Partial Purification of a Cell-Division Factor from Peas

Rogozińska, Janina; Helgeson, John P.; Skoog, Folke; Lipton, S. H.; Strong, F. M.

doi:10.1104/pp.40.3.469

Cited by 32 publications

(11 citation statements)

References 19 publications

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“…mediated through the stimulation of endogenous ethylene production. Indeed, it has been demonstrated previously that one possible mode of action of daminozide on celery seeds is to stimulate the production of endogenous cytokinins, which themselves appear to play an important role in the breaking of thermodormancy [26] , Although in the present experiments the promotive effect of BA was apparently inhibited by AgNOa, there is a possibility that this might have been due to inactivation of the cytokinin through precipitation, a phenomenon which has been utilized in isolating cytokinins from plant tissues [21] , If the early germination events are dependent on ethylene it would be anticipated that pretreatment of germinating celery seeds with inhibitors of ethylene synthesis or action would inhibit germination even in the light. However, neither AVG nor AgN03 were particularly inhibitory, except for high, possibly phytotoxic, concentrations of AgNOa .…”

Section: Discussioncontrasting

confidence: 48%

Possible control of gibberellin-induced release of temperature-dependent primary dormancy in seeds of celery (Apium graveolens L.) by transmembrane ion fluxes

Thomas¹,

Sambrooks²

1985

Plant Growth Regul

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Abstract. The temperature-dependent primary dormancy of cv Florida 683 celery seeds in darkness was broken by GA,, (2 X lo-' M) alone but other growth regulators such as BA, ethephon or daminozide were necessary to break dormancy of cv Lathom Blanching seeds in the presence of GA,, at this concentration. Although AgNO, partially inhibited both the ethephon-and BA-induced germination of cv Lathom Blanching seeds in the presence of GA.,,, in the dark it did not affect the promotive action of daminozidc. Ethephon did not overcome the inhibitory action of high concentrations of AgNO, in thelight. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) did not inhibit the germination of cv Lathom Blanching seeds induced by growth regulators in the dark or in the absence of growth regulators in the light. Fusicoccin (FC) did not break celery seed dormancy unless applied in the presence of GA,, , . Germination of cv Lathom Blanching celery seeds treated with GA,,, at 16 "C in the dark was inhibited by the K+ ionophore benzo-18-crown-C-6 (18-C-6) and In the presence of Cal+ by the Ca*+ ionophore A23 187; the 18-C-6 inhibition was reversed by BA.It is concluded that the involvement of gibberellin in celery seed dormancy is not dependent on endogenous ethylene and is directly or indirectly controlled through the action of other hormones on transmembranc ion fluxes.

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Section: Discussioncontrasting

confidence: 48%

Possible control of gibberellin-induced release of temperature-dependent primary dormancy in seeds of celery (Apium graveolens L.) by transmembrane ion fluxes

Thomas¹,

Sambrooks²

1985

Plant Growth Regul

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“…Cytokinin activity was determined in the tobacco bioassay as described (10,19) and activity is expressed as kinetin equivalents (KE, the Ag of kinetin [6-furfurylaminopurine] required to give the same growth response as the test sample under the specified bioassay conditions).…”

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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“…Pea seedlings are well suited for investigating the natural occurrence of cytokinins in roots, as Carr and Burrows (4) demonstrated that cytokinins are present in the xylem exudate of the field pea. Rogozinska et al (28) and Zwar and Skoog (38) found that extracts from young pea seedlings exhibited cytokinin activity. Furthermore, Babcock and Morris (1) identified N6-(A2-isopentenyl)adenosine2 and the cis-isomer of zeatin ribonucleoside in tRNA hydrolysates of young seedling roots of pea.…”

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confidence: 99%

Cytokinins in Seedling Roots of Pea

1972

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The natural occurrence of cytokinins existing both in a free form and as a constituent of transfer RNA was examined in serial segments of young seedling roots of pea. Purified ethanol extracts of root apices were resolved into four factors capable of inducing soybean callus tissue proliferation. The most active factor was identified as zeatin or some closely related compound; it produced polyploid divisions and tracheary element differentiation when tested on cultured pea root segments. 2Abbreviations: IPA: 6-(3-methyl-2-butenylamino)-9-,3-D-ribofuranosylpurine or isopentenyladenosine; 2iP: 6-(3-methyl-2-butenylamino)purine or isopentenyladenine; ZR: 6-(4-hydroxy-3-methyl-2-cisbutenylamino)-9-,8-Dribofuranyosylpurine or zeatin ribonucleoside; Z: 6-(4-hydroxy-3-methyl-2-transbutenylamino)purine or zeatin. Extraction of Free Cytokinins. Immediately after harvesting, the different fractions of pea root segments were immersed separately in precooled absolute ethanol at -72 C. The frozen segments were collected by filtration on Whatman No. 1 filter paper and homogenized in an all-glass homogenizer in cold 80% (v/v) ethanol. The homogenized tissue was extracted for 3 hr in two portions of 80% ethanol and centrifuged at 10,000g for 15 min at 4 C. The original absolute ethanol fraction and the two 80% ethanol extracts were combined and the alcohol evaporated in vacuo. The water phase was adjusted to pH 8.0 and extracted with three equal volumes of 1-butanol which were then combined and taken to dryness in vacuo. The residue was redissolved in about 100 ml of water and adjusted to pH 2.0 with concentrated H2SO,. Fifteen milliliters of cold saturated AgNO, solution were added, and the mixture constantly was stirred for 12 hr at 4 C and centrifuged at 7000g for 15 min. The precipitate was washed with cold 2% AgNO, solution and stirred with 20 ml of 0.2 N HCl at 50 C for 30 min before being centrifuged at 7000g. This procedure was repeated twice. The combined acidic supernatants were adjusted to pH 2.5 with concentrated NaOH and percolated through a Dowex 50W-X8, H+ (200-400 mesh) column, 1.0 cm X 25.0 cm, which was then washed with 100 ml of water and eluted with 300 ml 3 N NH,OH. The eluate was evaporated in vacuo to about 50 ml and extracted at pH 8.0 with three equal volumes of 1-butanol which were combined and taken to dryness in vacuo. The resulting residue was dissolved in 80% ethanol and applied as a streak on Whatman No. 1 paper and developed with solvent system A, 1-butanol-acetic acid-water (12: 3:5 v/v). In addition, the following solvents were employed: B: 30 mM borate buffer at pH 8.4; C: ethyl methyl ketone saturated with water; D: l-butanol-1 N ammonium hydroxide (10:7 v/v); E: 1-butanol saturated with water. Each chromatogram was thoroughly air dried and cut into 10 equal RF strips and eluted with 80% ethanol. The eluates were reduced in volume and tested for cytokinin activity using the soybean callus bioassay.

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Partial Purification of a Cell-Division Factor from Peas

Cited by 32 publications

References 19 publications

Possible control of gibberellin-induced release of temperature-dependent primary dormancy in seeds of celery (Apium graveolens L.) by transmembrane ion fluxes

Possible control of gibberellin-induced release of temperature-dependent primary dormancy in seeds of celery (Apium graveolens L.) by transmembrane ion fluxes

Cytokinins in Corynebacterium fascians Cultures

Cytokinins in Seedling Roots of Pea

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