J Plant Growth Regul (2015) 34:723–739 DOI 10.1007/s00344-015-9543-4 Tracking the Story of Cytokinin Research Miroslav Kamı´nek1 Received: 9 June 2015 / Accepted: 14 September 2015 / Published online: 12 October 2015 Ó Springer Science+Business Media New York 2015 Abstract Existence of compound(s) inducing cell division in Monitoring the history of research in a special field of excised plant tissues was spotted more than 100 years ago. plant science represents a great adventure. It allows con- Since then research of cytokinins (CKs), plant hormones which frontation of the presumptions and speculations of scien- in cooperation with other phytohormones, namely auxin, con- tists about the role of what at that time were new findings trol cytokinesis, and a number of other physiological processes and the attempts of these scientists to fit their discoveries in plants, has advanced correspondingly to the progress in other into proposed functional system(s). In contrast to reviews fields of life sciences. This historical overview is focused on focused on the actual state of knowledge, the historical major topics of CK research including (1) discovery of CKs, (2) review gives us an opportunity to appreciate the power of search for natural CKs, (3) role of CKs in transfer RNA, (4) intellectual factors in the process of uncovering the secrets biosynthesis (5) metabolism, (6) signaling of CKs, and (7) of plant life. molecular probing of the physiological functions of CKs. Some Cytokinins (CKs) are a distinguished class of plant parts of these subjects can already be assessed within the hormones that in interaction/cooperation with other hor- context of an appropriate time span necessary for critical mones, especially auxin, control cell division, and a num- evaluation. I have used this opportunity to present also some ber of other fundamental biological processes, including personal recollections, namely those of Prof Folke Skoog, in lateral shoot formation, leaf senescence, formation and whose laboratory the first CK, kinetin, was discovered. strength of metabolic sinks, and others (see Mok 1994). Those occurring naturally are adenine derivatives bearing Keywords Cytokinin Á History Á Plant hormones Á either an isoprenoid or a ring substituent at the N6 position t-RNA Á Cytokinin biosynthesis Á Cytokinin metabolism (Fig. 1). Uncovering various aspects of CK formation, action and functions in plants has been a long-distance run, requiring application of various strategic and method- Introduction ological approaches. Taking this into account, as well as being limited by available space, this essay is focused only Oats, peas, beans and barley grow, on major topics of CK research. Some parts of these sub- Oats, peas, beans and barley grow, jects can already be assessed within the context of an Can you, or I, or anyone know appropriate time span necessary for critical evaluation. I How oats, peas, beans and barley grow? have used this opportunity to present also some personal (Old English Nursery Rhyme quoted by Went and recollections, namely those of Prof Folke Skoog. These are Thimann in ‘‘Phytohormones’’, 1937) not always related to science, but they may contribute to an understanding of this interesting person. & Miroslav Kamı´nek The story of CK research involves a long period of [email protected] presentiment of their existence, followed by identification of the chemical structures of kinetin and the first natural 1 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova 135, 165 02 Prague 6, CK, trans-zeatin (transZ). Biological functions of CKs Czech Republic have been gradually recognized and since the chemical 123 724 J Plant Growth Regul (2015) 34:723–739 Fig. 2 Prof. Folke Skoog (Symposium Physiology and Biochemistry of Cytokinins in Plants, Liblice, Czech Republic 1990) hormone, and the term ‘‘plant hormone’’ is used rather as a matter of convenience not only in this essay. The active cell division factor was identified 42 years later in the laboratory of Prof Folke Skoog at the University of Wisconsin, Madison, USA. Skoog became interested in this problem during studies of the stimulatory effects of external chemical factors on the initiation and growth of callus and buds in tobacco stem cuttings (Fig. 2). It was found that addition of adenine to the culture medium reversibly counteracted the inhibiting effect of auxin on bud formation and also enhanced growth of callus in the presence of auxin (Skoog and Tsui 1948, 1951). This effect could be related to limited but detectable incorporation of Fig. 1 Structures of kinetin and basic natural cytokinins radiolabeled adenine into CKs (Chen and Petschow 1978; Dickinson and others 1986). For a number of reasons, nature of CKs was determined, their biosynthesis, meta- tobacco stem pith (consisting of parenchyma cells without bolism, translocation, and signaling in plants have been to a vascular tissues), as well as the corresponding callus tis- great extent resolved. Moreover, the research of CKs pro- sues, appeared to be much more uniform materials than vided a number of direct or associated applications, not stem segments for testing the effects of exogenous chem- only in agriculture and plant biotechnology but also in ical factors on growth and differentiation of plant tissues. pharmacology and beauty cosmetics. Experiments with tobacco stem pith tissues showed that auxin was capable of inducing mitosis unaccompanied by Discovery of Cytokinins cytokinesis (Naylor and others 1954). Cell division was achieved by the addition of certain natural products such as The first experimental indication of the existence of CKs coconut milk (introduced by van Overbeek and others was reported by the intellectual aristocrat Gottlieb Haber- 1941) to mineral medium containing auxin. Evidently these landt (1913), who noted that non-dividing potato par- products contained bioactive factor(s) that in the presence enchyma cells can be induced to divide in the presence of of auxin were capable of maintaining continuous cell phloem sap from various plant species. He suggested that a division. The stage for the hunt for the ‘‘elixir’’ of cell soluble material occurring in phloem was responsible for proliferation was set. the induction of cell division. In accordance with what was A substantial role in this chase was played by Carlos then the new concept of hormones as specific chemical Miller, a new post-doctorate fellow who joined Skoog’s signals coordinating activities and growth of different parts laboratory after receiving his PhD at Ohio State University. of the animal body (Starling 1905), Haberlandt assumed He found that an active factor, which was present in some that the inducing factor is of hormonal nature. Actually, preparations of yeast extract, could be precipitated by sil- CKs do not meet the technical criteria of the definition of a ver nitrate, indicating that it might be a purine or 123 J Plant Growth Regul (2015) 34:723–739 725 pyrimidine derivative. Considering the reported positive Availability of kinetin allowed preparation of com- effects of adenine on bud formation and callus growth, the pletely defined culture media for continuous cultivation of alternative that the growth factor could be a purine was plant tissues under in vitro conditions. To optimize the preferred. Testing of various purine derivatives and system, the mineral and organic components of the medium potential sources of purine compounds showed that com- of White (White 1943), commonly used at that time, were mercial preparations of herring sperm DNA, either old or fundamentally modified. The revised medium (Murashige artificially ‘‘aged’’ by autoclaving under acidic conditions, and Skoog 1962 and Linsmaier and Skoog 1965) was exhibited high cell division promoting activity. Following designated for use in bioassays of organic growth-pro- extraction of autoclaved DNA preparations with n-butanol moting compounds. However, it has been widely used not at pH 6.8, evaporation of the butanol fraction, and passing only for testing biological activity of CKs but also in plant the residual solution through the cation-exchanger Dowex biotechnology as indicated by the enormously high citation 50 (H?), the active compound was eluted by 1.5 N HCl. records, according to my knowledge, the highest in plant Entrapment of pooled active fractions on a similar column biology (39,334 and 2958, respectively, to June 5th, 2015 and elution with 1 N ammonium hydroxide provided the in WOS database). active compound in a crystalline form that was visible moving down the column. Its chemical characteristic and Search for Natural Cytokinin(s) properties, including the empirical chemical formula and an equivalent molecular weight, were determined with the Cytokinins Bearing N6-Isopentenyl Side Chain aid of Prof Frank Strong and associates from the Depart- ment of Biochemistry (Miller and others 1955a, b). Pre- Kinetin represented a powerful tool for investigation of cisely, the first crystals of kinetin were isolated on structure–activity relations and the biological functions of December 16, 1954 (Amasino 2005). The paper reporting CKs. However, it was not a natural plant hormone but a the isolation and chemical properties of the compound that secondary oxidative product of DNA (Hall and De Ropp was named kinetin with respect to its ability to promote 1955; Barciszewski and others 1997). The search for nat- cytokinesis was submitted to the Journal of the American ural cell division stimulant(s) was already under way in the Chemical Society in January 1955 (Miller and others laboratory of Prof F. C. Steward at Cornell University prior 1955a). On the basis of the chemical properties and reac- to the discovery of kinetin. In fact, the unwillingness of tivity of the isolated compound with different reagents, Steward to provide information about the procedure Miller correctly proposed the chemical structure of kinetin employed in his laboratory for purification of coconut milk as 6-furfurylaminopurine, which was confirmed by syn- prior to its use as a component of culture media evoked thesis (Miller and others 1955b; for details see Skoog 1994 Skoog to focus on a search for the ‘‘cell division factor’’ and Amasino 2005).