We have developed a N6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the N6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N6-(Δ2-isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM-1cm-2 (n = 4, R2 = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N6-(Δ2-isopentenyl) adenine, N6-(Δ2-isopentenyl) adenosine, cis-zeatin, trans-zeatin and trans-zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates.

• MeSH
• biosenzitivní techniky metody   MeSH
• cytokininy analýza   metabolismus   MeSH
• enzymy imobilizované chemie   metabolismus   MeSH
• isopentenyladenosin analogy a deriváty   analýza   metabolismus   MeSH
• kukuřice setá enzymologie   MeSH
• limita detekce MeSH
• oxidoreduktasy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Cytokinins are an important group of plant hormones that are also found in other organisms, including cyanobacteria. While various aspects of cytokinin function and metabolism are well understood in plants, the information is limited for cyanobacteria. In this study, we first experimentally confirmed a prenylation of tRNA by recombinant isopentenyl transferase NoIPT2 from Nostoc sp. PCC 7120, whose encoding gene we previously identified in Nostoc genome along with the gene for adenylate isopentenyl transferase NoIPT1. In contrast to NoIPT2, the transcription of NoIPT1 was strongly activated during the dark period and was followed by an increase in the cytokinin content several hours later in the light period. Dominant cytokinin metabolites detected at all time points were free bases and monophosphates of isopentenyladenine and cis-zeatin, while N-glucosides were not detected at all. Whole transcriptome differential expression analysis of cultures of the above Nostoc strain treated by cytokinin compared to untreated controls indicated that cytokinin together with light trigger expression of several genes related to signal transduction, including two-component sensor histidine kinases and two-component hybrid sensors and regulators. One of the affected histidine kinases with a cyclase/histidine kinase-associated sensory extracellular domain similar to the cytokinin-binding domain in plant cytokinin receptors was able to modestly bind isopentenyladenine. The data show that the genetic disposition allows Nostoc not only to produce free cytokinins and prenylate tRNA but also modulate the cytokinin biosynthesis in response to light, triggering complex changes in sensing and regulation.

• MeSH
• alkyltransferasy a aryltransferasy metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• bakteriální RNA metabolismus   MeSH
• cytokininy biosyntéza   MeSH
• Nostoc metabolismus   MeSH
• prenylace MeSH
• RNA transferová metabolismus   MeSH
• světlo * MeSH
• Publikační typ
• časopisecké články MeSH

Clonal propagation plays a critical integral role in the growth and success of a global multi-billion dollar horticulture industry through a constant supply of healthy stock plants. The supply chain depends on continuously improving the micropropagation process, thus, understanding the physiology of in vitro plants remains a core component. We evaluated the influence of exogenously applied cytokinins (CKs, N6-benzyladenine = BA, isopentenyladenine = iP, meta-topolin = mT, 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine = mTTHP) in Murashige and Skoog (MS)-supplemented media on organogenic response and accumulation of endogenous CK and indole-3-acetic acid (IAA) metabolites. The highest shoot proliferation (30 shoots/explant) was obtained with 20 μM mT treatment. However, the best quality regenerants were produced in 10 μM mT treatment. Rooting of Amelanchier alnifolia in vitro plantlets was observed at the lowest CK concentrations, with the highest root proliferation (3 roots/explant) in 1 μM mTTHP regenerants. Similar to the organogenic response, high levels of endogenous bioactive CK metabolites (free bases, ribosides, and nucleotides) were detected in mT and mTTHP-derived regenerants. The level of O-glucosides was also comparatively high in these cultures. All CK-treated plants had high levels of endogenous free IAA compared to the control. This may suggest an influence of CKs on biosynthesis of IAA.

• MeSH
• cytokininy farmakologie   MeSH
• ovoce růst a vývoj   MeSH
• Rosaceae růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH

A better understanding of phytohormone physiology can provide an essential basis to coherently achieve a conservation drive/strategy for valuable plant species. We evaluated the distribution pattern of cytokinins (CKs) and phenolic compounds in different organs of 1-year-old greenhouse-grown Tulbaghia simmleri pre-treated (during micropropagation) with three aromatic CKs (benzyladenine = BA, meta-topolin = mT, meta-topolin riboside = mTR). The test species is highly valuable due to its medicinal and ornamental uses. Based on UHPLC-MS/MS quantification, mT and mTR pre-treated plants had the highest total CK, mostly resulting from the isoprenoid CK-type, which occurred at highest concentrations in the roots. Although occurring in much lower concentrations when compared to isoprenoid CKs, aromatic CKs were several-fold more abundant in the root of mT pre-treated plants than with other treatments. Possibly related to the enhanced aromatic CKs, free bases and ribonucleotides, plants pre-treated with mT generally displayed better morphology than the other treatments. A total of 12 bioactive phenolic compounds, including four hydroxybenzoic acids, five hydroxycinnamic acids and three flavonoids at varying concentrations, were quantified in T. simmleri. The occurrence, distribution and levels of these phenolic compounds were strongly influenced by the CK pre-treatments, thereby confirming the importance of CKs in phenolic biosynthesis pathways.

• MeSH
• cytokininy metabolismus   farmakologie   MeSH
• fenoly metabolismus   MeSH
• flavonoidy metabolismus   MeSH
• hydroxybenzoáty metabolismus   MeSH
• kořeny rostlin účinky léků   metabolismus   MeSH
• kyseliny kumarové metabolismus   MeSH
• liliovité účinky léků   metabolismus   MeSH
• regulátory růstu rostlin metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In the current study, we evaluated the effect of α-naphthaleneacetic acid (NAA) individually or in combination with different cytokinins (CKs) including benzyladenine (BA), meta-topolin (mT) and isopentenyladenine (iP) on organogenesis, auxin and CK content in Eucomis autumnalis subspecies autumnalis (EA) and Eucomis zambesiaca (EZ). These species were used as model plants due to their ornamental and medicinal properties. Three leaf explants were inoculated in screw-cap jars containing 30mL Murashige and Skoog (MS) media supplemented with 5μM NAA alone or in combination with 5μM CK (BA, mT or iP). After 10 weeks (EA) or 15 weeks (EZ), parameters including shoot and root growth as well as plant fresh weight were recorded. For analysis of auxin and CK content, whole plantlets were harvested, pooled and freeze-dried for the different treatments. In both species, shoot and root proliferation as well as plant biomass were generally higher when NAA was combined with the individual CK than in NAA or CK treatment. The highest concentration of indole-3-acetic acid (IAA, 619pmolg(-1) DW) and 2-oxindole-3-acetic acid (OxIAA, 2381pmolg(-1) DW) were observed in EA-treated with NAA alone while mT treatment (without NAA) had the most abundant indole-3-acetyl-l-aspartic acid (IAAsp, 904 and 582pmolg(-1) DW for EA and EZ, respectively) in both species. A significant concentration of total endogenous CK accumulated in both Eucomis regenerants from mT and mT+NAA when compared to the other treatments. The majority of the detected CKs were of the aromatic CK-type, mainly free bases. The potential physiological roles of these quantified phytohormones in relation to the observed morphological responses are discussed.

• MeSH
• Asparagaceae účinky léků   růst a vývoj   metabolismus   MeSH
• biomasa MeSH
• biotechnologie MeSH
• cytokininy aplikace a dávkování   metabolismus   MeSH
• druhová specificita MeSH
• kořeny rostlin účinky léků   růst a vývoj   metabolismus   MeSH
• kyseliny indoloctové aplikace a dávkování   metabolismus   MeSH
• kyseliny naftalenoctové aplikace a dávkování   metabolismus   MeSH
• lékové interakce MeSH
• regulátory růstu rostlin metabolismus   farmakologie   MeSH
• výhonky rostlin účinky léků   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

The moss Physcomitrella patens is part of an early divergent clade of land plants utilizing the plant hormone cytokinin for growth control. The rate-limiting step of cytokinin biosynthesis is mediated by isopentenyltransferases (IPTs), found in land plants either as adenylate-IPTs or as tRNA-IPTs. Although a dominant part of cytokinins in flowering plants are synthesized by adenylate-IPTs, the Physcomitrella genome only encodes homologues of tRNA-IPTs. This study therefore looked into the question of whether cytokinins in moss derive from tRNA exclusively. Targeted gene knockout of ipt1 (d|ipt1) along with localization studies revealed that the chloroplast-bound IPT1 was almost exclusively responsible for the A37 prenylation of tRNA in Physcomitrella. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based cytokinin profiling demonstrated that the total amount of all free cytokinins in tissue was almost unaffected. However, the knockout plants showed increased levels of the N (6) -isopentenyladenine (iP)- and trans-zeatin (tZ)-type cytokinins, considered to provide active forms, while cis-zeatin (cZ)-type cytokinins were reduced. The data provide evidence for an additional and unexpected tRNA-independent cytokinin biosynthetic pathway in moss. Comprehensive phylogenetic analysis indicates a diversification of tRNA-IPT-like genes in bryophytes probably related to additional functions.

• MeSH
• alkyltransferasy a aryltransferasy genetika   metabolismus   MeSH
• biologická evoluce * MeSH
• chloroplasty enzymologie   genetika   MeSH
• cytokininy biosyntéza   MeSH
• fylogeneze * MeSH
• genový knockout MeSH
• mechy klasifikace   enzymologie   genetika   MeSH
• molekulární sekvence - údaje MeSH
• RNA transferová genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• rostliny klasifikace   enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Light and cytokinins are known to be the key players in the regulation of plant senescence. In detached leaves, the retarding effect of light on senescence is well described; however, it is not clear to what extent is this effect connected with changes in endogenous cytokinin levels. We have performed a detailed analysis of changes in endogenous content of 29 cytokinin forms in detached leaves of Arabidopsis thaliana (wild-type and 3 cytokinin receptor double mutants). Leaves were kept under different light conditions, and changes in cytokinin content were correlated with changes in chlorophyll content, efficiency of photosystem II photochemistry, and lipid peroxidation. In leaves kept in darkness, we have observed decreased content of the most abundant cytokinin free bases and ribosides, but the content of cis-zeatin increased, which indicates the role of this cytokinin in the maintenance of basal leaf viability. Our findings underscore the importance of light conditions on the content of specific cytokinins, especially N6 -(Δ2 -isopentenyl)adenine. On the basis of our results, we present a scheme summarizing the contribution of the main active forms of cytokinins, cytokinin receptors, and light to senescence regulation. We conclude that light can compensate the disrupted cytokinin signalling in detached leaves.

• MeSH
• Arabidopsis metabolismus   účinky záření   MeSH
• chlorofyl metabolismus   MeSH
• cytokininy metabolismus   MeSH
• fotosystém II - proteinový komplex metabolismus   MeSH
• listy rostlin metabolismus   účinky záření   MeSH
• malondialdehyd metabolismus   MeSH
• peroxidace lipidů MeSH
• stárnutí metabolismus   účinky záření   MeSH
• světlo MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free-growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather-dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.

• MeSH
• biosyntetické dráhy genetika   MeSH
• chlorofyl metabolismus   MeSH
• cytokininy metabolismus   MeSH
• down regulace genetika   MeSH
• glukosidy metabolismus   MeSH
• listy rostlin metabolismus   fyziologie   MeSH
• počasí MeSH
• Populus genetika   metabolismus   fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• roční období MeSH
• rostlinné geny MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH

Given the close relationship between cytokinins (CKs), photosynthesis and nitrogen metabolism, this study assessed the effect of arsenic (As) contamination on these metabolic components in the As-hyperaccumulators Pteris cretica L. var. Albo-lineata (Pc-A) and var. Parkerii (Pc-P) as well as the As-non-hyperaccumulator Pteris straminea Mett. ex Baker (Ps). The ferns were cultivated in a pot experiment for 23 weeks in soil spiked with As at the levels 20 and 100 mg·kg-1. For the purpose of this study, the CKs were placed into five functionally different groups according to their structure and physiological roles: bioactive forms (bCKs; CK free bases); inactive or weakly active forms (dCKs; CK N-glucosides); transport forms (tCKs; CK ribosides); storage forms (sCKs; O-glucosides); and primary products of CK biosynthesis (ppbCKs; CK nucleotides). An important finding was higher CKs total content, accumulation of sCKs and reduction of dCKs in As-hyperaccumulators in contrast to non-hyperaccumulator ferns. A significant depletion of C resources was confirmed in ferns, especially Ps, which was determined by measuring the photosynthetic rate and chlorophyll fluorescence. A fluorescence decrease signified a reduction in the C/N ratio, inducing an increase of bioactive CKs forms in Pc-P and Ps. The impact of As on N utilization was significant in As-hyperaccumulators. The glutamic acid/glutamine ratio, an indicator of primary N assimilation, diminished in all ferns with increased As level in the soil. In conclusion, the results indicate a large phenotypic diversity of Pteris species to As and suggest that the CKs composition and the glutamic acid/glutamine ratio can be used as a tool to diagnose As stress in plants.

• MeSH
• aminokyseliny metabolismus   MeSH
• arsen toxicita   MeSH
• biomasa MeSH
• cytokininy metabolismus   MeSH
• dusík metabolismus   MeSH
• fotosyntéza účinky léků   MeSH
• látky znečišťující půdu toxicita   MeSH
• listy rostlin účinky léků   růst a vývoj   metabolismus   MeSH
• Pteris účinky léků   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytokinin ribosides (N(6)-substituted adenosine derivatives) have been shown to have anticancer activity both in vitro and in vivo. This study presents the first systematic analysis of the relationship between the chemical structure of cytokinins and their cytotoxic effects against a panel of human cancer cell lines with diverse histopathological origins. The results confirm the cytotoxic activity of N(6)-isopentenyladenosine, kinetin riboside, and N(6)-benzyladenosine and show that the spectrum of cell lines that are sensitive to these compounds and their tissues of origin are wider than previously reported. The first evidence that the hydroxylated aromatic cytokinins (ortho-, meta-, para-topolin riboside) and the isoprenoid cytokinin cis-zeatin riboside have cytotoxic activities is presented. Most cell lines in the panel showed greatest sensitivity to ortho-topolin riboside (IC(50)=0.5-11.6 microM). Cytokinin nucleotides, some synthesized for the first time in this study, were usually active in a similar concentration range to the corresponding ribosides. However, cytokinin free bases, 2-methylthio derivatives and both O- and N-glucosides showed little or no toxicity. Overall the study shows that structural requirements for cytotoxic activity of cytokinins against human cancer cell lines differ from the requirements for their activity in plant bioassays. The potent anticancer activity of ortho-topolin riboside (GI(50)=0.07-84.60 microM, 1st quartile=0.33 microM, median=0.65 microM, 3rd quartile=1.94 microM) was confirmed using NCI(60), a standard panel of 59 cell lines, originating from nine different tissues. Further, the activity pattern of oTR was distinctly different from those of standard anticancer drugs, suggesting that it has a unique mechanism of activity. In comparison with standard drugs, oTR showed exceptional cytotoxic activity against NCI(60) cell lines with a mutated p53 tumour suppressor gene. oTR also exhibited significant anticancer activity against several tumour models in in vivo hollow fibre assays.

• MeSH
• adenosin analogy a deriváty   chemie   farmakologie   MeSH
• antitumorózní látky chemie   farmakologie   MeSH
• cytokininy analýza   chemie   metabolismus   MeSH
• geny p53 účinky léků   genetika   MeSH
• inhibiční koncentrace 50 MeSH
• isopentenyladenosin analogy a deriváty   chemie   farmakologie   MeSH
• kinetin chemie   farmakologie   MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• molekulární struktura MeSH
• National Cancer Institute (U.S.) MeSH
• regulátory růstu rostlin farmakologie   MeSH
• stereoizomerie MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Spojené státy americké MeSH