Metabolism and physiological function of cytokinins
We proposed a simplified model illustrating metabolic regulation of cytokinins in plant cells. It elucidates mechanisms of establishment of transient imbalance in concentration ratios between cytokinins and other phytohormones, especially auxins, leading to induction of specific morphogenic processes (differentiation of organs) and subsequent re-establishment and maintenance of hormonal homeostasis required for further development and termination of induced physiological events (Kamínek et al., Physiologia Plantarum 1997; 2003). In collaboration with the Group of Mathematical Modelling we have recently also proposed a complex model of cytokinin metabolic conversions comprising all so far known enzymatic pathways identified for cytokinins in Arabidopsis thaliana plants (Fig.1). Parameters of this model are further optimized and its applicability is considered to probe different hypotheses associated with cytokinin biosynthesis and metabolism in plants.
Fig 1. Proposed model illustrating cytokinin metabolic conversions comprising all so far known enzymatic pathways identified for cytokinins in Arabidopsis thaliana plants (according to Hošek and Hoyerová). iP = N6-(∆2-isopentenyl)adenine; tZ = trans-zeatin; DHZ = dihydrozeatin; cZ = cis-zeatin; -R = –9-riboside; -RP = –9-riboside-5´-phosphate; -OG = -O-glucoside; -N7G = -N7-glucoside; -N9G = -N9-glucoside; DMAPP = dimethylallyl diphosphate; CKX = cytokinin oxidase/dehydrogenase, IPT = isopentenyl transferase; APT = adenine phosphoribosyltransferase; LOG = cytokinin phosphoribohydrolase „Lonely guy“; ADK = adenosine kinase; CYP = cytochrome P450 mono-oxygenase; UGT – N-glucosyltransferase
We have been verifying the validity of both models using in vitro cultures of plant cells and tissues as well as intact plants, in which the content of native cytokinins was experimentally modified either by exogenously applied natural or synthetic cytokinins (Motyka et al., Physiologia Plantarum 2003) or by expression of genes encoding enzymes participating in regulation of levels of precursors (farnesyl diphosphate synthase; Manzano et al., Plant Molecular Biology 2006), biosynthesis (isopentenyl transferase; Galichet and Hoyerová et al., Plant Physiology 2008; Sýkorová et al., Journal of Experimental Botany 2008; Yevdakova et al., J Plant Growth Regul 2008; Žižková et al., BMC Plant Biology 2015) , degradation (cytokinin oxidase/dehydrogenase; Werner et al., PNAS 2001 ; Werner et al., Plant Cell 2003; von Schwartzenberg et al., Plant Physiology 2007; Mýtinová and Motyka et al. Biologia Plantarum 2010; and Journal of Plant Growth Regulation 2011; Raspor and Motyka et al., Journal of Plant Growth Regulation 2012; Trifunović et al., Plant Cell Tissue and Organ Culture 2013; Trifunović and Motyka et al., Plant Cell Tissue and Organ Culture 2015) of cytokinins and cytokinin signal transduction (Frank et al., Plant Physiology 2000). Using mutants of model plants (moss Physcomitrella, Arabidopsis) we have also confirmed that phenotypic changes reflect proportions between cytokinin and auxin levels as well as changes in corresponding gene expression and enzyme activities (Yevdakova et al., Journal Plant Growth Regulation 2008; Dwivedi et al., Plant Growth Regulation 2010).
We also pay attention to the study of distribution, metabolism and physiological functions of so far rather overlooked and ignored group of cytokinins, cis-zeatins. We have shown the abundance of these cytokinin forms across the plant kingdom (Gajdošová and Spíchal et al., Journal of Experimental Botany 2011; Záveská Drábková et al., PLoS One 2015) and demonstrated their biological activities and metabolic conversions (Gajdošová and Spíchal et al., Journal of Experimental Botany 2011) as well as conceivable physiological functions in regulation of plant development (Stirk et al., Journal of Plant Growth Regulation 2012 a Journal of Plant Physiology 2012) and modulation of plant defense responses against abiotic stresses (Havlová et al., Plant Cell and Environment 2008; Dobrá et al., Journal of Plant Physiology 2010; Kosová et al., Journal of Plant Physiology 2012; Macková et al., Journal of Experimental Botany 2013) and pathogen infection (Behr and Motyka et al., Molecular Plant-Microbe Interactions 2012).
in collaboration with the Analytical Unit of the Laboratory of Hormonal Regulations in Plants we have developed a novel extraction and purification procedure allowing separation of auxin and abscisic acid from cytokinins (Dobrev a Kamínek, Journal of Chromatography A 2002) and their reliable identification and quantification by 2D-HPLC (Dobrev et al., Journal of Chromatography A 2005). We have evaluated efficiency of different methods of cytokinin extraction and purification and optimized the procedure by reducing the contents of interfering substances and thus enhancing responses of labelled internal standards during cytokinin quantification by mass spectrometry (Hoyerová et al., Phytochemistry 2006). We have been involved in miniaturization of the analytical procedure allowing simultaneous determination of a great number of phytohormones such as auxins, abscisic acid, salicylic acid, gibberellins and jasmonates (including their metabolites and precursors) together with cytokinins in very small quantities of plant samples (tens of milligrams) (Djilianov and Dobrev et al., Journal of Plant Growth Regulation 2013). We have also developed a method for simultaneous determination of cytokinin oxidase/dehydrogenase and zeatin reductase enzyme activities in plant tissues and clarified their roles in maintenance of hormonal homeostasis (Gaudinová et al., Journal of Plant Growth Regulation 2005).
Using various model plants we have extended knowledge concerning physiological significance of cytokinins in control of plant organogenesis (Sriskandarajah et al., Journal of Plant Growth Regulation 2006; Klemš et al., Plant Growth Regulation 2011; Ćosić and Motyka et al., Plant Cell Tissue and Organ Culture 2015), leaf senescence (Ananieva et al., Physiologia Plantarum 2004a; 2004b; 2008 and Plant Growth Regulation 2011; Conrad et al., Physiologia Plantarum 2007 and South African Journal of Botany 2015; Rubia et al., Journal of Plant Growth Regulation 2014; Uzelac et al., Protoplasma 2015), seed germination (Stirk et al., Journal of Plant Physiology 2012 and Journal of Plant Growth Regulation 2012) as well as in regulation of uptake and utilization of mineral nitrogen (Sýkorová et al., Journal of Experimental Botany 2008).
Together with other members of the Laboratory of Hormonal Regulations in Plants we presented a proposal of the system of abbreviations of purine cytokinins (Kamínek et al., Plant Growth Regulation 2000) and contributed to the preparation of the nomenclature for members of the two-component pathway in plants involved e.g. in cytokinin signaling (Heyl et al., Plant Physiology 2013).
In addition to the above mentioned activities our research is also focused on understanding and elucidation of potential control of cytokinin contents and activities in plants during the root-to-shoot transport (Kašík et al., Anal Bioanal Chem 2010), the floral organ development (Quinet et al., Journal of Experimental Botany 2014) and the formation and modifications of plant defense mechanisms against bacterial pathogens (Hann et al., New Phytologist 2014). Last but not least we are involved in exploration of regulatory functions of transcriptional factors in relation to phytohormones in control of plant growth, development and salinity response (Hichri et al., Plant Physiology 2014 and Plant Cell and Environment 2015). Attention is also paid to exploitation of our findings to increase resistance (Zhang et al., Journal of Integrative Plant Biology 2010) and productivity (Sýkorová et al., Journal of Experimental Botany 2008; Rubia et al., Journal of Plant Growth Regulation 2014) of crop plants.
We collaborate with many scientific institutions such as Institute of Plant Sciences, ETH Zurich, Switzerland (Prof. W. Gruissem); National Laboratory of Plant Molecular Genetics, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China (Prof. P. Zhang); Biozentrum Klein Flottbek, Universität Hamburg, Germany (Dr. K. von Schwartzenberg); Research Centre for Plant Growth and Development, University of KwalaZulu-Natal Pietermaritzburg, South Africa (Dr. W. Stirk, Prof. J. van Staden); Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria (Dr. K. Danova); Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria (Dr. K. Ananieva, Prof. E. D.Ananiev); Institut de la terre et de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium (Prof. S. Lutts, Dr. I. Hichri, Dr. M. Quinet); Institute for Biological Research, University of Belgrade, Serbia (Dr. I. Dragićević); Interdisziplinäres Zentrum für Nutzpflanzenforschung, Martin-Luther-Universität Halle-Wittenberg, Germany (Dr. S. Wirsel) and with other colleagues in the Czech Republic and abroad.