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Cytokinins

Cytokinins are a class of plant growth hormones that promote cell division and differentiation.
They are involved in a variety of physiological processes, including shoot and root development, leaf senescence, and stress response.
Cytokinins are derived from adenine and are classified into different types based on their side chain structure.
They exert their effects by binding to cytokinin receptors and activating downstream signaling pathways.
Cytokinin research is crucial for understanding plant growth and development, as well as for developing strategies to improve crop productivity and stress tolerance.
Optimizing cytokinin research through AI-driven protocol comparisons can enhance reproducibility and productivity in this important field of plant biology.

Most cited protocols related to «Cytokinins»

Phytohormone Analysis by HPLC-MS

Cytokinins (zeatin, Z, and zeatin riboside, ZR), indole-3-acetic acid (IAA), and abscisic acid (ABA) were extracted and purified according to the method of Dobrev and Kaminek (2002) (link). One gram of fresh plant material (leaf or root) was homogenized in liquid nitrogen and placed in 5 ml of cold (–20 °C) extraction mixture of methanol/water/formic acid (15/4/1 by vol., pH 2.5). After overnight extraction at –20 °C solids were separated by centrifugation (20 000 g, 15 min) and re-extracted for 30 min in an additional 5 ml of the same extraction solution. Pooled supernatants were passed through a Sep-Pak Plus †C₁₈ cartridge (SepPak Plus, Waters, USA) to remove interfering lipids and plant pigments and evaporated to dryness. The residue was dissolved in 5 ml of 1 M formic acid and loaded on an Oasis MCX mixed mode (cation-exchange and reverse phase) column (150 mg, Waters, USA) preconditioned with 5 ml of methanol followed by 5 ml of 1 M formic acid. To separate different CK forms (nucleotides, bases, ribosides, and glucosides) from IAA and ABA, the column was washed and eluted stepwise with different appropriate solutions indicated in Dobrev and Kaminek (2002) (link). ABA and IAA were analysed in the same fraction. After each solvent was passed through the columns, they were purged briefly with air. Solvents were evaporated at 40 °C under vacuum. Samples then dissolved in a water/acetonitrile/formic acid (94.9:5:0.1 by vol.) mixture for HPLC/MS analysis. Analyses were carried out on a HPLC/MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a μ-well plate autosampler and a capillary pump, and connected to an Agilent Ion Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an electrospray (ESI) interface. Prior to injection, 100 μl of each fraction extracted from tissues or a similar volume of xylem sap were filtered through 13 mm diameter Millex filters with 0.22 μm pore size nylon membrane (Millipore, Bedford, MA, USA). 8 μl of each sample, dissolved in mobile phase A, was injected onto a Zorbax SB-C18 HPLC column (5 μm, 150×0.5 mm, Agilent Technologies, Santa Clara, CA, USA), maintained at 40 °C, and eluted at a flow rate of 10 μl min⁻¹. Mobile phase A, consisting of water/acetonitrile/formic acid (94.9:5:0.1 by vol.), and mobile phase B, consisting of water/acetonitrile/formic acid (10:89.9:0.1 by vol.), were used for the chromatographic separation. The elution programme maintained 100% A for 5 min, then a linear gradient from 0% to 6% B in 10 min, followed by another linear gradient from 6% to 100% B in 5 min, and finally 100% B maintained for another 5 min. The column was equilibrated with the starting composition of the mobile phase for 30 min before each analytical run. The UV chromatogram was recorded at 280 nm with a DAD module (Agilent Technologies, Santa Clara, CA, USA). The mass spectrometer was operated in the positive mode with a capillary spray voltage of 3500 V, and a scan speed of 22 000 m/z s⁻¹ from 50–500 m/z. The nebulizer gas (He) pressure was set to 30 psi, whereas the drying gas was set to a flow of 6.0 l min⁻¹ at a temperature of 350 °C. Mass spectra were obtained using the DataAnalysis program for LC/MSD Trap Version 3.2 (Bruker Daltonik GmbH, Germany). For quantification of Z, ZR, ABA, and IAA, calibration curves were constructed for each component analysed (0.05, 0.075, 0.1, 0.2, and 0.5 mg l⁻¹) and corrected for 0.1 mg l⁻¹ internal standards: [²H₅]trans-zeatin, [²H₅]trans-zeatin riboside, [²H₆]cis,trans-abscisic acid (Olchemin Ltd, Olomouc, Czech Republic), and [¹³C₆]indole-3-acetic acid (Cambridge Isotope Laboratories Inc., Andover, MA, USA). Recovery percentages ranged between 92% and 95%.
ACC (1-aminocyclopropane-1-carboxylic acid) was determined after conversion into ethylene by gas chromatography using an activated alumina column and a FID detector (Konik, Barcelona, Spain). ACC was extracted with 80% (v/v) ethanol and assayed by degradation with alkaline hypochlorite in the presence of 5 mM HgCl₂ (Casas et al., 1989 ). A preliminary purification step was performed by passing the extract through a Dowex 50W-X8, 50–100 mesh, H⁺-form resin and later recovered with 0.1 N NH₄OH. The conversion efficiency of ACC into ethylene was calculated separately by using a replicate sample containing 2.5 nmol of ACC as an internal standard and used for the correction of data.

Albacete A., Ghanem M.E., Martínez-Andújar C., Acosta M., Sánchez-Bravo J., Martínez V., Lutts S., Dodd I.C, & Pérez-Alfocea F. (2008). Hormonal changes in relation to biomass partitioning and shoot growth impairment in salinized tomato (Solanum lycopersicum L.) plants. Journal of Experimental Botany, 59(15), 4119-4131.

Publication 2008

1-aminocyclopropane-1-carboxylic acid Abscisic Acid acetonitrile Capillaries Centrifugation Chaperone-Mediated Autophagy Chromatography cis-acid Cold Temperature CREB3L1 protein, human Cytokinins DNA Replication Dowex Ethanol Ethylenes formic acid Gas Chromatography Glucosides High-Performance Liquid Chromatographies Hypochlorite indoleacetic acid Isotopes Lipids Mass Spectrometry Mercuric Chloride Methanol Nebulizers Nitrogen Nucleotides Nylons Oxide, Aluminum Pigmentation Plant Leaves Plant Roots Plants Pressure Radionuclide Imaging Resins, Plant Sep-Pak C18 Solvents Strains Tissue, Membrane Tissues Vacuum Xylem Zeatin zeatin riboside

Embryogenesis Induction and Transcript Profiling

The preparation of embryogenic suspension cells and RNA extractions for the suppression subtractive hybridization (SSH) library constructions from the 30-day proliferation cycle and after 16 days of liquid pretreatment to initiate somatic embryogenesis was performed and described previously [17 (link)]. In addition, a portion of pretreated embryogenic suspension cells initiated to undergo somatic embryogenesis was plated on solid agar plates containing the basal medium with or without 6-benzylaminopurine (synthetic cytokinin) for further somatic embryo development and collected after 7 days for RNA extractions and SSH library constructions. The material collected for the shoot apex, female and male inflorescences, and zygotic embryos for the unnormalized library constructions was described previously [16 (link)]. The material for the normal and abnormal male inflorescences SSH libraries was described and performed previously [15 (link)]. The zygotic embryos (3-5.5 months of development) were isolated from tenera palm seeds collected from trees (Deli x La Mé origin) cultivated at CRAPP Pobé Station, Benin.

Tranbarger T.J., Kluabmongkol W., Sangsrakru D., Morcillo F., Tregear J.W., Tragoonrung S, & Billotte N. (2012). SSR markers in transcripts of genes linked to post-transcriptional and transcriptional regulatory functions during vegetative and reproductive development of Elaeis guineensis. BMC Plant Biology, 12, 1.

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Publication 2012

Agar Arecaceae benzylaminopurine butocin cDNA Library Cells Cytokinins Diploid Cell Embryo Embryonic Development Females Inflorescence Males Plant Embryos Subtractive Hybridization Techniques Trees Zygote

Optimizing Cytokinin Purification Using StageTips

To test the selectivity, affinity, and capacity of different StageTip sorbent combinations, samples containing ³H-labelled CK standards ([³H]cZ, [³H]tZR, [³H]iPR) were used. Firstly, to characterize the potential sorbents (C18, SDB-RPS, and Cation-SR), the single-StageTip experiments were performed by pipetting 50 μl of the selected ³H-CK standard (10⁵ dpm) in Bieleski buffer directly onto a PT-SPE column. Secondly, different combinations of sorbents (multi-StageTips) were tested using the same spiked Bieleski buffer with and without plant matrices (1.0 and 2.0 mg FW) to monitor the loading capacity and extraction recovery. For all samples, the yields of ³H-CKs after passage through StageTips filled with different sorbents and their combinations were determined. Thirdly, non-spiked and spiked (with 0.1, 1 and 10 pmol of unlabelled CK standards) samples of 10-day old Arabidopsis thaliana seedlings (1–5 mg FW) were used to verify the reproducibility, sensitivity and accuracy of the method. Aliquots of the extracts were processed using the developed purification procedure (Figure 2), then analyzed by UHPLC-ESI(+)-MS/MS. The intraday reproducibility was evaluated by repeating the process for three times within one day, and the interday reproducibility was investigated on three successive days. The recovery of added authentic cytokinin standards were then determined from each series of extracts, based on the amounts of endogenous compounds, calculated from non-spiked samples, which subsequently served as reference levels.

Svačinová J., Novák O., Plačková L., Lenobel R., Holík J., Strnad M, & Doležal K. (2012). A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction. Plant Methods, 8, 17.

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Publication 2012

Arabidopsis thalianas Buffers Cytokinins Genetic Selection Hypersensitivity Plants Seedlings SpeA protein, Streptococcus pyogenes Tandem Mass Spectrometry

Cloning Promoter-Gene Constructs for Cotton

For the proTCS::iaaM and proDR5::IPT constructs, the cytokinin-inducible promoter proTCS (Müller and Sheen, 2008 (link)) and the auxin-inducible promoter proDR5 (Ulmasov et al., 1997 (link)) were synthesized with flanking HindIII and XbaI restriction sites. iaaM (Comai and Kosuge, 1982 (link)) and IPT sequences (Medford et al., 1989 (link)) were amplified by PCR (Novoprotein, China) from Agrobacterium tumefaciens with flanking KpnI and EcoRI restriction sites. After digestion (ThermoFisher Scientific), proTCS linked with iaaM, and proDR5 linked with IPT were separately inserted into the binary vector p5 (Luo et al., 2007 (link)). For the proTCS::GUS construct, the proCaMV35S (pro35S) promoter in the binary vector pBI121 was replaced by the proTCS promoter through the HindIII and XbaI restriction sites. For the proGhPIN3a::GUS construct, the proGhPIN3a promoter was amplified from upland cotton (Gossypium hirsutum cultivar ‘Jimian 14’) with flanking HindIII and BamHI restriction sites. After digestion, the proGhPIN3a promoter was placed upstream of the GUS reporter gene in a modified pCambia2300 binary vector. For the proPV::antisenseGhCKX3 construct, an ovule-specific promoter proPV (Goossens et al., 1999 (link); Zhang et al., 2011 ) was amplified from common bean (Phaseolus vulgaris) with flanking HindIII and BamHI restriction sites, and the partial antisense sequence of GhCKX3 (antisenseGhCKX3) was amplified from the cotton ‘Jimian 14’ with flanking BamHI and EcoRI restriction sites. proPV linked with antisenseGhCKX3 was inserted into the binary vector p5. For the proBAN::GhPIN3a-RNAi construct, 3´-UTR of GhPIN3a was amplified and inserted to a terminal of the first intron of GA20ox1 with SalI and XbaI sites, and inversely inserted to the other terminal with NotI and EcoRI sites in a vector pUCm-T. The RNAi construct was then placed downstream of the ovule epidermis-specific promoter proBAN isolated from Arabidopsis (Debeaujon et al., 2003 (link); Zhang et al., 2011 ) in a binary vector pLGN modified from pCambia2300. In the T-DNA region of pLGN there is a neomycin phosphotransferase II (NPTII) selection marker fused with a GUS reporter gene under the control of a pro35S promoter. For the pro35S::GhPIN3a::YFP construct, YFP without the start and stop codons was inserted into the GhPIN3a coding sequence (CDS) behind position 1413 bp relative to the start codon through amplifying the fragment with flanking SpeI and SalI restriction sites. The generated GhPIN3a::YFP was placed downstream of a pro35S promoter in the pLGN vector. Information for all constructs including primer sequences and fragment sizes are listed in Supplementary Table S1 at JXB online. These constructs were transformed into cotton ‘Jimian 14’ using A. tumefaciens strain LBA4404 (Luo et al., 2007 (link)). Kanamycin-resistant and GUS-positive plantlets were screened out and grown in a greenhouse receiving natural daylight.

Zeng J., Zhang M., Hou L., Bai W., Yan X., Hou N., Wang H., Huang J., Zhao J, & Pei Y. (2019). Cytokinin inhibits cotton fiber initiation by disrupting PIN3a-mediated asymmetric accumulation of auxin in the ovule epidermis. Journal of Experimental Botany, 70(12), 3139-3151.

Publication 2019

3' Untranslated Regions Agrobacterium tumefaciens Arabidopsis Auxins Cloning Vectors Codon, Initiator Codon, Terminator Cytokinins Deoxyribonuclease EcoRI Digestion Epidermis Genes, Reporter Gossypium Introns Kanamycin Neomycin Phosphotransferase II Oligonucleotide Primers Open Reading Frames Ovule Phaseolus vulgaris RNA Interference Strains

Quantification of Tritium-Labeled Cytokinins

The radioactivity of tritium-labelled cytokinin standards was measured after addition of 1 ml liquid scintillation cocktail Ultima Gold^TM on a multi-purpose scintillation counter LS 6500 (Beckman Coulter, Brea, CA, USA). An Acquity UPLC® System (Waters, Milford, MA, USA), including a Binary solvent manager and Sample manager was linked simultaneously to a 2996 PDA detector (Waters) and a triple quadrupole mass spectrometer Xevo^TM TQ MS (Waters MS Technologies, Manchester, UK) equipped with an electrospray interface (ESI) and the unique performance of a collision cell (ScanWave^TM). This hyphenated technique was used for analysis of unlabelled and stable isotope-labelled cytokinins. All MS data were processed by MassLynx^TM software with TargetLynx^TM program (version 4.2., Waters, Milford, MA, USA).

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Publication 2012

Cytokinins Isotopes Scintillation Counters Solvents Tritium

Most recents protocols related to «Cytokinins»

Plant hormone profiling via HPLC

The contents of auxin (indole-3-acetic acid, IAA), gibberellin (GA₃), cytokinin (CTK), and abscisic acid (ABA) in roots were determined by high performance liquid chromatography (Pan et al., 2010 (link)).

Chen W., Mou X., Meng P., Chen J., Tang X., Meng G., Xin K., Zhang Y, & Wang C. (2023). Effects of arbuscular mycorrhizal fungus inoculation on the growth and nitrogen metabolism of Catalpa bungei C.A.Mey. under different nitrogen levels. Frontiers in Plant Science, 14, 1138184.

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Publication 2023

Abscisic Acid Auxins Cytokinins Gibberellins High-Performance Liquid Chromatographies indoleacetic acid Plant Roots

Establishing M. baccata Stress Response Model

M. baccata tissue culture plantlets was put on Murashige and Skoog (MS) medium containing 0.55 mg/L cytokinin (6-BA) and 0.6 mg/L indole butyric acid (IBA) for rapid propagation. After one month, selected robust tissue culture plantlets and transfered them to rooting medium (1.2 mg/L IBA) (Yao et al., 2022b (link)). After rooting, seedlings were put into Hoagland hydroponic culture solution for growth. The room temperature and humidity of tissue culture were maintained at about 25°C and 80%. Changed the hydroponic culture solution regularly. When 7-8 fully developed mature leaves and new strong roots grow out of the hydroponic seedlings, seedlings with roughly the same growth conditions were selected and grouped, 10 seedlings in each group, a total of 5 groups, one of which was the control group without any treatment. The other 4 groups were treated as follows respectively, and the hydroponic seedlings were placed in the 4°C tissue culture room for low temperature stress; The Hoagland hydroponic culture solution with 200 mM NaCl was used for salt stress; Hoagland hydroponic medium containing 20% PEG6000 was used for water stress; The hydroponic seedlings were put into the tissue culture room at 37°C for high temperature stress. The young leaves, mature leaves, roots and stems of all seedlings were sampled after treatment for 0, 1, 3, 6, 9 and 12 h, and stored at -80°C after liquid nitrogen quick freezing (Han et al., 2018b (link)).

Liu W., Wang T., Wang Y., Liang X., Han J, & Han D. (2023). MbMYBC1, a M. baccata MYB transcription factor, contribute to cold and drought stress tolerance in transgenic Arabidopsis. Frontiers in Plant Science, 14, 1141446.

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Publication 2023

Aftercare Cold Temperature Cytokinins Fever Growth Disorders Humidity indolebutyric acid Nitrogen Plant Roots Polyethylene Glycol 6000 Salt Stress Seedlings Sodium Chloride Stem, Plant Tissues Water Stress

Hormonal Influence on REEs Accumulation in G. sulphuraria

To study the effect of hormones on REEs accumulation, one set of asynchronous cultures of G. sulphuraria was cultivated with CFL and hormones for 24 h. For this study two synthetic plant hormones, 6-Benzylaminopurine (BAP-Cytokinin family) and 1-Naphthaleneacetic acid (NAA-Auxin family) (Sigma-Aldrich) were used at a final concentration of 5 mg L⁻¹ for this study. At the end of the experiment, cultures with and without plant hormones were harvested by centrifugation (3,000 rpm, 5 min), freeze dried and analyzed by ICP-MS.

Singh A., Čížková M., Náhlík V., Mezricky D., Schild D., Rucki M, & Vítová M. (2023). Bio-removal of rare earth elements from hazardous industrial waste of CFL bulbs by the extremophile red alga Galdieria sulphuraria. Frontiers in Microbiology, 14, 1130848.

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Publication 2023

1-naphthaleneacetic acid Auxins benzylaminopurine Centrifugation Cytokinins Freezing Hormones Plant Growth Regulators

Axillary Shoot Regeneration and Root Formation

Five Petri dishes and eight whole zygotic embryos per Petri dish, cytokinin type, and light treatment were cultured in HLP. When the axillary shoots were isolated and cultured individually in the elongation medium, the EFS, the NS/E, and percentage of (PSR) out of the total number of shoots produced per explant were calculated. A logistic regression model was used to analyze the effect of the cytokinin type and light treatment on survival and EFS. When necessary, Tukey’s post hoc test (α = 0.05) was used for multiple comparisons.
The confirmation of the homogeneity of variances and normality of the data on the NS/E and PSR were performed, and PSR was

\sqrt{x}

transformed to meet homocedasticity. Data for the NS/E and PSR were analyzed by analysis of variance (ANOVA). When necessary, multiple comparisons were made using Tukey’s post hoc test (α = 0.05).
A completely randomized design was carried out using six to twenty plantlets per cytokinin type and light treatment per each auxin treatment. The RI percentage (RI), the mean NR/E, and the LLR (cm) were recorded after six weeks of culture in a root elongation medium.
To assess the effect of the cytokinin and light treatment on the RI, a logistic regression was performed. Data for NR/E and LLR were analyzed by ANOVA. When necessary, multiple comparisons were made using Tukey’s post hoc test (α = 0.05). The acclimatization percentage was calculated after four weeks under ex vitro conditions. The data were analyzed using R Core Team software^® (version 4.2.1, Vienna, Austria).

Rojas-Vargas A., Castander-Olarieta A., do Nascimento A.M., Vélez M.L., Pereira C., Martins J., Zuzarte M., Canhoto J., Montalbán I.A, & Moncaleán P. (2023). Testing Explant Sources, Culture Media, and Light Conditions for the Improvement of Organogenesis in Pinus ponderosa (P. Lawson and C. Lawson). Plants, 12(4), 850.

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Publication 2023

Acclimatization Auxins Axilla Cytokinins Embryo Hyperostosis, Diffuse Idiopathic Skeletal Light Plant Roots Zygote

Structural Analysis of Plant Tissues After Elongation

For structural analysis after eighteen weeks in the elongation medium, 16 needles were used following the procedure described in Giacomolli et al. [67 (link)]. Two needles per cytokinin and light treatment were fixed in 0.2 M phosphate buffer (pH 7.2) and 2.5% paraformaldehyde for 48 h at 4 °C. Then, the samples were washed twice with 0.1 M phosphate buffer (pH 7.2) for 15 min and then dehydrated in ethanolic series (30%, 50%, 70%, 80%, 90%, 95%, and 100% v/v) (1 h each).
Subsequently, the samples were embedded in paraffin by means of dehydration with ethanol and Clear Rite™ and the consecutive immersion in liquid paraffin at 65 °C, as described in Rossi et al. [68 (link)]. The dehydration process was the following: ethanol 70% (v/v) for 120 min; ethanol 90% (v/v) for 90 min (2×); ethanol 95% (v/v) for 90 min; ethanol 100% (v/v) for 90 min (×2); ethanol: Clear Rite™ (1:1 v/v) and pure Clear Rite™ for 90 min (2×). Then, samples were embedded in paraffin wax at 65 °C for 120 min (2×) before the inclusion in paraffin molds. After paraffin inclusion (HistoDream EW, Milestone Medical Sorisole, Italy), sections of 8–10 µm were obtained in a rotary microtome (Microm HM 340E, Thermo Scientific, Waltham, USA) and transferred to microscope slides previously prepared with albumin glycerol and kept at 30 °C for 12 h. Next, the deparaffinization was performed with Clear Rite™ for 20 min, 100% ethanol for 4 min, and washing under running water. The samples were stained with an Astra Blue (0.15%) and Safranin (0.04%) water solution for 10 min; and finally, they were observed in an optical microscope (Leica DM 4000 B, Mannheim, Germany) and photos were taken with a Leica camera (Leica Application Suite version 4.13).

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Publication 2023

Albumins Astra Blue Buffers Cytokinins Dehydration Ethanol Fungus, Filamentous Glycerin Light Light Microscopy Microscopy Microtomy Needles Oil, Mineral Paraffin Paraffin Embedding paraform Phosphates safranine T Submersion

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More about "Cytokinins"

Cytokinins, also known as plant growth hormones or phytohormones, are a class of compounds that play a crucial role in regulating various physiological processes in plants.
These adenine-derived molecules are involved in promoting cell division, differentiation, and development, as well as influencing shoot and root growth, leaf senescence, and stress responses.
Cytokinin research is a vital area of plant biology, as understanding the mechanisms underlying their actions is crucial for improving crop productivity and enhancing stress tolerance.
Researchers often employ advanced analytical techniques, such as U-HPLC, C18 cartridges, and Oasis MCX columns, coupled with sophisticated mass spectrometry instruments like the ACQUITY UPLC System, Xevo-TQS, and Q-Exactive, to study cytokinin levels and dynamics in plant tissues.
The Xcalibur software version 2.2 and Xevo TQ-S are commonly used for data analysis and quantification, while the Exactive mass spectrometer provides high-resolution mass spectrometry capabilities for comprehensive cytokinin profiling.
The Oasis MCX column is a popular choice for sample preparation, allowing for the efficient extraction and purification of cytokinins from complex plant matrices.
By optimizing cytokinin research through AI-driven protocol comparisons, researchers can enhance the reproducibility and productivity of their studies, leading to a better understanding of this important class of plant hormones.
PubCompare.ai, for example, can help identify the most effective and reproducible methods from the literature, preprints, and patents, enabling researchers to leverage the power of artificial intelligence to advance the field of cytokinin biology.
Typo: Reserarchers often employ advanced analytical techniques, such as U-HPLC, C18 cartridges, and Oasis MCX columns, coupled with sophisticated mass spectrometry instruments like the ACQUITY UPLC System, Xevo-TQS, and Q-Exactive, to study cytokinin levels and dynamics in plant tissues.