The effect of exogenous 24-epibrassinolide on the ecdysteroid content in the leaves of Spinacia oleracea L. Marek Kamlar a , Olga Rothova b , Sarka Salajkova c,f , Dana Tarkowska d , Pavel Drasar c , Marie Kocova b , Juraj Harmatha e , Dana Hola b , Ladislav Kohout e , Tomas Macek a,⇑ a Dept. of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, 16628, Czech Republic b Dept. of Genetics and Microbiology, Faculty of Science, Charles University in Prague, 12843, Czech Republic c Dept. of Natural Products, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, 16628, Czech Republic d Laboratory of Growth Regulators, Palacky Univ. and Institute of Experimental Botany ASCR, Olomouc 78371, Czech Republic e Institute of Organic Chemistry and Biochemistry ASCR, Prague 16610, Czech Republic f Present address: Biomedical Research Center, University Hospital Hradec Kralove, 500 05 Hradec Kralove, Czech Republic article info Article history: Received 16 July 2014 Received in revised form 10 December 2014 Accepted 29 December 2014 Available online xxxx Keywords: Ecdysteroids Spinach 24-Epibrassinolide 20-Hydroxyecdysone abstract The aim of this study was to show whether/how the application of exogenous 24-epibrassinolide can affect the content of ecdysteroids in spinach leaves. Brassinosteroids and ecdysteroids, structurally related phytosterols, show effect on a range of processes in plants. Brassinosteroids increase biomass yield in some species, photosynthesis and resistance to stress, and ecdysteroids show effect on proteins responsible for binding of CO 2 or water cleavage. The mutual interaction of these sterols in plants is unclear. The UPLC–(+)ESI-MS/MS analyses of extracts of treated and untreated spinach (Spinacia oleracea L.) leaves show that the application of exogenous 24-epibrassinolide does influence the ecdysteroid con- tent in plant tissues. The response differs for the major ecdysteroids and also differs from that for the minor ones and is dependent on the developmental stage of the leaves within the same plant or the 24-epibrassinolide concentration applied. Ó 2015 Published by Elsevier Inc. 1. Introduction The aim of this work was to find out if application of the steroi- dal plant hormone 24-epibrassinolide (24-epiBL) induces changes of the content of main ecdysteroids in spinach leaves. Ecdysteroids (ES) have close structural similarity with brassinosteroids (BS) and recently discovered closeness with them in some physiological effects. Phytoecdysteroids are present in many plant species [1], but their biological role is still far from being clear [2,3]. While ecdys- teroids as insect hormones have been investigated thoroughly, knowledge of their function and the mechanism of action in plants and other organisms are not so detailed, except the antifeeding activity against herbivorous insects [4]. This is despite the fact that their concentration in plants can be even two orders higher than in arthropods. Currently over 400 of them are known, but the most common phytoecdysteroid in plants is probably 20-hydroxyecdy- sone (20-E) followed by polypodine B [1]. Brassinosteroids (BS) discovered in 1979 [5] were proved to be plant hormones [6,7] and are well known as playing important roles in controlling gene expression, cell division and expansion, responses to light and dark, and fertility, as reviewed in [8]. The described effects of exogenous application of the main representa- tive of BS hormones, 24-epibrassinolide (24-epiBL) at concentra- tions between 10 À4 M and 10 À10 M range from influence on growth and photosynthesis yield to increased stress tolerance, e.g. salt stress [9]. Sterols in general are required in the control of plant pattern formation, morphogenesis and cell polarity, influenc- ing at least in part the functionality of key regulatory proteins [10]. http://dx.doi.org/10.1016/j.steroids.2014.12.024 0039-128X/Ó 2015 Published by Elsevier Inc. Abbreviations: BS, brassinosteroids; ES, ecdysteroids; 24-epiBL, 24-epibrassin- olide; 20-E, 20-hydroxyecdysone; PolB, polypodine B; StaC, stachysterone C; AjuC, ajugasterone C; PonA, ponasterone A; RuBisCO, ribulose 1,5-bisphosphate carbox- ylase/oxygenase; PsbP, oxygen-evolving enhancer protein 2-1; FW, fresh weight; UPLC, ultra performance liquid chromatography. ⇑ Corresponding author at: Dept. of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, 16628 Prague 6, Czech Republic. Tel.: +420 732826353. E-mail addresses: marek.kamlar@gmail.com (M. Kamlar), olga.rothova@natur. cuni.cz (O. Rothova), salajkova.kac@gmail.com (S. Salajkova), tarkowska@ueb.cas.cz (D. Tarkowska), Pavel.Drasar@vscht.cz (P. Drasar), kocova@natur.cuni.cz (M. Kocova), harmatha@uochb.cas.cz (J. Harmatha), dana.hola@natur.cuni.cz (D. Hola), ladislavkohout@seznam.cz (L. Kohout), tomas.macek@vscht.cz (T. Macek). Steroids xxx (2015) xxx–xxx Contents lists available at ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids Please cite this article in press as: Kamlar M et al. The effect of exogenous 24-epibrassinolide on the ecdysteroid content in the leaves of Spinacia oleracea L.. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2014.12.024