Folke Skoog

Cytokinins: Structure/activity relationships☆

Abstract Sixty-nine compounds, mostly purine derivatives and closely related substances, were tested for promotion of growth and regulation of organ formation in the tobacco bioassay to determine relationships between chemical structure and cytokinin activity. Forty-three substances were synthesized in this study, and 13 of these were reported for the first time. N 6 -Alkyladenines (I) varied in activity over a wide concentration range depending on the length of the alkyl chain. Starting with adenine, detectable at ⩾200 μM, activity increased with the chain length to an optimum for 6-pentylaminopurine detectable at ca. 0–001 μM, and then decreased to reach a barely detectable level for 6-decylaminopurine. The result of the incorporation of polar groups in the side chain was not necessarily reduction in activity. One hydroxyl group, as in zeatin (Id), improved the activity of 6-(γ,γ-dimethylallylamino)purine (Ib) if it affected it at all; two hydroxyl groups, as in 6-(2,3-dihydroxy-3-methylbutylamino)purine strongly reduced activity. Comparisons of 6-isoamylaminopurine with 6-(γ,γ-dimethylallylamino)purine and of other closely related pairs of compounds showed that a double bond in the side chain greatly increased cytokinin activity. Adenine derivatives with cyclic substituents in the N 6 -position (benzyl-Ic), cyclohexyl-, etc.) showed the same general range of activity, potentiation by unsaturation, and variation in activity with substituent size, etc. as did the alkyl derivatives. Heteroatoms in or on the substituent groups decreased activity (in the case of N or Cl) or had little effect (S for O in furfuryl). Of the mono-substituted adenines only the N 6 -derivatives definitely possessed cytokinin activity. The 1-(III), 3-(II), or 9-substituted adenines probably are inactive but could be activated by conversion to the N 6 -isomers. Except for slight activity in tests of high concentrations, which could be ascribed to contaminants, 7-substituted adenines were completely inactive. Modification in the adenine moiety lowered the cytokinin activity, often by 95 per cent or more. Substitution of N for the 8-C atom in kinetin and in 6-benzylaminopurine or S for the 6-amino N atom in 6-(γ,γ-dimethylallylamino)purine did not eliminate but drastically reduced activity in the tobacco bioassay. Elimination of the 6-amino group without substituting another group completely removed activity; thus, the purine derivatives, 1-benzylpurine and 1-(γ,γ-dimethylallyl)purine, were inactive in tests where the 1-adenine derivatives could be activated to give a positive response. Addition of a second substituent on the 1-or 3-position of N 6 -substituted adenines drastically reduced or eliminated cytokinin activity. It is suggested that the 1-position and possibly also the 3-position must be free. A second substituent in the N 6 -, 7-, or 9-position of N 6 -substituted adenine derivatives lowered but did not eliminate activity. Also, the disubstituted 1-adenine derivatives, 1,9-dibenzyladenine and 1,7-dibenzyladenine were active, presumably after rearrangement to the corresponding N 6 -substituted isomers.

Isolation of cytokinins from tRNA

Abstract A method is described for the isolation of cytokinins from tRNA. The cytokinin active ribosides are recovered from enzymatic hydrolysates of tRNA by extraction with ethyl acetate. The extracts are fractionated by chromatography on Sephadex LH-20 columns in 35% ethanol. The method appears to be quantitative and resolves individual cytokinins such as the ribosides 6-(3-methyl-2-butenylamino)-9-β d -ribofuranosylpurine (2iPA) and 6-(3-methyl-2-butenylamino)-2-methylthio-9-β- d -ribofuranosylpurine (ms2iPA).

Cytokinin from Soluble RNA of Escherichia coli: 6-(3-Methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine

We have isolated a compound responsible for the cytokinin activity of soluble RNA from Escherichia coli. The structure, indicated as 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuranosylpurine, C16H23N504S, on the basis of low-and high-reso!ution mass spectrometry, was established by unequivocal synthesis. The mass spectra, chromatographic behavior, and ultraviolet spectra of the compounds from natural and synthetic sources were identical.

Cell Contents of Nitrogen and Phosphorous as a Measure of Their Availability for Growth of Microcystis Aeruginosa

Problems arising from excessive growths of phytoplankton in fresh water lakes have become intensified through the discharge of nutrients in sewage effluents and industrial wastes into these bodies of water. It has been proposed that the amount of growth might be reduced by removal of critical essential elements before the effluents are released. The effectiveness of removing a particular essential element will depend on the extent to which it is normally a limiting or critical factor for algal growth, as obviously removal of a nutrient that is already present in a lake in abundant supply cannot produce the desired restilts. Reliable measurements on the degree to which a specific element is actually limiting the growth of algae in nature are difficult to obtain. They must be based on determinations of the availahility of the element to individual species in relation to their quantitative nutritional requirements. Granting that algae are capable of absorbing elements from water to the limits of detection by chemical procedures, still the simple method of estimating nutrient supplies by analysis of water collected from the area in which the algae are present may be of little value in this connection for the following reasons: (1) the total supply of a nutrient element is dependent on the total volume as well as the concentration of the solution from which it is absorbed, and this effective volume will vary with the extent to which the algae are moved about and come in contact with different volumes of water. This fact becomes particularly important when it is considered that the composition of the water may vary greatly front the top to bottom layers, especially in lakes that stratify, in a particular area and from one area to another, and that the algae collected in one place may be derived from areas scattered widely with respect to both depth and surface location. (2) The concentration of nutrients in the water 1 This work was supported in part by a research grant from the Division of Research Grants and Fellowships of the National Institute of Health, United States Public Health Service, and in part by the University Research Committee on funds from the Wisconsin Alumni Research Foundation. The authors wish to thank Miss Betty Robinson, Technical Assistant, and Mrs. Margaret Hirozawa, Technical Assistant, for their aid in carrying out these experiments may not reflect the total supply in that water over a period of time but rather the level reached as a result of continuous withdrawal by organisms and renewal by inflow and release from less available forms, or, in the case of nitrogen, perhaps also from biological fixation by certain organisms. These difficulties in determining available supplies of nutrients for algal growth are essentially similar to those encountered in determining nutrient availability to crops in terms of soil analyses but in some respects are accentuated by the high degree of independent movement of both the organisms and the water. In recent years, methods of evaluating nutrient availability to plants in terms of tissue composition have been developed (Lundegatrdh, 195 1; Ulrich, 1952; Thomas, 1945; Goodall and Gregory, 1947) and have been extensively employed for crop production. It has been found that, regardless of the composition of the external medium and other factors which may affect the uptake of a certain element, a fairly definite concentration (critical level) of that element must be maintained in the tissue to permit optimum growth. The tissue content of the element may continue to increase far above this critical level when it is in abundant supply, but the excess (luxury consumption) has little effect on growth. This paper presents a comparable procedure for evaluating the supply of nitrogen and phosphorus, the elements generally considered most likely to limit phytoplankton growth, to Microcystis aeruginosa in lake waters and includes preliminary results obtained in its application.

Cytokinin Activity: Localization in Transfer RNA Preparations

Transfer RNA from yeast, liver, and Escherichia coli has cytokinin activity in the tobacco callus bioassay, whereas ribosomal RNA from yeast is inactive. In contrast to fractions of yeast transfer RNA rich in serine acceptor and cytokinin activity, preparations (70 to 90 percent pure) of arginine transfer RNA2, glycine transfer RNA, phenylalanine transfer RNA, and valine transfer RNA1 and of highly purified alanine transfer RNA from yeast were inactive at concentrations of 20 to 2500 micrograms per liter. One molecule of 6-(γ,γ-dimethylallylamino) purine per 20 molecules of yeast tRNA would account for the observed cytokinin activity. The number of major molecular species contributing to cytokinin activity of transfer RNA, therefore, must be small.

Cytokinin antagonists: Synthesis and physiological effects of 7-Substituted 3-Methylpyrazolo[4,3-d]Pyrimidines

Abstract A series of sixteen pyrazolo[4,3- d ]pyrimidine derivatives has been synthesized and tested for cytokinin antagonist activity in the tobacco bioassay

Cytokinins: Synthesis and growth-promoting activity of 2-substituted compounds in the N6-isopentenyladenine and zeatin series

Abstract Fourteen compounds were tested for relative promotion of cell division and growth (cytokinin) activity in the tobacco bioassay. The results suggested that 2-substituted- N 6 -(hydroxy)isopentenylaminopurines were generally less active than their unsubstituted couterparts. Thus, a 2-OH substituent greatly lowered cytokinin activity in both the isopentenyl and hydroxyisopentenyl (zeatin) series, while 2-NH 2 and 2-SCH 3 groups had a lesser effect on activity and a 2-Cl substituent had a negligible effect. Mass spectra were determined for all of the 9-β- d -ribofuranosides and for a number of the purines as well; the fragmentation patterns were consistent and characteristic, providing a possible systematic approach to the identification of new 2-substituted- N 6 -(hydroxy)isopentenyladenines.

Autoradiographic and microspectrophotometric studies of DNA synthesis in excised tobacco pith tissue

SummaryPith tissue was cultured on modified White’s nutrient medium supplemented, except for controls, with 2 mg/l of IAA and/or 0,5 mg/l of kinetin. For autoradiographs sections were used from tissue grown on medium containing tritiated thymidine.Nuclear DNA contents (Feulgen) were measured by the microspectrophotometric two-wavelengths method. No fading of Feulgen dye in nuclei was found in 11 weeks, in contrast to considerable fading observed in earlier work when a different batch of basic fuchsin had been employed.Counts of radioactive nuclei in autoradiographs agreed well with microspectrophotometric results on the occurrance of DNA synthesis.In control cultures, with or without tritiated thymidine, DNA doubling took place in about 20% of the nuclei during the first two days but in few, if any, thereafter.It was confirmed that kinetin, as well as IAA, increases the frequency of nuclei undergoing DNA synthesis. However, IAA, in contrast with kinetin, still induced considerable DNA doubling after two days. Continued cell reproduction was maintained only in the presence of both substances.

Comparison of cytokinin activities of naturally occurring ribonucleosides and corresponding bases

Abstract Cytokinin activities in the tobacco bioassay have been determined for four adenosine derivatives known to be components of wheat germ t RNA: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β- d -ribofuranosylpurine, 6-(3-methyl-2-butenylamino)-9-β- d -ribofuranosylpurine, 6-(4-hydroxy-3-methyl-2-butenylamino)- 2-methylthio-9-β- d -ribofuranosylpurine, and 6-(3-methyl-2-butenylamino)-2-methylthio-9-β- d -ribofuranosylpurine. Also determined and compared with the four natural components of t RNA were the activities of the four 3-methylbutylamino analogs of the naturally occurring species and the eight substituted purines corresponding to both sets of ribonucleosides. The systematic structural modifications within this group of sixteen compounds were reflected in the variations in cytokinin activity with the level of modification.

Cytokinin of wheat germ transfer RNA: 6-(4-Hydroxy-3-methyl-2-butenylamino) -2-methylthio-9-β-D-ribofuranosylpurine

A new modified nucleoside is responsible, in part, for the cytokinin activity of transfer RNA from wheat germ. The structure as judged by mass spec-trometry is 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-D-ribofuran-osylpurine. Unequivocal synthesis afforded material having ultraviolet, mass spectral, and chromatographic properties identical with those of the natural product.