Fiziol. rast. genet. 2021, vol. 53, no. 4, 346-368, doi: https://doi.org/10.15407/frg2021.04.346

Profiling of cytokinins in plant tissues: sampling, qualitative and quantitative analysis

Shcherbatiuk М.М.1, Voytenko L.V.1, Kharkhota М.A.2, Коsakivska І.V.1

  1. M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine 2 Tereshchenkivska St., Кyiv, 01004, Ukraine
  2. D.K. Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine 154 Akademika Zabolotnogo St., Кyiv, 03143, Ukraine

The profiling of plant hormones is a complex analytical task, as these compounds are contained in tissues in nanoquantities together with numerous interfering substances of higher concentration. Cytokinins, which are involved in the regulation of all vital plant functions, including cell division, meristems formation, responses to environmental influences, photosynthesis, aging, absorption of macro- and micronutrients, form one of the most important classes of phytohormones. The publication describes the phytohormones of cytokinin nature, and makes a brief review into the history of these compounds identification by analytical methods. The importance of methodological developments in the determination of cytokinins in plant tissues for solving problems of modern plant physiology is emphasized. The new method of simultaneous quantitative analysis of five forms of cytokinins is presented in detail. This sensitive method of quantitative analysis based on chromatographic separation of analytes and single-quadrupole mass spectrometry with an «electrospray» ionization interface is characterized. The analytical HPLC/EC-MS method developed by us is based on efficient gradient chromatographic separation of substances from aliquots of samples in combination with appropriate extraction and purification procedures of samples. It is noted that the extraction in the solvent system, followed by purification on two cartridges of solid-phase extraction, allow efficient and rapid isolation of cytokinins from plant material. The developed HPLC/EC-MS method is characterized by high resolution and selectivity, has sufficient sensitivity to determine the location of cytokinins and elucidate their physiological effects in individual organs and tissues. The studied cytokinins were defined as the dominant molecular ions [M+H]+ with a detection limit below 0.5 ng per ml of extract. Using the advantages of high-performance chromatographic separation of substances and sensitive mass spectrometric detection, the presented method of quantitative analysis is a useful tool for plant physiologists and biochemists in studying the participation of cytokinin hormones in the regulation of growth and development, formation of plant adaptation strategy under impact of abiotic and biotic stressors.

Keywords: cytokinins, solid phase extraction, high performance liquid chromatography, mass spectrometry, profiling

Fiziol. rast. genet.
2021, vol. 53, no. 4, 346-368

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