Cytokinins are plant hormones, derivatives of adenine with a side chain at the N6-position. They are involved in many physiological processes. While the metabolism of trans-zeatin and isopentenyladenine, which are considered to be highly active cytokinins, has been extensively studied, there are others with less obvious functions, such as cis-zeatin, dihydrozeatin, and aromatic cytokinins, which have been comparatively neglected. To help explain this duality, we present a novel hypothesis metaphorically comparing various cytokinin forms, enzymes of CK metabolism, and their signalling and transporter functions to the comics superheroes Hulk and Deadpool. Hulk is a powerful but short-lived creation, whilst Deadpool presents a more subtle and enduring force. With this dual framework in mind, this review compares different cytokinin metabolites, and their biosynthesis, translocation, and sensing to illustrate the different mechanisms behind the two CK strategies. This is put together and applied to a plant developmental scale and, beyond plants, to interactions with organisms of other kingdoms, to highlight where future study can benefit the understanding of plant fitness and productivity.

• Klíčová slova
• Hulk/Deadpool, aromatic cytokinins, cis-zeatin, cytokinin biosynthesis, cytokinin oxidase/dehydrogenase, cytokinin signalling, cytokinin transport, cytokinins, isopentenyl transferase,
• MeSH
• Arabidopsis metabolismus   MeSH
• biologické modely MeSH
• biologický transport MeSH
• biotest MeSH
• cytokininy metabolismus   MeSH
• fyziologie rostlin * MeSH
• glykosylace MeSH
• hydrolýza MeSH
• kinetika MeSH
• kinetin metabolismus   MeSH
• oxidoreduktasy metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostliny metabolismus   MeSH
• signální transdukce * MeSH
• vazba proteinů MeSH
• zeatin analogy a deriváty   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• dihydrozeatin MeSH Prohlížeč
• kinetin MeSH
• oxidoreduktasy MeSH
• regulátory růstu rostlin MeSH
• zeatin MeSH

Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

• Klíčová slova
• Arabidopsis, N-glucosides, Zea mays, [2-3H]tZ9G, cis-zeatin, cytokinin, cytokinin oxidase/dehydrogenase, oat, senescence, trans-zeatin, β-D-glucosidase Zm-p60.1,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• cytokininy genetika   MeSH
• glukosidy genetika   MeSH
• glukosyltransferasy genetika   MeSH
• kukuřice setá genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• oxidoreduktasy genetika   MeSH
• regulace genové exprese u rostlin genetika   MeSH
• regulátory růstu rostlin genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• glukosidy MeSH
• glukosyltransferasy MeSH
• oxidoreduktasy MeSH
• regulátory růstu rostlin MeSH

Modifying the cytokinin content in plants is a means of improving plant productivity. Here, we report the development and biological activity of compound TD-K (1-(furan-2-ylmethyl)-3-(1,2,3-thiadiazol-5-yl)urea)which is related to thidiazuron. TD-K-which exhibited extremely high antisenescence activity in the wheat leaf bioassay-and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine)-a plant growth regulator reported to inhibit cytokinin oxidase/dehydrogenase (CKX), an enzyme involved in the degradation of the plant hormone cytokinin-were selected for investigation of their effects on the model plant Rapid Cycling Brassica rapa (RCBr). We monitored the expression of BrCKX and isopentenyl transferase (BrIPT), which codes for the key cytokinin biosynthesis enzyme, in developing leaves following INCYDE and TD-K application. Growth room experiments revealed that INCYDE increased RCBr seed yield per plant, but only when applied multiple times and when grown in 5 mM KNO3. Expression in control leaves showed transient, high levels of expression of BrCKX and BrIPT at true leaf appearance. Following INCYDE application, there was a rapid and strong upregulation of BrCKX3, and a transient downregulation of BrIPT1 and BrIPT3. Interestingly, the upregulation of BrCKX3 persisted in a milder form throughout the course of the experiment (16 days). TD-K also upregulated BrCKX3. However, in contrast to INCYDE, this effect disappeared after two days. These results suggest that both compounds (CKX inhibitor and cytokinin TD-K) influenced cytokinin homeostasis in RCBr leaves, but with different mechanisms.

• Klíčová slova
• CKX, INCYDE, IPT, TD-K, cytokinin, cytokinin oxidase/dehydrogenase, isopentenyl transferase, thidiazuron,
• Publikační typ
• časopisecké články MeSH

Drought and salinity reduce seed germination, seedling emergence, and early seedling establishment, affect plant metabolism, and hence, reduce crop yield. Development of technologies that can increase plant tolerance of these challenging growth conditions is a major current interest among plant scientists and breeders. Seed priming has become established as one of the practical approaches that can alleviate the negative impact of many environmental stresses and improve the germination and overall performance of crops. Hormopriming using different plant growth regulators has been widely demonstrated as effective, but information about using cytokinins (CKs) as priming agents is limited to only a few studies using kinetin or 6-benzylaminopurine (BAP). Moreover, the mode of action of these compounds in improving seed and plant fitness through priming has not yet been studied. For many years, BAP has been one of the CKs most commonly applied exogenously to plants to delay senescence and reduce the impact of stress. However, rapid endogenous N 9-glucosylation of BAP can result in negative effects. This can be suppressed by hydroxylation of the benzyl ring or by appropriate N 9 purine substitution. Replacement of the 2' or 3' hydroxyl groups of a nucleoside with a fluorine atom has shown promising results in drug research and biochemistry as a means of enhancing biological activity and increasing chemical or metabolic stability. Here, we show that the application of this chemical modification in four new N 9-substituted CK derivatives with a fluorinated carbohydrate moiety improved the antisenescence properties of CKs. Besides, detailed phenotypical analysis of the growth and development of Arabidopsis plants primed with the new CK analogs over a broad concentration range and under various environmental conditions revealed that they improve growth regulation and antistress activity. Seed priming with, for example, 6-(3-hydroxybenzylamino)-2'-deoxy-2'-fluoro-9-(β)-D-arabinofuranosylpurine promoted plant growth under control conditions and alleviated the negative effects of the salt and osmotic stress. The mode of action of this hormopriming and its effect on plant metabolism were further analyzed through quantification of the endogenous levels of phytohormones such as CKs, auxins and abscisic acid, and the results are discussed.

• Klíčová slova
• Arabidopsis, abiotic stress, antisenescence, cytokinin analogs, hormopriming, plant biostimulant characterization index,
• Publikační typ
• časopisecké články MeSH

Cytokinin (CK) N-glucosides are the most abundant group of CK metabolites in many species; however, their physiological role in planta was for a long time perceived as irreversible storage CK forms only. Recently, a comprehensive screen showed that only vascular plants form CK N-glucosides in contrast to mosses, algae, and fungi. The formation of CK N-glucosides as biologically inactive CK conjugates thus represents an evolutionarily young mechanism for deactivation of CK bases. Even though CK N-glucosides are not biologically active themselves due to their inability to activate the CK perception system, new data on CK N-glucoside metabolism show that trans-zeatin (tZ) N7- and N9-glucosides are metabolized in vivo, efficiently releasing free CK bases that are most probably responsible for the biological activities observed in a number of bioassays. Moreover, CK N-glucosides' subcellular localization as well as their abundance in xylem both point to their possible plasma membrane transport and indicate a role also as CK transport forms. Identification of the enzyme(s) responsible for the hydrolysis of tZ N7- and N9-glucosides, as well as the discovery of putative CK N-glucoside plasma membrane transporter, would unveil important parts of the overall picture of CK metabolic interconversions and their physiological importance.

• Klíčová slova
• UGT, cytokinin N-glucoside, cytokinin metabolism, cytokinin transport, isopentenyladenine N7-glucoside, isopentenyladenine N9-glucoside, zeatin N7-glucoside, zeatin N9-glucoside,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies. Cytokinin hormones regulate a wide range of essential processes in plants. Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, TDZ), formerly registered as a cotton defoliant, is a well known inhibitor of cytokinin oxidase/dehydrogenase (CKX), an enzyme catalyzing the degradation of cytokinins. TDZ thus increases the lifetime of cytokinins and their effects in plants. We used in silico modeling to design, synthesize and characterize twenty new TDZ derivatives with improved inhibitory properties. Two compounds, namely 1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl)phenyl]-3-(1,2,3-thiadiazol-5-yl)urea (HETDZ), displayed up to 15-fold lower IC 50 values compared with TDZ for AtCKX2 from Arabidopsis thaliana and ZmCKX1 and ZmCKX4a from Zea mays. Binding modes of 3FMTDZ and HETDZ were analyzed by X-ray crystallography. Crystal structure complexes, solved at 2.0 Å resolution, revealed that HETDZ and 3FMTDZ bound differently in the active site of ZmCKX4a: the thiadiazolyl ring of 3FMTDZ was positioned over the isoalloxazine ring of FAD, whereas that of HETDZ had the opposite orientation, pointing toward the entrance of the active site. The compounds were further tested for cytokinin activity in several cytokinin bioassays. We suggest that the combination of simple synthesis, lowered cytokinin activity, and enhanced inhibitory effects on CKX isoforms, makes 3FMTDZ and HETDZ suitable candidates for in vivo studies.

• Klíčová slova
• Crystal structure, Cytokinin, Cytokinin oxidase/dehydrogenase, Molecular docking, Organic synthesis, Thidiazuron,
• MeSH
• cytokininy metabolismus   MeSH
• fenylmočovinové sloučeniny chemie   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• oxidoreduktasy antagonisté a inhibitory   MeSH
• thiadiazoly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• fenylmočovinové sloučeniny MeSH
• inhibitory enzymů MeSH
• oxidoreduktasy MeSH
• thiadiazoly MeSH
• thidiazuron MeSH Prohlížeč

Plant hormones cytokinins (CKs) are one of the major mediators of physiological responses throughout plant life span. Therefore, a proper homeostasis is maintained by regulation of their active levels. Besides degradation, CKs are deactivated by uridine diphosphate glycosyltransferases (UGTs). Physiologically, CKs active levels decline in senescing organs, providing a signal to nutrients that a shift to reproductive tissues has begun. In this work, we show CK glucosides distribution in Arabidopsis leaves during major developmental transition phases. Besides continuous accumulation of N-glucosides we detected sharp maximum of the glucosides in senescence. This is caused prevalently by N7-glucosides followed by N9-glucosides and specifically also by trans-zeatin-O-glucoside (tZOG). Interestingly, we observed a similar trend in response to exogenously applied CK. In Arabidopsis, only three UGTs deactivate CKs in vivo: UGT76C1, UGT76C2 and UGT85A1. We thereby show that UGT85A1 is specifically expressed in senescent leaves whereas UGT76C2 is activated rapidly in response to exogenously applied CK. To shed more light on the UGTs physiological roles, we performed a comparative study on UGTs loss-of-function mutants, characterizing a true ugt85a1-1 loss-of-function mutant for the first time. Although no altered phenotype was detected under standard condition we observed reduced chlorophyll degradation with increased anthocyanin accumulation in our experiment on detached leaves accompanied by senescence and stress related genes modulated expression. Among the mutants, ugt76c2 possessed extremely diminished CK N-glucosides levels whereas ugt76c1 showed some specificity toward cis-zeatin (cZ). Besides tZOG, a broader range of CK glucosides was decreased in ugt85a1-1. Performing CK metabolism gene expression profiling, we revealed that activation of CK degradation pathway serves as a general regulatory mechanism of disturbed CK homeostasis followed by decreased CK signaling in all UGT mutants. In contrast, a specific regulation of CKX7, CKX1 and CKX2 was observed for each individual UGT mutant isoform after exogenous CK uptake. Employing an in silico prediction we proposed cytosolic localization of UGT76C1 and UGT76C2, that we further confirmed by GFP tagging of UGT76C2. Integrating all the results, we therefore hypothesize that UGTs possess different physiological roles in Arabidopsis and serve as a fine-tuning mechanism of active CK levels in cytosol.

• Klíčová slova
• Arabidopsis, GFP subcellular localization, cytokinin, glycosyltransferase, senescence,
• Publikační typ
• časopisecké články MeSH

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

• MeSH
• biologická evoluce MeSH
• cytokininy metabolismus   MeSH
• Escherichia coli enzymologie   růst a vývoj   MeSH
• fylogeneze MeSH
• geneticky modifikované rostliny genetika   růst a vývoj   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• Nostoc enzymologie   genetika   MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• prenyltransferáza genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• rekombinantní proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• tabák enzymologie   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• oxidoreduktasy MeSH
• prenyltransferáza MeSH
• rekombinantní proteiny MeSH

Cytokinins (CKs) are well-established as important phytohormonal regulators of plant growth and development. An increasing number of studies have also revealed the function of these hormones in plant responses to biotic and abiotic stresses. While the function of certain CK classes, including trans-zeatin and isopentenyladenine-type CKs, have been studied in detail, the role of cis-zeatin-type CKs (cZs) in plant development and in mediating environmental interactions is less well defined. Here we provide a comprehensive summary of the current knowledge about abundance, metabolism and activities of cZs in plants. We outline the history of their analysis and the metabolic routes comprising cZ biosynthesis and degradation. Further we provide an overview of changes in the pools of cZs during plant development and environmental interactions. We summarize studies that investigate the role of cZs in regulating plant development and defence responses to pathogen and herbivore attack and highlight their potential role as 'novel' stress-response markers. Since the functional roles of cZs remain largely based on correlative data and genetic manipulations of their biosynthesis, inactivation and degradation are few, we suggest experimental approaches using transgenic plants altered in cZ levels to further uncover their roles in plant growth and environmental interactions and their potential for crop improvement.

• Klíčová slova
• Abiotic stress, c-io6A37-tRNA, cis-zeatin, herbivory, pathogen, plant growth, prenylated tRNA.,
• MeSH
• býložravci MeSH
• fyziologie rostlin * MeSH
• potravní řetězec * MeSH
• regulace genové exprese u rostlin * MeSH
• regulátory růstu rostlin metabolismus   MeSH
• vývoj rostlin MeSH
• zeatin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• regulátory růstu rostlin MeSH
• zeatin MeSH