Abstract
Brassinosteroids (BRs) are a class of steroid plant hormones that participate with other plant hormones in the regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, and seed germination. A characteristic feature of all plant hormones, including BRs, is that their concentration is extremely low in plant tissues and, therefore, the methods dealing with their determination belong to ultra-trace analysis, for which very sensitive analytical tools are needed. The analysis of natural BRs is essential when their functions and roles in plant growth and development are to be elucidated. Here, we describe a reliable protocol for high-throughput extraction and purification of BRs. The procedure consists of two solid-phase extraction steps and provides selective enrichment and efficient cleanup of these compounds from complex plant extracts. The protocol is designed for sensitive liquid chromatography-tandem mass spectrometry-based method for simultaneous detection of 22 naturally occurring BRs, including their biosynthetic precursors and most of their biologically active metabolites, without need for derivatization.
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References
Bajguz A (2011) Brassinosteroids—occurrence and chemical structures in plants. In: Hayat S, Ahmad A (eds) Brassinosteroids: a class of plant hormone. Springer, Dordrecht, pp 1–27
Tarkowská D, Novák O, Floková K et al (2014) Quo vadis plant hormone analysis? Planta 240:55–76
Swaczynová J, Novák O, Hauserová E et al (2007) New techniques for the estimation of naturally occurring brassinosteroids. J Plant Growth Regul 26:1–14
Takatsuto S, Ying B, Morisaki M et al (1982) Microanalysis of brassinolide and its analogues by gas chromatography and gas chromatography-mass spectrometry. J Chromatogr 239:233–241
Yokota T, Sato T, Takeuchi Y et al (2001) Roots and shoots of tomato produce 6-deoxo-28-norcathasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone, possible precursors of 28-norcastasterone. Phytochemistry 58:233–238
Wu Q, Wu D, Shen Z et al (2013) Quantification of endogenous brassinosteroids in plant by on-line two-dimensional microscale solid phase extraction-on column derivatization coupled with high performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 1297:56–63
Ding J, Mao L-J, Yuan B-F et al (2013) A selective pretreatment method for determination of endogenous active brassinosteroids in plant tissues: double layered solid phase extraction combined with boronate affinity polymer monolith microextraction. Plant Methods 9:13
Ding J, Wu J-H, Liu J-F et al (2014) Improved methodology for assaying brassinosteroids in plant tissues using magnetic hydrophilic material for both extraction and derivatization. Plant Methods 10:39
Tarkowská D, Novák O, Oklestková J et al (2016) The determination of 22 natural brassinosteroids in a minute sample of plant tissue by UHPLC–ESI–MS/MS. Anal Bioanal Chem 408:6799–6812
Polko JK, Pierik R, van Zanten M et al (2013) Ethylene promotes hyponastic growth through interaction with ROTUNDIFOLIA3/CYP90C1 in Arabidopsis. J Exp Bot 64:613–624
Singh AP, Fridman Y, Friedlander-Shani L et al (2014) Activity of the brassinosteroid transcription factors BRASSINAZOLE RESISTANT1 and BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1/BRASSINAZOLE RESISTANT2 blocks developmental reprogramming in response to low phosphate availability. Plant Physiol 166:678–688
Stirk WA, Bálint P, Tarkowská D et al (2013) Hormone profiles in microalgae: Gibberellins and brassinosteroids. Plant Physiol Biochem 70:348–353
Stirk WA, Tarkowská D, Turečková V et al (2014) Abscisic acid, gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima. J Appl Physiol 26:561–567
Acknowledgments
The authors thank Sees-editing Ltd. for critical reading and editing of the manuscript. The work was supported by the Grant Agency of the Czech Republic (14-34792S) and by the Ministry of Education, Youth and Sport of the Czech Republic through National Program of Sustainability (LO 1204).
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Tarkowská, D., Strnad, M. (2017). Protocol for Extraction and Isolation of Brassinosteroids from Plant Tissues. In: Russinova, E., Caño-Delgado, A. (eds) Brassinosteroids. Methods in Molecular Biology, vol 1564. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6813-8_1
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DOI: https://doi.org/10.1007/978-1-4939-6813-8_1
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6811-4
Online ISBN: 978-1-4939-6813-8
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