Cytokinin metabolism is very complex and reflects the existence of dozens of native compounds found in plant tissues (Fig.1). Over the last few decades, the study of cytokinin metabolic pathways has focused almost exclusively on „active“ cytokinin bases that, however, represent only a small portion of these compounds. More than twenty years ago, we proposed a simplified model illustrating metabolic regulation of „active“ cytokinin levels in plant cells and elucidating fundamental mechanisms affecting hormonal homeostasis subsequently leading to induction of specific morphogenic processes and further development and termination of induced physiological events (Kamínek et al., 1997). Only recently, in collaboration with the Group of Mathematic Modelling we contributed to the introduction of a complex model of cytokinin metabolic conversions comprising all so far known enzymatic pathways identified for cytokinins (including also „non-active“ forms such as ribosides, N– and O-glucosides, cis-zeatins, etc.) and providing estimates of crucial metabolic reaction rates in Arabidopsis thaliana (Hošek et al., online first). Parameters of this model are further optimized and its applicability is considered to probe different hypotheses associated with cytokinin biosynthesis and metabolism in plants.

Integrating phylogenetic, physiological, biochemical and molecular approaches, we have performed as yet the most comprehensive characterization of specific and so far rather overlooked and ignored CK forms, cis-zeatins.  We have shown that cis-zeatin-type cytokinins occur ubiquitously throughout the plant kingdom in cyanobacteria, algae (Žižková et al. 2017), mosses (Záveská Drábková et al. 2015) as well as in vascular plants including ferns (Zemanová et al. 2019) and seed plants (Gajdošová et al. 2011). We have demonstrated involvement of cis-zeatins in control of numerous plant developmental processes and environmental responses; theirputative physiological functions have been presented indicating regulation of plant development (Stirk et al. 2012a, b) and modulation of plant defense responses against abiotic stresses (Havlová et al. 2008; Dobrá et al. 2010; Kosová et al. 2012; Macková et al. 2013) and pathogen infections (Behr and Motyka et al. 2012; Trdá et al. 2017). Altogether, we have implied that the cis-zeatins have much higher impact for cytokinin biology being more relevant and prevalent in plants than previously supposed.

Using analogous approaches, we have been studying numerous aspects of N-glucosyltransferase pathways that are supposed to be involved in irreversible inactivation of cytokinins. The products of this pathway, cytokinin N-glucosides, biosynthesized by specific glucosyltransferases encoded by UGT76C1 and UGT76C2 genes were found ubiquitous in seed plants in contrast to their mostly rather low levels or a total absence in non-vascular plants and ferns (Záveská Drábková et al. 2015; Žižková et al. 2017; Zemanová et al. 2019). We have identified and characterized orthologs of AtUGT76C1 and AtUGT76C2 in tomato (SlUGT76C1 and SlUGT76C2) and reported apparent effects of their overexpression on cytokinin homeostasis (Žižková et al. 2015). In contradiction to a generally accepted hypothesis that N-glucosylation inactivates cytokinins irreversibly we have shown cytokinin N-glucosides to be subject to metabolic conversions that differ between N7- and N9-glucosides in oats (Doležálková 2019, diploma thesis; Pokorná et al., under review) and between N-glucosides of N⁶-(∆²-isopentenyl)adenine and trans-zeatin in Arabidopsis (Hošek et al., online first).

Additionally, we have focused on evolutionary aspects of cytokinin O-glucosyltransferase pathway. In collaboration with the Laboratory of Pollen Biology, we have performed a phylogeny reconstruction of zeatin O-glucosyltranferase (ZOG) gene(s) that expanded during transition from algae to vascular plants. The phylogenetic analyses revealed that the ZOG gene seems to be unique in angiosperms, however, being absent in Arabidopsis thaliana and other members of order Brassicales (Záveská Drábková et al., under review).

Research in UGT76C1, UGT76C2 and ZOG evolution and involvement in hormonal homeostatic mechanisms as well as structural and functional manifestation of N– and O-glucosyltransferase pathways and their biological significance within evolution of higher plants are currently being a subject of further intensive study. As a part of this study, distinct priorities in glucosylation of trans-zeatin (glucosylated preferentially at the N9 position) and cis-zeatin (forming mainly O-glucosides) have been reported in maize in our collaborative work with colleagues from Palacký University in Olomouc (Hluska et al. 2016).

Over a long period, we have been exploring the role of cytokinin oxidase/dehydrogenase (CKX) catalysed down-regulating pathway in the complex process of hormonal homeostasis in plants (Motyka et al. 1996; Werner et al. 2001; Motyka et al. 2003; Werner et al. 2003; Gaudinová et al. 2005). Very recently we have unraveled, according to our knowledge for the first time in the fungal kingdom, the CKX activity in the pathogenic fungus Leptosphaeria maculans (Trdá et al. 2017). Using functional genomics, enzymatic and/or feeding assays we have also demonstrated activities of isopentenyl transferase in plants and fungi (Žižková et al. 2015; Trdá et al. 2017, respectively) and CK-related adenosine kinase in fungi (Trdá et al. 2017).

Using various model plants we have demonstrated a major role of cytokinins in plant-microbe interactions (Hann et al. 2014) and extended current knowledge concerning involvement of cytokinins (in association with other hormones) in control of somatic embryogenesis in conifers (Vondráková et al. 2018; Gautier et al. 2019), plant organogenesis (Ćosić and Motyka et al. 2015; Trifunović-Momčilov and Motyka et al. 2016; Danova et al. 2018) and in natural leaf senescence of wild-growing (Conrad et al. 2015) as well as in vitro cultured plants (Uzelac et al. 2016). Additionally, we have reported different tendencies in endogenous cytokinin and auxin levels in response to inoculation with arbuscular mycorrhizae compared to endophytic fungi (Schmidt et al. 2017).

In collaboration with the group of Prof. Lutts (Université catholique de Louvain, Belgium) we have significantly contributed to revealing molecular mechanisms engaged in hormonal control of plant development and plant responses to salinity. Using cultivated and wild relative tomato species (Solanum lycopersicum, Solanum chilense) we have demonstrated the involvement of phytohormones in these processes and specified the roles of SlIPT3 and SlIPT4 genes coding for CK biosynthesis (Žižková et al. 2015), transcription factors SlZF2, SlDREB2 and SlWRKY3 (Hichri et al. 2014, 2016 and 2017, respectively) and inorganic ions as osmotica (Gharbi et al. 2017) in salt resistance.Within this collaboration, we have also contributed to understanding phytohormone function in plant floral and fruit development (Quinet et al. 2014 and 2019, respectively) and in the transition from primary to secondary growth and lignification in the hemp plants (Behr et al. 2016 and 2019).

We highly appreciate the existing collaboration with other scientific institutions such as

• USDA-ARS-SASL, Beltsville Agricultural Research Center, Beltsville, MD, USA (Autar Mattoo); 
• Department of Biological Sciences, Auburn University, Auburn, AL, USA (Aaron Rashotte);
• Institut de la terre et de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium (Stanley Lutts, Muriel Quinet);    
• Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria (Kalina Danova);   
• Institute for Biological Research “Siniša Stanković”, University of Belgrade, Serbia (Dragan Vinterhalter, Ivana Dragićević)                                                           

and with other numerous colleagues in the Czech Republic as well as abroad.