UHPLC–MS/MS based target profiling of stress-induced phytohormones Kristy ´ na Floková, Danuše Tarkowská, Otto Miersch, Miroslav Strnad, Claus Wasternack, Ondr ˇej Novák ⇑ Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany AS CR & Palacky ´ University, Šlechtitelu ˚ 11, CZ-78371 Olomouc, Czech Republic article info Article history: Received 19 February 2014 Received in revised form 12 May 2014 Available online xxxx Keywords: Stress-induced phytohormones Jasmonates Abscisic acid Salicylic acid Indole-3-acetic acid Arabidopsis thaliana Solid-phase extraction (SPE) Ultra-high performance liquid chromatography (UHPLC) Tandem mass spectrometry (MS/MS) abstract Stress-induced changes in phytohormone metabolite profiles have rapid effects on plant metabolic activ- ity and growth. The jasmonates (JAs) are a group of fatty acid-derived stress response regulators with roles in numerous developmental processes. To elucidate their dual regulatory effects, which overlap with those of other important defence-signalling plant hormones such as salicylic acid (SA), abscisic acid (ABA) and indole-3-acetic acid (IAA), we have developed a highly efficient single-step clean-up procedure for their enrichment from complex plant matrices that enables their sensitive quantitative analysis using hyphenated mass spectrometry technique. The rapid extraction of minute quantities of plant material (less than 20 mg fresh weight, FW) into cold 10% methanol followed by one-step reversed-phase poly- mer-based solid phase extraction significantly reduced matrix effects and increased the recovery of labile JA analytes. This extraction and purification protocol was paired with a highly sensitive and validated ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method and used to simultaneously profile sixteen stress-induced phytohormones in minute plant material sam- ples, including endogenous JA, several of its biosynthetic precursors and derivatives, as well as SA, ABA and IAA. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Plants have evolved a number of unique defense mechanisms to adapt to changing ambient conditions. This ability to alter their growth and development is regulated by signalling of several phytohormones. Although the individual roles of jasmonates (JAs), abscisic acid (ABA) and salicylic acid (SA) in plants’ responses to various biotic and abiotic stresses are quite well established, stress-related JAs signalling in the modulation of numerous developmental processes remains to be clarified (Li et al., 2001; Linkies and Leubner-Metzger, 2012). The jasmonates are short- chain alkylcyclopentenone and alkylcyclopentanone carboxylates that are formed via the lipoxygenase pathway. The de novo synthesis of jasmonic acid (JA) and its subsequent metabolism are both crucial in controlling the level of the bioactive hormone (Wasternack and Kombrink, 2010). JA accumulates very rapidly in both local and distal sites of wounded model plant Arabidopsis leaf tissues: its level increases noticeably within around two min- utes after injury (Glauser et al., 2009). Simultaneously, its volatile methyl ester (MeJA) is generated to enable the rapid transmission of JA signalling after its demethylation to free JA in target tissues (Stitz et al., 2011). Crucial is the conjugation of primarily synthe- sized (+)-7-iso-JA, the initial product of JA biosynthesis. This com- pound is conjugated with the amino acid isoleucine (Ile) to form the most active JA compound and the ligand of the JA-receptor, (+)-7-iso-JA-Ile, which plays a key role in JA signalling (Fonseca et al., 2009; Sheard et al., 2010). Other JA conjugates with primarily non-polar amino acids including leucine (Leu), valine (Val), phen- ylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and methionine (Met) have also been detected in various plant species and may have roles that go beyond environmental stress responses (Knöfel and Sembdner, 1995; Tamogami and Kodama, 1997). For example, JA-Trp acts as a potential regulator of auxin homeostasis via an unknown mechanism during root growth (Staswick, 2009; Guttierrez et al., 2012). In addition, a wide range of JA metabolites with different physiological functions have been described, includ- ing 11-OH-JA, 12-OH-JA, and 12-glucosylated or sulfonylated JA derivatives (Wasternack and Hause, 2013). Finally, both the bio- synthetic precursor of JA, cis-(+)-12-oxo-phytodienic acid (OPDA), and its 16-carbon homolog dinor-OPDA (dn-OPDA) have been also shown to accumulate in wounded leaves (Stintzi et al., 2001). Modern target profiling analyses involve the simultaneous mea- surement of several phytohormonal classes in order to determine their individual significance and control mechanisms. Two main conventional hyphenated techniques, liquid chromatography–mass http://dx.doi.org/10.1016/j.phytochem.2014.05.015 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +420 58563 4853; fax: +420 58563 4870. E-mail address: novako@ueb.cas.cz (O. Novák). Phytochemistry xxx (2014) xxx–xxx Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Please cite this article in press as: Floková, K., et al. UHPLC–MS/MS based target profiling of stress-induced phytohormones. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.05.015