Mendel University in Brno 

Keynote Lecture 9: Phytohormone profiling at the cellular and subcellular levels

Ondřej Novák [a]

[a] Laboratory of Growth Regulators, Institute of Experimental Botany, The Czech Academy of Sciences & Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic 

Phytohormones play crucial role in the control of various physiological processes. The identification and quantification of plant hormones in plant tissues are necessary for physiological studies of their metabolism and mode of action. The major problem associated with plant hormone 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. The application of targeted metabolomics has been shown to be the 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). 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. We have also developed a new method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for targeted profiling of more than 100 analytes, members of the main classes of plant hormones like cytokinins, auxins, brassinosteroids, gibberellins, jasmonates and abscisates and salicylic acid. Use of our procedures can allow the quantification of plant hormones and their derivatives in very limited amounts of plant material.

By employing novel methods, we are able to gain a much better understanding of how genetic and experimental manipulations affect hormone levels in plant tissues and organs. However, our knowledge of the distribution and compartmentation of phytohormones and their metabolites at the cellular level is still very limited. We focus on the efficient cell and organelle isolation, combining different approaches such as a density gradient ultracentrifugation or a fluorescence-assisted cell/organelle sorting (FACS/FAOS) with an ultra-sensitive tandem mass spectrometry techniques. New analytical tools will provide comprehensive insights into the phytohormone regulatory networks, such as detailed distribution of plant hormones in specific cells and organelles.