Zeatin and zeatinriboside were identified from the Coconut Milk

Identification of Zeatin and Zeatinriboside in Coconut Milk. Zeatin and zeatinriboside were identified from the milk of mature Cocos nucifera fruits. It would appear as if the two compounds are present in roughly equal proportions. Ethyl acetate extraction in a liquid-liquid extractor at an alkaline pH (8.0) proved to be a very efficient method of extracting zeatinriboside. Partitioning with water-saturated n-butanol proved to be the best way of extracting zeatin.  
Author Information : Departments of Botany and Chemistry, University of Natal, Pietermaritzburg, South Africa
How to Cite STADEN, J. V. and DREWES, S. E. (1975), Identification of Zeatin and Zeatinriboside in Coconut Milk. Physiologia Plantarum, 34: 106–109. doi:0.1111/j.1399-3054.1975.tb03801.x
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Concentration Gradients of Zeatin Riboside(in the Maize Stem), Measurement by a Specific Enzyme Immunoassay

A sensitive, specific enzyme immunoassay (EIA) for trans-zeatin riboside (ZR) and trans-zeatin (Z) in the 0.3 to 30 picomole range has been described. The reliability of the method for measuring ZR + Z in partially purified extracts of Zea mays L. tissues was verified by highperformance liquid chromatography. EIA measurements showed that there was a concentration gradient of ZR + Z along the length of the Zea stem. The topmost internodes, internodes 7 and 8 counting from the coleoptilar node, had the highest concentration ([similar, equals]130 picomoles per gram fresh weight). Moving basipetally, the concentration dropped [similar, equals]10-fold to a minimum at internode 4, and then increased slightly in internodes 2 and 3. There were also gradients within each internode. The five lowest internodes contained the highest concentrations toward their apical end, the region which included the node; this asymmetry was less pronounced near the top of the plant. Source : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1067032/ Download Full text - Concentration Gradients of trans-Zeatin Riboside and trans-Zeatin in the Maize Stem, Link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1067032/pdf/plntphys00578-0089.pdf Plant Physiol. 1984 August; 75(4): 959–963. PMCID: PMC1067032

tRNA is the source of low-level trans-zeatin production in Methylobacterium spp

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered.

trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo.

In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.

Determination of cytokinins in plant samples by polymer monolith microextraction coupled with hydrophilic interaction chromatography-tandem mass spectrometry

Source: http://pubs.rsc.org/en/Content/ArticleLanding/2010/AY/C0AY00334D
Issued: Analytical Methods,Issue 11, 2010 ,Early applied demonstrations of new analytical methods with clear societal impact
In this study, a sensitive assay of cytokinins was developed using polymer monolith microextraction/hydrophilic interaction chromatography/electrospray ionization-tandem mass spectrometry (PMME/HILIC/ESI-MS/MS). The extraction was realized on a poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-ethylene dimethacrylate) (poly(AMPS-co-EDMA)) capillary monolith and the subsequent separation was carried out on a Luna silica column.
Several parameters of PMME and HILIC were optimized to obtain the optimum results. After optimizing the extraction conditions, 10 mM ammonium formate of pH 2.5 was chosen as the matrix solution to obtain the highest extraction efficiency. The MS sensitivity for cytokinins investigated could be enhanced 3-fold by the use of hydrophilic interaction chromatography with the mobile phase of 85% acetonitrile with 0.01% (v/v) formic acid and 15% water with 0.01% (v/v) formic acid, when compared to the use of conventional reversed phase liquid chromatography (RPLC). Good linearities were obtained for five cytokinins with correlation coefficients (R2) > 0.9962. The LODs (S/N = 3) for the targets were found to be 0.0028–0.068 ng mL−1. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations (RSDs) less than 12.7% and the recoveries in plant samples ranged from 70.3% to 113.3%. The method was applied to the determination of cytokinins in Oryza sativa, Arabidopsis thaliana and oil seed rape tissues.