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Cytokinin is an indispensable phytohormone responsible for physiological processes ranging from root development to leaf senescence. The term "cytokinin" refers to several dozen adenine-derived compounds occurring naturally in plants. Cytokinins (CKs) can be divided into various classes and forms; base forms are generally considered to be active while highly abundant cytokinin-N-glucosides (CKNGs), composed of a CK base irreversibly conjugated to a glucose molecule, are considered inactive. However, results from early CK studies suggest CKNGs do not always lack activity despite the perpetuation over several decades in the literature that they are inactive. Here we show that exogenous application of trans-Zeatin-N-glucosides (tZNGs, a specific class of CKNGs) to Arabidopsis results in CK response comparable to the application of an active CK base. These results are most apparent in senescence assays where both a CK base (tZ) and tZNGs (tZ7G, tZ9G) delay senescence in cotyledons. Further experiments involving root growth and shoot regeneration revealed tZNGs do not always have the same effects as tZ, and have largely distinct effects on the transcriptome and proteome. These data are in contrast to previous reports of CKNGs being inactive and raise questions about the function of these compounds as well as their mechanism of action.
Plants face temporal and spatial variation in nitrogen (N) availability. This includes heterogeneity in soil nitrate (NO3-) content. To overcome these constraints, plants modify their gene expression and physiological processes to optimize N acquisition. This plasticity relies on a complex long-distance root-shoot-root signaling network that remains poorly understood. We previously showed that cytokinin (CK) biosynthesis is required to trigger systemic N signaling. Here, we performed split-root experiments and used a combination of CK-related mutant analyses, hormone profiling, transcriptomic analysis, NO3- uptake assays, and root growth measurements to gain insight into systemic N signaling in Arabidopsis thaliana By comparing wild-type plants and mutants affected in CK biosynthesis and ABCG14-dependent root-to-shoot translocation of CK, we revealed an important role for active trans-zeatin (tZ) in systemic N signaling. Both rapid sentinel gene regulation and long-term functional acclimation to heterogeneous NO3- supply, including NO3- transport and root growth regulation, are likely mediated by the integration of tZ content in shoots. Furthermore, shoot transcriptome profiling revealed that glutamate/glutamine metabolism is likely a target of tZ root-to-shoot translocation, prompting an interesting hypothesis regarding shoot-to-root communication. Finally, this study highlights tZ-independent pathways regulating gene expression in shoots as well as NO3- uptake activity in response to total N deprivation.
- MeSH
- Arabidopsis metabolismus MeSH
- dusík metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny metabolismus MeSH
- signální transdukce MeSH
- výhonky rostlin metabolismus MeSH
- zeatin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis↔trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.
- MeSH
- Arabidopsis enzymologie růst a vývoj MeSH
- biokatalýza MeSH
- biologický transport MeSH
- biotest MeSH
- časové faktory MeSH
- fylogeneze MeSH
- isomerie MeSH
- kultivované buňky MeSH
- listy rostlin metabolismus MeSH
- oves metabolismus MeSH
- oxidoreduktasy metabolismus MeSH
- protein - isoformy MeSH
- rostliny enzymologie metabolismus MeSH
- semena rostlinná metabolismus MeSH
- signální transdukce MeSH
- substrátová specifita MeSH
- tabák cytologie enzymologie MeSH
- tritium metabolismus MeSH
- zeatin genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Studies of vitality/mortality of cortex cells, as well as of the concentrations of ethylene (ETH), gibberellins (GAs), indolic compounds/auxins (ICs/AUXs) and cytokinins (CKs), were undertaken to explain the hormonal background of kinetin (Kin)-regulated cell death (RCD), which is induced in the cortex of the apical parts of roots of faba bean (Vicia faba ssp. minor) seedlings. Quantification was carried out with fluorescence microscopy, ETH sensors, spectrophotometry and ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC‒MS/MS). The results indicated that Kin was metabolized to the transport form, i.e., kinetin-9-glucoside (Kin9G) and kinetin riboside (KinR). KinR was then converted to cis-zeatin (cZ) in apical parts of roots with meristems, to cis-zeatin riboside (cZR) in apical parts of roots without meristems and finally to cis-zeatin riboside 5'-monophosphate (cZR5'MP), which is indicated to be a ligand of cytokinin-dependent receptors inducing CD. The process may be enhanced by an increase in the amount of dihydrozeatin riboside (DHZR) as a byproduct of the pathway of zeatin metabolism. It seems that crosstalk of ETH, ICs/AUXs, GAs and CKs with the cZR5'MP, the cis-zeatin-dependent pathway, but not the trans-zeatin-dependent pathway, is responsible for Kin-RCD, indicating that the process is very specific and offers a useful model for studies of CD hallmarks in plants.
Plant survival in temperate zones requires efficient cold acclimation, which is strongly affected by light and temperature signal crosstalk, which converge in modulation of hormonal responses. Cold under low light conditions affected Arabidopsis responses predominantly in apices, possibly because energy supplies were too limited for requirements of these meristematic tissues, despite a relatively high steady-state quantum yield. Comparing cold responses at optimal light intensity and low light, we found activation of similar defence mechanisms-apart from CBF1-3 and CRF3-4 pathways, also transient stimulation of cytokinin type-A response regulators, accompanied by fast transient increase of trans-zeatin in roots. Upregulated expression of components of strigolactone (and karrikin) signalling pathway indicated involvement of these phytohormones in cold responses. Impaired response of phyA, phyB, cry1 and cry2 mutants reflected participation of these photoreceptors in acquiring freezing tolerance (especially cryptochrome CRY1 at optimal light intensity and phytochrome PHYA at low light). Efficient cold acclimation at optimal light was associated with upregulation of trans-zeatin in leaves and roots, while at low light, cytokinin (except cis-zeatin) content remained diminished. Cold stresses induced elevation of jasmonic acid and salicylic acid (in roots). Low light at optimal conditions resulted in strong suppression of cytokinins, jasmonic and salicylic acid.
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
The maize β-D-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-β-D-glucopyranoside versus the trans-zeatin-O-β-D-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-β-D-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.
- MeSH
- aminokyseliny metabolismus MeSH
- beta-glukosidasa chemie genetika MeSH
- cytokininy metabolismus MeSH
- glukosidy metabolismus MeSH
- hydrolýza MeSH
- isomerie MeSH
- kukuřice setá chemie enzymologie genetika MeSH
- molekulární konformace MeSH
- mutace MeSH
- mutageneze cílená metody MeSH
- rostlinné geny MeSH
- rostlinné proteiny chemie genetika MeSH
- sekvence aminokyselin MeSH
- substrátová specifita MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zeatin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The endogenous auxin and cytokinin contents of in vitro regenerated Tulbaghia simmleri maintained on applied plant growth regulators in Murashige and Skoog (MS) medium were investigated using UHPLC-MS analysis. The highest number of shoots (27.6 per leaf) were produced in MS medium supplemented with 2.5 μM thidiazuron. A higher number of these shoots were rooted with 10 μM 6-(2-hydroxy-3-methylbenzylamino) purine (PI-55, cytokinin antagonist). Production of somatic embryos (SEs: 16.4-4.6, globular to cotyledonary stages) improved significantly with liquid MS medium containing 2.5 μM picloram, 2.5 μM phloroglucinol (PG) and 1.5 μM gibberellic acid or 1.5 μM PI-55 and 1.0 μM trans-zeatin. SEs (torpedo and cotyledonary stages) germinated (100%) in plant growth regulator free MS medium. The plantlets were acclimatized and all survived in the greenhouse. Higher levels of endogenous auxin, 2-oxindole-3-acetic acid (oxIAA, 371.52 pmol/g DW) and indole-3-acetylaspartate (IAAsp, 141.56 pmol/g DW) were detected in shoots from PG treatments. The roots of garden-grown mother plants possessed the highest level of indole-3-acetic acid (IAA, 630.54 pmol/g DW) and oxIAA (515.26 pmol/g DW). Cytokinins [CKs: trans-zeatin-O-glucoside (tZOG), cis-zeatin (cZ) and N6-isopentenyladenosine-5'-monophosphate (iPRMP)] levels were relatively high in shoots and roots of plantlets in vitro. However, PI-55 treatments influenced the development of plantlets promoting a higher biosynthesis level of iPRMP (418.06 pmol/g DW in root) and cZRMP (904.61 pmol/g DW in roots and 1427.83 pmol/g DW in shoots). The presented protocols offer organogenesis and somatic organogenesis systems for rapid plant regeneration of T. simmleri. In addition, the importance of exogenous and endogenous hormonal effects on in vitro plant growth and development as well as endogenous hormone metabolism signalling and transport related to the physiological processes of CK metabolism and transport are illustrated for in vitro development of T. simmleri.
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.
- 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
The biologically active molecules karrikinolide (KAR1) and trimethylbutenolide (TMB) present in wildfire smoke play a key role in regulating seed germination of many plant species. To elucidate the physiological mechanism by which smoke-water (SW), KAR1, and TMB regulate seed germination in photosensitive 'Grand Rapids' lettuce (Lactuca sativa), we investigated levels of the dormancy-inducing hormone abscisic acid (ABA), three auxin catabolites, and cytokinins (26 isoprenoid and four aromatic) in response to these compounds. Activity of the hydrolytic enzymes α-amylase and lipase along with stored food reserves (lipids, carbohydrate, starch, and protein) were also assessed. The smoke compounds precisely regulated ABA and hydrolytic enzymes under all light conditions. ABA levels under red (R) light were not significantly different in seeds treated with TMB or water. However, TMB-treated seeds showed significantly inhibited germination (33%) compared with water controls (100%). KAR1 significantly enhanced total isoprenoid cytokinins under dark conditions in comparison with other treatments; however, there was no significant effect under R light. Enhanced levels of indole-3-aspartic acid (an indicator of high indole-3-acetic acid accumulation, which inhibits lettuce seed germination) and absence of trans-zeatin and trans-zeatin riboside (the most active cytokinins) in TMB-treated seeds might be responsible for reduced germination under R light. Our results demonstrate that SW and KAR1 significantly promote lettuce seed germination by reducing levels of ABA and enhancing the activity of hydrolytic enzymes, which aids in mobilizing stored reserves. However, TMB inhibits germination by enhancing ABA levels and reducing the activity of hydrolytic enzymes.
- MeSH
- furany farmakologie MeSH
- fytochrom metabolismus MeSH
- gama-butyrolakton analogy a deriváty farmakologie MeSH
- klíčení účinky léků MeSH
- kouř * MeSH
- lékové interakce MeSH
- pyrany farmakologie MeSH
- regulátory růstu rostlin metabolismus MeSH
- salát (hlávkový) účinky léků metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
