Gas-Liquid Chromatographic Isolation of Cytokinins from Natural Sources

Upper, Christen D.; Helgeson, John P.; Kemp, John D.; Schmidt, C. J.

doi:10.1104/pp.45.5.543

Cited by 74 publications

(19 citation statements)

References 21 publications

(24 reference statements)

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“…The syntheses and properties of authentic c-io6Ade, t-io6Ade, c-ms2io6Ade, and t-ms2io6Ade used as standards in the identification of cytokinins in this study have been described (17,27).…”

Section: Methodsmentioning

confidence: 99%

Cytokinins in Corynebacterium fascians Cultures

et al. 1976

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In addition to the four cytokinins, 6-(3-methyl-2-butenylamino)purine, 6-methylaminopunne and the cis and trans isomers of 6-(4-hydroxy-3-methyl-2-butenylamino)purine, reported earlier from our laboratories, three cytokinin-active fractions have been obtained from the aqueous medium of 6-day-old Corynebacterium fascians cultures. One of these has been identified as 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine (2-methylthio-cis-zeatin, c-ms2io6Ade).The elution volumes of the other two frctions correspond to those of authentic 6-(4-hydroxy-3-methyl-2-butenylamino)-9-p-D-ribofuranosylpunne and 6-(3-methyl-2-butenylamino)-9-j3-D-ribofuranosylpurine, indicating the presence of trace amounts of these two ribonudeosides.Fasciation and modified growth of seedlings closely resembling symptoms of infection with Corynebacterium fascians have been induced in Pisum by soaking the seeds in high concentrations of kinetin (20). That in fact these symptoms of infection by the bacterium might be due to its production of cytokinin was indicated by the ability of extracts of pure cultures to release lateral buds of Pisum from apical dominance in the same manner as kinetin (25). Isolation of cytokinin-active material from aqueous cultures of the same stock5 was achieved by solvent extraction and chromatography. Three crystalline fractions with cytokinin activity as monitored in the tobacco bioassay were obtained (8, 10). The most active fraction was identified as 6(3-methyl-2-butenylamino)purine (i6Ade) (6), known at that time only as a synthetic, highly active cytokinin (5, 9). The second fraction contained nicotinamide. Inasmuch as synthetic nicotinamide was inactive in the tobacco bioassay, the activity of the isolated material was ascribed to a contaminant. This cytokinin was recently identified as 6-(4-hydroxy-3-methyl-cis-2-buteny- for the activity of this fraction in the tobacco bioassay. Hence, another more potent cytokinin was assumed to be present as a contaminant and to be mainly responsible for the cytokinin activity in this fraction. We now report the purification and characterization of this cytokinin from Corynebacterium fascians culture medium as 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine (2-methylthio-cis-zeatin, c-ms2io6Ade). Paper Chromatography. Whatman No. 1 filter paper, washed as described (21), was used. Cytokinin samples were dissolved in small volumes of 95% ethanol and applied to the paper as 5-cm streaks. The chromatograms were developed by ascending chromatography over 30 cm. UV-absorbipg bands were recovered from the chromatograms in small volumes (about 0.5-1 ml) by 749 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from MATERIALS AND METHODS Growth of Corynebacterium fascians

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“…The syntheses and properties of authentic c-io6Ade, t-io6Ade, c-ms2io6Ade, and t-ms2io6Ade used as standards in the identification of cytokinins in this study have been described (17,27).…”

Section: Methodsmentioning

confidence: 99%

Cytokinins in Corynebacterium fascians Cultures

et al. 1976

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“…The extracts were combined and evaporated to dryness in a microsilylation vessel with a stream of N2. The resulting sample was freed of any residual water by repeatedly suspending it in an acetone-benzene mixture (1:4, v/v) and evaporating to dryness with a stream of N2 (19). The sample was trimethylsilylated by incubating at 60 C for 45 min in 10 ,1u of dry pyridine and 40 ,ul of bis(trimethylsilyl)trifluoro acetamide (4, 18).…”

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confidence: 99%

Kinetin Incorporated into Tobacco Callus Ribosomal RNA and Transfer RNA Preparations

Murai¹,

Taller²,

Armstrong³

et al. 1977

Plant Physiol.

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Kinetin, N6-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) caUus RNA isolated from rapidly growing tissue cultured in the presence of N6-furfuryladenine-8-14C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N6-furfuryladenosine (frA) in the rRNA and tRNA preparations from the tobacco cafus. The rRNA contained over 90% of these fr6A moieties. The extent of kinetin incorporation was four times greater than that observed for N6-benzyladenine. The radiochemical purity of the recovered fr6A was confirmed by three successive chromatographic prifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 elated with water). A cytokinin-active ribonadeoside with elation volumes corresponding to frWA was isolated from the tobacco caflus rRNA preparation. This compound was analyzed by gasliquid chromatogrphy and rtorously chacterized as N6-furfuyladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative.The occurrence, biosynthesis, and function of the cytokininactive ribonucleosides in tRNA have been examined in detail in Escherichia coli (9,14,16). N6-(A2-Isopentenyl)adenosine and ms2i6A3 have been identified as cytokinin-active ribonucleosides from E. coli tRNA. They have been found to be located next to the 3'-end of the anticodon of tRNA species responding to codons beginning with uridine. The cytokinin i6A is synthesized by the transfer of the isopentenyl moiety from A2-isopentenyl pyrophosphate to the specific adenosine residues in this location N6-furfuryladenosine or 6-furfurylamino-9-f>-D-ribofuranosylpurine; i6A: N6-(A2-isopentenyl)adenosine or 6-(3-methyl-2-butenylamino)-9-,D3--ribofuranosylpurine; ms2i6A: 6-(3-methyl-2-butenylamino)-2-methylthio-9-f3-D-ribofuranosylpurine; c-io6A: 6-(cis-4-hydroxy-3-methyl-2-butenylamino)-9-,-D-ribofuranosylpurine; t-io6A: 6-(trans-4-hydroxy-3-methyl-2-butenylamino)-9-,-E.nribofuranosylpurine; c-ms2io6A: 6-(cis-4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-/-E>-ribofuranosylpurine; t-ms2io6A: 6-(trans-4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-3-n-ribofuranosylpurine; CTAB: cetyltrimethylammonium bromide; TEAB: tetraethylammonium bromide; DEAE-cellulose: diethylaminoethyl-cellulose; 8HQ: 8-hydroxyquinoline; NSA: naphthalene-1,5-disulfonic acid disodium salt.in preformed tRNA molecules. Evidence that the cytokininactive components of tRNA function to insure proper codonanticodon interaction of the mRNA-tRNA complex on ribosomes has also been obtained in in vitro E. coli systems.The subsequent identification of cytokinins in tRNA preparations from various plant sources stimulated interest in the possibility that the hormonal regulation of plant growth and development by cytokinins might be due to their control of the synthesis and/or function of particular tRNA species (16). The exact location of the cytokinin moieties in nucleotide sequences of particular plant tRNA species...

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“…One to 5 ,ul of each TMS derivative was injected into a Varian Aerograph GLC (Model 1200) equipped with a flame ionization detector, a temperature programmer, and a stainless steel column (3 mm X 150 cm) packed with 3% QF-l on 60/80 mesh Chrom-Q. The GLC was operated under the following conditions: hydrogen flow, 30 ml/min; air flow, 200 ml/min; nitrogen carrier gas flow, 30 ml/min; injector temperature, 200 C; and detector temperature, 275 C. Temperature programming started at 100 C and increased linearly to 250 C at the rate of 4 Radioactivity Studies. The following radioactive hormones were used for "spiking" the plant tissue: ABA-2-"C, specific radioactivity 26.3 mCi/mmole (26); kinetin-8-"C, specific radioactivity 20 ,uCi/mmmole (Calbiochem); gibberellic acid-C, specific radioactivity 5.29 tCi/mmole (gift from Dr. C. W. Coggins, Jr.); and indole-3-(acetic acid-I-`C), specific radioactivity 183 1.Ci/mmole (gift from Dr. L. N. Lewis).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, development of GLC has provided a physicalchemical assay of ABA (11,19), auxins (13,29), cytokinins (30), and gibberellins (8), demonstrating the feasibility of qualitative as well as quantitative measurements of naturally occurring plant hormones. Gas-liquid chromatography-mass spectrometry has been used for further identification of some of these hormones (15,21,22,31).…”

Section: Introductionmentioning

confidence: 99%

Identification of Plant Hormones from Cotton Ovules

Shindy

Smith²

1975

Plant Physiol.

162

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An extract from 8-day-old cotton ovules (Gossypiumii hirsutuint L.) was partitioned into three fractions and each fraction was derivatized and analyzed separately. Gas-liquid chromatography and computer-controlled gas-liquid chromatography-mass spectrometry were used to separate, measure, and identify the naturally occurring plant hormones. A single extract contained abscisic acid, indoleacetic acid, and gibberellins A1, A3, A4. A7, A9, and A13 in the first fraction; ethyl indole-3-acetate and indole-3-aldehyde in the second fraction; and the cytokinins 6-(3-methyl-4-hydroxybutylamino)purine (dihydrozeatin), 6-(4-hydroxy-3-methyl-2-trans-butenylamino) purine (zeatin), 6-(3-methyl-2-butenylamino)purine (2iP), 6-(3-methyl-2-butenylamino) .9 -A -D -ribofuranosylpurine (2iPA), and 6-(4-hydroxy-3-methyl-2-trans-butenylamino) -9-/3-D-ribofuranosylpurine (zeatin riboside) in the third fraction.Many research workers have attempted to identify and measure hormone levels in cotton plants by using various bioassays (1,6,7,9,24,25,28). Because plant hormones are present in microgram quantities, the usefulness of bioassays has been limited because of interference of excessive quantities of impurities in extracts containing the hormones (2,14).Recently, development of GLC has provided a physicalchemical assay of ABA (11,19), auxins (13,29), cytokinins (30), and gibberellins (8), demonstrating the feasibility of qualitative as well as quantitative measurements of naturally occurring plant hormones. Gas-liquid chromatography-mass spectrometry has been used for further identification of some of these hormones (15,21,22,31).Computer-controlled GLC-MS4 has been used for identifica- 6-(4-hydroxy-3-methyl-2-trans-butenylamino)purine; dihydrozeatin: 6-(3-methyl-4-hydroxybutylamino)purine; zeatin riboside: 6-(4-hydroxy-3-methyl-2-trans-butenylamino)-9-PB-D-ribofuranosylpurine; 2iP: 6-(3-methyl-2-butenylamino)purine; 2iPA: 6-(3-methyl-2-butenylamino)-9-fl-D-ribofuranosylpurine: 3iPA: 6-(3-methyl-3-butenylamino)-9-I3-D-ribofuranosylpurine.tion of cytokinins (31), gibberellins, and ABA (21). Abscisic acid (11) and IAA (32) have been identified from cotton tissues by GLC but we have found no report in which ABA, gibberellins, cytokinins, and auxins were measured in a single extract by physical-chemical assays.The objective of this investigation was to utilize GLC and computer-controlled GLC-MS to separate, identify, and measure individual plant hormones of four major groups from a single extract of ovules from 8-day-old cotton fruit. MATERIALS AND METHODSCotton plants (Gossypiu,n hirsutum L., cv. Acala SJ-1) were grown in a greenhouse. Cotton flowers were tagged at anthesis and bolls were harvested 8 days thereafter. The developing ovules were separated from the fruits, frozen immediately on Dry Ice, and stored at -20 C until the time of extraction. Extraction Procedure. In different experiments, 2 to 5 g of frozen ovules were ground in cold 80% (v/v) aqueous methanol with a mortar and pestle. The macerate was transferred to a fla...

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Gas-Liquid Chromatographic Isolation of Cytokinins from Natural Sources

Cited by 74 publications

References 21 publications

Cytokinins in Corynebacterium fascians Cultures

Cytokinins in Corynebacterium fascians Cultures

Kinetin Incorporated into Tobacco Callus Ribosomal RNA and Transfer RNA Preparations

Identification of Plant Hormones from Cotton Ovules

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