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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sakakibara H (2006) Cytokinins: activity, biosynthesis, and translocation. Annu Rev Plant Biol 57:431–449
Kieber JJ, Schaller GE (2014) Cytokinins. The Arabidopsis Book 12:e0168
Mok DWS, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Biol 52:89–118
Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases. Plant Cell Physiol 42:677–685
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 32:12–30
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 66:863–878
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 65:6081–6095
Dodd IC, Davies WJ (1996) The relationship between leaf growth and ABA accumulation in the grass leaf elongation zone. Plant Cell Environ 19:1047–1056
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 59:4119–4131
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 59:3039–3050
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 37:720–729
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 311:91–94
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 104:19631–19636
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 56:613–626
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 37:592–603
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 62:125–140
Pan X, Wang X (2009) Profiling of plant hormones by mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 877:2806–2813
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 69:2214–2224
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 50:1201–1214
Pieterse CMJ, Leon-Reyes A, Van Der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5:308–316
Du F, Ruan G, Liu H (2012) Analytical methods for tracing plant hormones. Anal Bioanal Chem 403:55–74
Tarkowski P, Ge L, Yong JWH, Tan SN (2009) Analytical methods for cytokinins. TrAC Trends Anal Chem 28:323–335
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 67:1151–1159
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 950:21–29
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 1260:67–80
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Albacete, A. (2017). Quantification of Cytokinin Levels and Responses in Abiotic Stresses. In: Dandekar, T., Naseem, M. (eds) Auxins and Cytokinins in Plant Biology. Methods in Molecular Biology, vol 1569. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6831-2_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6831-2_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6829-9
Online ISBN: 978-1-4939-6831-2
eBook Packages: Springer Protocols