Hormones play a pivotal role in most physiological processes in plants. These structurally diverse compounds that act usually at nanomolar levels include five groups of the so-called "classic" hormones, comprising auxins, cytokinins, gibberellins (GA), abscisic acid (ABA) and ethylene, and several other plant growth regulators, including jasmonates, salicylates, brassinosteroids, polyamines or the very recently discovered strigolactones, which fit several of the criteria to be considered hormones [1–3]. Furthermore, the list of plant hormones is expected to increase due to a better understanding of plant growth and development and stress responses, and the use of technological advances in analytical methods.
Recent studies support the contention that hormone actions build a signaling network and mutually regulate several signaling and metabolic systems, such as auxins and GAs in growth regulation , CKs, auxins, ABA and strigolactones in apical dominance [2, 5], auxins and brassinosteroids in cell expansion [6, 7], ethylene and cytokinins in the inhibition of root and hypocotyl elongation , ethylene, ABA and GAs in some plant stress responses [9, 10], or SA, JA and auxin in plant responses to pathogens [11, 12] to name just a few of the reported hormonal interactions. Therefore, focusing on a single endogenous plant hormone to evaluate hormone-regulated physiological or developmental biological problems is not sufficient anymore .
In order to understand better the network regulation of hormone action influencing plant growth and development as well as the distribution of several hormones at the organ, cellular and sub-cellular levels, an ideal analytical method should provide a measure of multiple hormone concentrations (hormonal profiling) from a single experimental sample. Therefore several methods for the simultaneous quantification of multiple plant hormones using mass spectrometry with multiple reaction monitoring (MRM) have been developed recently. It has been reported a multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique for the simultaneous analysis of SA, JA, IAA, ABA and OPDA in Arabidopsis thaliana. However, GC-MS is limited to volatile compounds and as a result it is necessary to purify and derivatize hormones prior to analysis. Another potential downside in GC-MS procedures apart from the purification and derivatization is the use of high temperatures, which can degrade thermal labile compounds .
An alternative to GC-MS is liquid chromatography coupled to mass spectrometry (LC-MS). A high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous analysis of 15 plant hormones and metabolites from four different hormone classes (auxins, cytokinins, GAs and ABA) has been reported to analyze hormone regulation of thermodormancy of lettuce seeds . Also, a HPLC/ESI-MS/MS method to analyze seven major classes of plant hormones including auxins, cytokinins, GAs, ABA, jasmonates, brassinosteriods and SA in Arabidopsis thaliana has been developed . Furthermore, an ultrahigh-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) technique to analyze cytokinins, auxins, ABA and GAs in rice has been described . To improve the detection limit of the negatively charged compounds they derivatized auxin, ABA and GAs with bromocholin and analyzed all compounds in the positive ion mode. However, at present this method is limited and cannot target other plant hormones such as JA and SA.
Plant hormones are structurally diverse compounds with diverse physiochemical properties. The question as to whether all plant hormones can be extracted equally well has not yet been answered. The choice of extraction methods depends not only on the target analysts but also on the matrix of the analyzed tissues. The requirements on the extraction method increase with the complexity of the sample matrix. In the literature diverse extraction solvents such as methanol, methanol-water mixtures, isopropanol, or isopropanol-water mixtures have been used with one or two extraction steps [14, 15, 17–19]. In addition, time-consuming multiple steps of sample preparation procedures, including sample purification, drying of sample under N2 and re-suspension of the residues have been reported for plant hormone extraction [14, 20] which may increase the risk of hormone loss. However, the application of internal standards can provide corrections for hormone loss during sample preparation and chromatographic separation.
Here we developed a new method which allows to analyze dynamic changes in endogenous concentrations of major plant hormones and to study plant development processes or plant responses to biotic and abiotic stresses in complex sample matrices. An example is shown in which rosemary (Rosmarinus officinalis), an aromatic Mediterranean perennial shrub rich in secondary metabolites and epicuticular waxes, was exposed to salt stress. Soil salinity is one of the most serious environmental threats for plant survival and affects many undesirable changes in plants such as hyperionic and hyperosmotic effects, increase in reactive oxygen species and metabolic toxicity. These changes lead to growth reduction, changes in biomass allocation and phenology, leaf senescence, and finally to plant death [21–23]. It has been shown that senescence induced by salinity follows at least in part similar physiological events as drought-induced senescence . Plant hormones such as ABA, ethylene and cytokinins are involved in different plant strategies to overcome the damaging effects of salinity, however, the complex hormonal response is only partly known [25, 26]. The present work reports a sensitive and rapid method to quantify 17 plant hormones from seven plant classes including auxins, cytokinins, GAs, ABA, ACC (the ethylene precursor), SA and JA in complex tissues using ultra-performance liquid chromatography mass spectrometry (UPLC/ESI-MS/MS) with multiple reaction monitoring (MRM). This method allows obtaining a hormonal profiling in 6 min. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized.