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
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1. Davies, P.J. (2010). The Plant Hormones: Their Nature, Occurrence, and Functions. Davies, P.J. Plant Hormones, Springer, Dordrecht, pp. 1-15. https://doi.org/10.1007/978-1-4020-2686-7_1
2. Dun, E.A., Brewer, P.B. & Beveridge, C.A. (2009). Strigolactones: discovery of the elusive shoot branching hormone. Trends Plant Sci., 14, No. 7, pp. 364-372. https://doi.org/10.1016/j.tplants.2009.04.003
3. Pan, X., Welti, R. & Wang, X. (2008). Simultaneous quantification of major phytohormones and related compounds in crude plant extracts by liquid chromatography tandem mass spectrometry. Phytochemistry, 69, No. 8, pp. 1773-1781. https://doi.org/10.1016/j.phytochem.2008.02.008
4. Vedenicheva, N.P. & Kosakivska, I.V. (2017). Cytokinins as regulators of plant ontogenesis under different growth conditions. Nash format, Kyiv [in Ukrainian].
5. Schaller, G.E., Street, I.H. & Kieber, J.J. (2014). Cytokinins and the cell cycle. Current Opinion in Plant Biology, 21. pp. 7-15. https://doi.org/10.1016/j.pbi.2014.05.015
6. Kurepa, J., Shull, T.E. & Smalle, J.A. (2019). Antagonistic activity of auxin and cytokinin in shoot and root organs. Plant Direct, 3, pp. 1-9. https://doi.org/10.1002/pld3.121
7. Honig,M., Plihalova, L., Husickova, A., Nisler, J. & Dolezal, K. (2018). Role of Cytokinins in Senescence, Antioxidant Defence and Photosynthesis. Int. J. Mol. Sci., 19, pp. 4045. https://doi.org/10.3390/ijms19124045
8. Pavlu, J., Novak, J., Koukalova, V., Luklova, M., Brzobohaty, B. & Cerny, M. (2018). Cytokinin at the crossroads of abiotic stress signaling pathways. Int. J. Mol. Sci., 19, pp. 2450. https://doi.org/10.3390/ijms19082450
9. Bielach, A., Hrtyan, M. & Tognetti, V.B. (2017). Plants under stress: Involvement of auxin and cytokinin. Int. J. Mol. Sci., 18, No. 7, pp. 1427. https://doi.org/10.3390/ijms18071427
10. Cortleven, A., Leuendorf, J.E., Frank, M., Pezzetta D., Bolt, S. & Schmulling, T. (2019). Cytokinin action in response to abiotic and biotic stress in plants. Plant Cell Environ., 42, No. 3, pp. 998-1018. https://doi.org/10.1111/pce.13494
11. Werner, T. & Schmulling, T. (2009). Cytokinin action in plant development. Current Opinion in Plant Biology, 12, No. 5, pp. 527-538. https://doi.org/10.1016/j.pbi.2009.07.002
12. Ha, S., Vankova, R., Yamaguchi-Shinozaki, K., Shinozaki, K. & Tran, L-S.P. (2012). Cytokinins: metabolism and function in plant adaptation to environmental stresses. Trends Plant Science, 17, No. 3, pp. 172-179. https://doi.org/10.1016/j.tplants.2011.12.005
13. Diopan, V., Adam, V., Havel, L. & Kizek, R. (2009). Phytohormones as important biologically active molecules - their simple simultaneous detection. Molecules, 14, No. 5, pp. 1825-1839. https://doi.org/10.3390/molecules14051825
14. Romanov, G.A., Lomin, S.N. & Schmulling, T. (2018). Cytokinin signaling: from the ER or from the PM? That is the question! New Phytologist, 218, No. 1, pp. 41-53. https://doi.org/10.1111/nph.14991
15. Frebort, I., Kowalska, M., Hluska, T., Frebortova, J. & Galuszka, P. (2011). Evolution of cytokinin biosynthesis and degradation. J. Exp. Bot., 62, No. 8, pp. 2431-2452. https://doi.org/10.1093/jxb/err004
16. Vedenicheva, N.P. & Kosakivska, I.V. (2016). Modern aspects of cytokinins studies: evolution and crosstalk with other phytohormones. Fiziol. rast. genet., 48, No. 1, pp. 3-19 [in Ukrainian]. https://doi.org/10.15407/frg2016.01.003
17. Sakakibara, H. (2006). Cytokinins: Activity, biosynthesis and translocation. Ann. Rev. Plant Biology, 57, pp. 431-449. https://doi.org/10.1146/annurev.arplant.57.032905.105231
18. Voytenko, L.V. & Kosakivska, I.V. (2016). Polyfunctional phytohormone abscisic acid. The Bulletin of Kharkiv national agrarian university, 1, No. 37. pp. 27-41 [in Ukrainian].
19. Chernyad'ev, I.I. (2009). The protective action of cytokinins on the photosynthetic machinery and productivity of plants under stress (review). Appl. Biochem. Microbiol., 45, pp. 351-362. https://doi.org/10.1134/S0003683809040012
20. Cutler, S.R., Rodriguez, P.L., Finkelstein, R.R. & Abrams, S.R. (2010). Abscisic Acid: Emergence of a Core Signaling Network. Ann. Rev. Plant Biol., 61, pp. 651-679. https://doi.org/10.1146/annurev-arplant-042809-112122
21. Dobrev, P.I. & Vankova, R. (2012). Quantification of Abscisic Acid, Cytokinin, and Auxin Content in Salt-Stressed Plant Tissues. Shabala, S., Cuin, T. (Eds.) Plant Salt Tolerance (Methods and Protocols), Humana Press, Totowa, NJ, pp. 251-261. https://doi.org/10.1007/978-1-61779-986-0_17
22. Netting, A.G. (2000). pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations. J. Exp. Bot., 51, No. 343, pp. 147-158. https://doi.org/10.1093/jexbot/51.343.147
23. Tarkowska, D., Novak, O., Flokova, K., Tarkowski, P., Tureckova, V., Gruz, J., Rolcik, J. & Strnad, M. (2014). Quo vadis plant hormone analysis? Planta, 240, pp. 55-76. https://doi.org/10.1007/s00425-014-2063-9
24. Kurakawa, T., Ueda, N.; Maekawa, M., Kobayashi, K., Kojima, M., Nagato, Y., Sakakibara, H. & Kyozuka, J. (2007). Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature, 445, No. 7128, pp. 652-655. https://doi.org/10.1038/nature05504
25. Takei, K., Yamaya, T. & Sakakibara, H. (2004). Arabidopsis CYP735A1 and CYP735A2 encode cytokinin hydroxylases that catalyze the biosynthesis of trans-zeatin. J. Biol. Chem., 279, No. 40, pp. 41866-41872. doi: 10.1074/jbc.M406337200 https://doi.org/10.1074/jbc.M406337200
26. Mok, D.W.S. & Martin, R.C. (1994). Cytokinin metabolic enzymes. Mok, D.W.S., Mok, M.C. (Eds.) Cytokinins: chemistry, activity and function. Boca Raton: CRC Press, pp. 129-137. https://doi.org/10.1201/9781351071284-10
27. Kudo, T., Makita, N., Kojima, M., Tokunaga, H. & Sakakibara, H. (2012). Cytokinin activity of cis-zeatin and phenotypic alterations induced by overexpression of putative cis-zeatin-O-glucosyltransferase in rice. Plant Physiol., 160, pp. 319-331. https://doi.org/10.1104/pp.112.196733
28. Takagi, M., Yokota, T., Murofushi, N., Ota, Y. & Takahashi, N. (1985). Fluctuation of endogenous cytokinin contents in rice during its life cycle - quantification of cytokinins by selected ion monitoring using deuterium-labelled internal standards. Agr. Biol. Chem., 49, pp. 3271-3277. https://doi.org/10.1271/bbb1961.49.3271
29. Vyroubalova, S., Vaclavikova, K., Tureckova, V., Novak, O., Smehilova, M., Hluska, T., Ohnoutkova, L., Frebort, I. & Galuszka, P. (2009). Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels. Plant Physiol., 151, No. 1, pp. 433-447. https://doi.org/10.1104/pp.109.142489
30. Suttle, J.C. & Banowetz, G.M. (2000). Changes in cis-zeatin and cis-zeatin riboside levels and biological activity during potato tuber dormancy. Physiol. Plantarum., 109, No. 1, pp. 68-74. https://doi.org/10.1034/j.1399-3054.2000.100110.x
31. Emery, R.J.N., Leport, L., Barton, J.E., Turner, N.C. & Atkins, C.A. (1998). Cisisomers of cytokinins predominate in chickpea seeds throughout their development. Plant Physiol., 117, No. 4, pp. 1515-1523. https://doi.org/10.1104/pp.117.4.1515
32. Emery, R.J.N., Ma, Q. & Atkins, C.A. (2000). The forms and sources of cytokinins in developing white lupine seeds and fruits. Plant Physiol., 123, No. 4, pp. 1593-1604. https://doi.org/10.1104/pp.123.4.1593
33. Quesnelle, P.E. & Emery, R.J.N. (2007). Cis-cytokinins that predominate in Pisum sativum during early embryogenesis will accelerate embryo growth in vitro. Can. J. Bot., 85, pp. 91-103. https://doi.org/10.1139/b06-149
34. Dobra, J., Motyka, V., Dobrev, P., Malbeck, J., Prasil, I.T., Haisel, D., Gaudinova, A., Havlova, M., Gubis, J. & Vankova, R. (2010). Comparison of hormonal responses to heat, drought and combined stress in tobacco plants with elevated proline content. J. Plant Physiol., 167, No. 16, pp. 1360-1370. doi: 10.1016/j.jplph.2010.05.013 https://doi.org/10.1016/j.jplph.2010.05.013
35. Strnad, M. (1997). The aromatic cytokinins. Physiol. Plantarum, 101, No. 4, pp. 674-688. https://doi.org/10.1111/j.1399-3054.1997.tb01052.x
36. Galuszka, P., Frebort, I., Sebela, M., Sauer, P., Jacobsen, S., & Pec, P. (2001). Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals. Eur. J. Biochem., 268, No. 2, pp. 450-461. doi: 10.1046/j.1432-1033.2001.01910.x https://doi.org/10.1046/j.1432-1033.2001.01910.x
37. Imbault, N., Moritz, T., Nilsson, O., Chen, H-J., Bollmark, M. & Sandberg, G. (1993). Separation and identification of cytokinins using combined capillary liquid-chromatography mass-spectrometry. Biol. Mass Spectrom., 22, No. 3, pp. 201-210. https://doi.org/10.1002/bms.1200220310
38. Astot, C., Dolezal, K., Moritz, T. & Sandberg, G. (1998). Precolumn derivatization and capillary liquid chromatographic/frit-fast atom bombardment mass spectrometric analysis of cytokinins in Arabidopsis thaliana. J. Mass Spectrom., 33, No. 9, pp. 892-902. https://doi.org/ 10.1002/(SICI)1096-9888(199809)33:9<892::AID-JMS701>3.0.CO;2-N https://doi.org/10.1002/(SICI)1096-9888(199809)33:9<892::AID-JMS701>3.0.CO;2-N
39. Nordstrom, A., Tarkowski, P., Tarkowska, D., Dolezal, K., Astot, C., Sandberg, G. & Moritz, T. (2004). Derivatization for LC-Electrospray Ionization-MS: a tool for improving reversed-phase separation and ESI responses of bases, ribosides, and intact nucleotides. Anal. Chem., 76, pp. 2869-2877. https://doi.org/10.1021/ac0499017
40. Novak, O., Tarkowski, P., Tarkowska, D., Dolezal, K., Lenobel, R. & Strnad, M. (2003.) Quantitative analysis of cytokinins in plants by liquid chromatography-single-quadrupole mass spectrometry. Anal. Chim. Acta., 480, pp. 207-218. https://doi.org/10.1016/S0003-2670(03)00025-4
41. Prinsen, E., Redig, P., Van Dongen, W., Esmans, E.L. 7 Van Onckelen, H.A. (1995). Quantitative analysis of cytokinins by electrospray tandem mass spectrometry. Rapid Commun. Mass Spectrom., 9, No. 10, pp. 948-953. https://doi.org/10.1002/rcm.1290091016
42. Witters, E., Vanhoutte, K., Dewitte, W., Machackova, I., Benkova, E., Van Dongen, W., Esmans, E.L. & Van Onckelen, H.A. (1999). Analysis of cyclic nucleotides and cytokinins in minute plant samples using phase-system switching capillary electrospray-liquid chromatography-tandem mass spectrometry. Phytochem. Anal., 10, No. 3, pp. 143-151. https://doi.org/10.1002/(SICI)1099-1565(199905/06)10:3<143::AID-PCA441>3.0.CO;2-G
43. Yang, Y.Y., Yamaguchi, I., Kato, Y., Weiler, E.W., Murofuchi, N. & Takahashi, N. (1993). Qualitative and semi-quantitative analyses of cytokinins using LC/APCI-MS in combination with ELISA. J. Plant. Growth. Regul., 12, No. 21, pp. 21-25. https://doi.org/10.1007/BF00193674
44. Farrow, S.C. & Emery, R.J.N. (2012). Concurrent profiling of indole-3-acetic acid, abscisic acid, and cytokinins and structurally related purines by high-performance-liquid chromatography tandem electrospray mass spectrometry. Plant Methods., 8, p. 42. https://doi.org/10.1186/1746-4811-8-42
45. Muller, M. & Munne-Bosch, S. (2011). Rapid and Sensitive Hormonal Profiling of Complex Plant Samples by Liquid Chromatography Coupled to Electrospray Ionization Tandem Mass Spectrometry. Plant Methods, 7, pp. 37. https://doi.org/10.1186/1746-4811-7-37
46. Novak, O., Hauserova, E., Amakorova, P., Dolezal, K. & Strnad, M. (2008). Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry, 69, No. 11, pp. 2214-2224. https://doi.org/10.1016/j.phytochem.2008.04.022
47. Campell, B.R. & Town, C.D. (1991). Physiology of hormone autonomous tissue lines derived from radiation-induced tumors of Arabidopsis thaliana. Plant Physiol., 97, No. 3, pp. 1166-1173. https://doi.org/10.1104/pp.97.3.1166
48. Chory, J., Reinecke, D., Sim, S., Washburn, T. & Brenner, M. (1994). A role for cytokinins in de-etiolation in Arabidopsis (Det mutants have an altered response to cytokinins). Plant Physiol., 104, No. 2, pp. 339-347. https://doi.org/10.1104/pp.104.2.339
49. Nicander, B., Stahl, U., Bjorkman, P.O. & Tillberg, E. (1993). Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet spectrum detection. Planta, 189, pp. 312-320. https://doi.org/10.1007/BF00194427
50. Pacakova, V., Stulik, K., Vlasakova, V. & Brezinova, A. (1997). Capillary electrophoresis of cytokinins and cytokinin ribosides. J. Chromatography A, 764, pp. 331-335. https://doi.org/10.1016/S0021-9673(96)00927-2
51. Beres, T., Gemrotova, M., Tarkowski, P., Ganzera, M., Maier, V., Friedecky, D., Dessoyf, M.A., Wessjohannf, L.A., Spichal, L., Strnad, M. & Dolezal, K. (2012). Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction. Anal. Chim. Acta, 751, pp. 176-181. https://doi.org/10.1016/j.aca.2012.08.049
52. Ge, L., Yong, J.W., Tan, S.N. & Ong, E.S. (2006). Determination of cytokinins in coconut (Cocos nucifera L.) water using capillary zone electrophoresis-tandem mass spectrometry. Electrophoresis, 27, pp. 2171-2181. https://doi.org/10.1002/elps.200500465
53. Hernandez, L., Hernandez, P., Rica, M. & Galan, F. (1995). Determination of zeatin in plant extracts by square wave stripping polarography and differential pulse stripping polarography. Anal. Chim. Acta, 315, pp. 33-39. https://doi.org/10.1016/0003-2670(95)00290-G
54. Huskova, R., Pechova, D., Kotoucek, M. & Lemr, K. (2000). Voltammetric behavior and determination of some cytokinines on mercury electrode. Chem. Listy, 94, pp. 1004-1009.
55. Tarkowska, D., Kotoucek, M. & Dolezal, K. (2003). Electrochemical reduction of 6-benzylaminopurine at mercury electrodes and its analytical application. Collect. Czech Chem. Commun., 68, pp. 1076-1093. https://doi.org/10.1135/cccc20031076
56. Liu, Z., Wei, F. & Feng, Y.-Q. (2010). Determination of cytokinins in plant samples by polymer monolith microextraction coupled with hydrophilic interaction chromatography-tandem mass spectrometry. Anal. Methods, 2, pp. 1676-1685. https://doi.org/10.1039/c0ay00334d
57. Liu, X., Hegeman, A.D., Gardner, G. & Cohen, J.D. (2012). Protocol: high-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples. Plant Methods, 8, pp. 31. https://doi.org/10.1186/1746-4811-8-31
58. Svacinova, J., Novak, O., Plackova, L., Lenobel, R., Holik, J., Strnad, M. & Dolezal, K. (2012). A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction. Plant Methods, 8, p. 17. https://doi.org/10.1186/1746-4811-8-17
59. Liu, Z., Yuan, B.-F., Feng, Y.-Q. (2012). Tandem solid phase extraction followed by online trapping-hydrophilic interaction chromatography-tandem mass spectrometry for sensitive detection of endogenous cytokinins in plant tissues. Phytochem. Anal., 23, pp. 559-568. https://doi.org/10.1002/pca.2353
60. Du, F., Ruan, G., Liang, S., Xie, F. & Liu, H. (2012). Monolithic molecularly imprinted solid-phase extraction for the selective determination of trace cytokinins in plant samples with liquid chromatography-electrospray tandem mass spectrometry. Anal. Bioanal. Chem., 404, No. 2, pp. 489-501. https://doi.org/10.1007/s00216-012-6131-3
61. Ge, L.Y., Tan, S., Yong, J.W.H. & Tan, S.N. (2006). CE for cytokinin analyses: A review. Electrophoresis, 27, pp. 4779-4791. https://doi.org/10.1002/elps.200600195
62. Liu, J. & Li, S.F.Y. (1996). Separation and determination of auxins by capillary electrophoresis. J. Liq. Chromatogr. Relat. Technol., 19, pp. 1697-1713. https://doi.org/10.1080/10826079608013998
63. Olsson, J.C., Andersson, P.E., Karlberg, B. & Nordstrom, A.C. (1996). Determination of plant indoles by capillary electrophoresis with amperometric detection. J. Chromatography A, 755, No. 2, pp. 289-298. https://doi.org/10.1016/S0021-9673(96)00599-7
64. Zheng, B., Yang, H.X. & He, J.L. (1999). Quantitative analysis of plant hormones with capillary electrophoresis. Chin. J. Anal. Chem., 27, pp. 704-707.
65. Guidelines for the determination of phytohormones (1988). Kiev: Naukova Dumka [in Russian].
66. Kosakivska, I.V., Shcherbatiuk, M.M. & Voytenko, L.V. (2020). Profiling of hormones in plant tissues: history, modern approaches, use in biotechnology. Biotechnol Acta., 13, No. 4, pp. 14-25. https://doi.org/10.15407/biotech13.04.014
67. Shcherbatiuk, M.M., Voytenko, L.V., Vasyuk, V.A. & Kosakivska, I.V. (2020). Method for quantitative determination of phytohormones in plant tissues. Biol. Stud., 14, No. 2, pp. 117-136. [in Ukrainian]. https://doi.org/10.30970/sbi.1402.624
68. Sheikhian, L. & Bina, S. (2016). Simultaneous extraction and HPLC determination of 3-indole butyric acid and 3-indole acetic acid in pea plant by using ionic liquid-modified silica as sorbent. J. Chromatography B, 1009-1010. pp. 34-43. https://doi.org/10.1016/j.jchromb.2015.11.047
69. Annesley, T.M. (2003). Ion suppression in mass spectrometry. Clinical Chemistry, 49, pp. 1041-1044. https://doi.org/10.1373/49.7.1041