Cytokinins & Hormonomics

Head of Research Program: Ondřej Novák

The research in this group is focused on qualitative and quantitative determination of endogenous cytokinins (plant growth regulating substances) a powerful tool for increasing our understanding of the role of phytohormones during plant development. The main aims of our work are to optimize the isolation of phytohormones (especially cytokinins) from minute amounts of plant material (1-10 mg) and miniaturized solid-phase extraction. Immunoaffinity chromatography using both polyclonal and monoclonal antibodies is employed as an integral part of our purification procedures, resulting in highly purified samples prior to LC-MS analysis. This further enhances the detection limits of our analytical methods based on mass spectrometry (MS) coupled to ultra high-performance liquid chromatography (UHPLC). For quantitative analysis we are currently using triple-quadrupole MS, combining high sensitivity (<0.01 fmol) and a linear response over as much as five orders of magnitude. Qualitative analysis is usually performed using hybrid QqTOF MS technology which allows identification of compounds of interest using MS/MS spectra recorded in as little as 1 fmol; exact mass determination, giving the elemental composition of the analyte, is also possible.

The other main area of enquiry in our group is “hormonomics” – mutual estimation of 6 physiologically important groups of  plant hormones. We have developed several fast chromatographic separations and sensitive tandem mass spectrometry methods for simultaneous profiling of the majority of phytohormone metabolites (e.g. sixteen stress-induced phytohormones). Targeted metabolomics provides  an optimal method for phytohormonal screening in combination with  miniaturized purification and a highly sensitive MS-based detection. Use of our procedures can allow the quantification of plant hormones and their derivatives (more than 100 compounds) at the tissue and cellular level. This enables us to confirm the endogenous origin of analyzed compounds, to construct and confirm metabolic pathways and to determine the sites of their biosynthesis.