Plant hormone metabolite profiling on the tissue and cell level

Phytohormones play crucial role in the control of various physiological processes. The major problem associated with phytohormone analysis is that the amount of phytohormones present endogenously in plant tissues is very low, usually in the range of fmol to pmol/g fresh weight [1]. High-resolution measurements of phytohormones are therefore necessary for physiological studies of their mode of action. Application of targeted metabolomics shows an optimal method for phytohormonal screening. Our work is focused on the development of high-throughput miniaturized purification methods for minute amounts of plant tissue (1 – 10 mg). Two novel approaches, a simple one-step purification protocol based on in-tip microSPE (micro Solid-Phase Extraction) and a class-specific miniaturized immunoaffinity chromatography method were utilized. In order to verify the effect of complex sample matrix of the whole method as well as its efficiency and analytical accuracy, the appropriate stable isotope-labelled internal standards have been prepared by the organic synthesis. We have also developed several fast chromatographic separations (UHPLC) and highly sensitive tandem mass spectrometry (MS/MS) methods for simultaneous profiling of the phytohormone metabolites. Moreover, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with a highly sensitive LC-MS method for analysis of phytohormonal biosynthesis and homeostasis at cellular resolution.

Acknowledgement: Supported by the Czech Science Foundation (Nr. GA14 – 34792S), by the Ministry of Education, Youth and Sports of the Czech Republic – NPU I program with project LO1204 and the “Návrat” program LK21306.

References:

[1] Tarkowská D, Novák O, Floková K, Tarkowski P, Turečková V, Grúz J, Rolčík J, Strnad M. Quo vadis plant hormone analysis? Planta 2014; 240: 55 – 76