression of a putative AP2 transcription factor. Plant J 37:720–729CrossRefPubMedGoogle Scholar
  12. 12.
    Achard P, Cheng H, De Grauwe L, Decat J, Schoutteten H, Moritz T, Van Der Straeten D, Peng J, Harberd NP (2006) Integration of plant responses to environmentally activated phytohormonal signals. Science 311:91–94CrossRefPubMedGoogle Scholar
  13. 13.
    Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E (2007) Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl Acad Sci U S A 104:19631–19636CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Magome H, Yamaguchi S, Hanada A, Kamiya Y, Oda K (2008) The DDF1 transcriptional activator upregulates expression of a gibberellin-deactivating gene, GA2ox7, under high-salinity stress in Arabidopsis. Plant J 56:613–626CrossRefPubMedGoogle Scholar
  15. 15.
    Pérez-Alfocea F, Albacete A, Ghanem ME, Dodd IC (2010) Hormonal regulation of source-sink relations to maintain crop productivity under salinity: a case study of root-to-shoot signalling in tomato. Funct Plant Biol 37:592–603CrossRefGoogle Scholar
  16. 16.
    Ghanem ME, Albacete A, Smigocki AC, Frébort I, Pospisilová H, Martínez-Andújar C, Acosta M, Sánchez-Bravo J, Lutts S, Dodd IC, Pérez-Alfocea F (2011) Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants. J Exp Bot 62:125–140CrossRefPubMedGoogle Scholar
  17. 17.
    Pan X, Wang X (2009) Profiling of plant hormones by mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 877:2806–2813CrossRefPubMedGoogle Scholar
  18. 18.
    Novák O, Hauserová E, Amakorová P, Doležal K, Strnad M (2008) Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry 69:2214–2224CrossRefPubMedGoogle Scholar
  19. 19.
    Kojima M, Kamada-Nobusada T, Komatsu H, Takei K, Kuroha T, Mizutani M, Ashikari M, Ueguchi-Tanaka M, Matsuoka M, Suzuki K, Sakakibara H (2009) Highly sensitive and high-throughput analysis of plant hormones using ms-probe modification and liquid chromatographytandem mass spectrometry: an application for hormone profiling in Oryza sativa. Plant Cell Physiol 50:1201–1214CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Pieterse CMJ, Leon-Reyes A, Van Der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5:308–316CrossRefPubMedGoogle Scholar
  21. 21.
    Du F, Ruan G, Liu H (2012) Analytical methods for tracing plant hormones. Anal Bioanal Chem 403:55–74CrossRefPubMedGoogle Scholar
  22. 22.
    Tarkowski P, Ge L, Yong JWH, Tan SN (2009) Analytical methods for cytokinins. TrAC Trends Anal Chem 28:323–335CrossRefGoogle Scholar
  23. 23.
    Hoyerová K, Gaudinová A, Malbeck J, Dobrev PI, Kocábek T, Šolcová B, Trávníčková A, Kamínek M (2006) Efficiency of different methods of extraction and purification of cytokinins. Phytochemistry 67:1151–1159CrossRefPubMedGoogle Scholar
  24. 24.
    Dobrev PI, Kamı́nek M (2002) Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chromatogr 950:21–29CrossRefGoogle Scholar
  25. 25.
    Van Meulebroek L, Bussche JV, Steppe K, Vanhaecke L (2012) Ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant. J Chromatogr 1260:67–80CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Plant NutritionCEBAS-CSICMurciaSpain

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