Evidence in support of biosynthesis de novo of free cytokinins.

The four cytokinins in the tRNA from Lupinus luteus L. seeds have been purified and identified as ribosyl-cis-zeatin, 2-methylthio-ribosylzeatin, (Δ (2)-isopentenyl)adenosine and 2-methylthio-N(6)-(Δ (2)-isopentenyl)adenosine. These structures have been assigned on the basis of their chromatographic mobilities and the spectroscopic data of the parent materials and their silylated derivatives. The tRNA isolated from Populus x robusta Schneid. leaves contained four cytokinins with identical chromatographic properties to those identified in Lupinus luteus seed tRNA. No evidence was obtained for the presence, in tRNA, of the naturally occurring free cytokinins identified in these plant species, dihydrozeatin (Lupinus luteus) and N(6)-(2-hydroxybenzyl)adenosine (Populus x robusta). This is evidence in support of the possibility that free cytokinins can arise by biosynthesis de novo and are not exclusively by-products released intact during tRNA turnover.

Mode of action of N,N'-diphenylurea: The isolation and identification of the cytokinins in the transfer RNA from tobacco callus grown in the presence of N,N'-diphenylurea.

The tRNA from cytokinin-dependent tobacco callus (Nicotiana tabacum) grown on mineral medium containing N,N'-diphenylurea as the source of cytokinin was found to contain 3 cytokinin-active ribonucleosides. The 2 ribonucleosides present in the largest amounts were identified conclusively by their chromatographic properties, ultra-violet and low-resolution mass spectra as the naturally-occurring cytokinins 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-ß-D-ribofuranosylpurine and 6-(3-methyl-2-butenylamino)-9-ß-ribofuranosylpurine. A third ribonucleoside, present in smaller amounts, was identified as another naturally-occurring cytokinin 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-ß-D-ribofuranosylpurine on the basis of its chromatographic behaviour. No evidence was found to associate the mode of action of the non-purine cytokinin, N,N'-diphenylurea, with tRNA.

Identification of the cytokinin-active ribonucleosides in pure Escherichia coli tRNA species.

The distribution of the two cytokinin-active ribonucleosides 6-(3-methyl-2-butenylamino)-9-beta-D-ribofuranosylpurine (1) and 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine (2) in tRNA(Phe), tRNA(UUG) (Leu), tRNA(UCR) (Ser), tRNA(Tyr), tRNA(Cys), and tRNA(2) (Trp) from stationary phase Escherichia coli has been studied. Compound 2 predominated in all these species except tRNA(Phe), in which compound 1 was greatly in excess. Compound 1 was present in relatively small amounts in tRNA(UUG) (Leu), tRNA(Cys), and tRNA(2) (Trp), and was not detected in tRNA(UCR) (Ser) and tRNA(Tyr). Cells from the early logarithmic phase had the highest cytokinin content in their tRNA. Stationary phase tRNA had a slight increase in the amount of 1, which may be explained by the appearance of the new tRNA(Phe). No differences were observed between the chromatographic patterns of tRNAs from early-, mid-, and latelogarithmic phases.

Cytokinin of wheat germ transfer RNA: 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-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-beta-D-ribofuran-osylpurine. Unequivocal synthesis afforded material having ultraviolet, mass spectral, and chromatographic properties identical with those of the natural product.

Cytokinins: distribution in transfer RNA species of Escherichia coli.

This distribution of cytokinin activity in tRNA species of Escherichia coli has been determined. Cytokinin activity was restricted to tRNA species recognizing codons with the initial letter U. The following have been shown to contain a cytokinin: tRNA(Phe), tRNA(Leu) UUG, tRNA(Ser)UC(G) (A), tRNA(Tyr), and tRNA(Try). tRNA(Cys) was present in fractions with cytokinin activity but was not sufficiently pure to prove it to be active. One tRNA(Ser) UC(C) (U) species was inactive. All major tRNA species recognizing condons with initial letters other than U were devoid of cytokinin activity in the tobacco bioassay. The significance of these findings is discussed as providing a possible mechanism of quantitative modulation at the translation stage of gene-controlled protein biosynthesis.

Effects of flooding the root system of sunflower plants on the cytokin in content in the xylem sap.

The severe chlorosis observed in the lower most of flooded sunflower plants (Helianthus annuus L. cv. Tall Single) may lie initiated by a reduction in the import of cytokinins by the stoot from the flooded root system. Experiment indicates that during 12 hours following the release of flooding, plants previously flooded for 72 hours or less recover their ability to exude sap when the root systems are aerated, and the root systems synthesize and export amino-acids to the shoot. Plants flooded for longer periods lose these abilities. The metabolic activity of the root apices declines parallel with the decline in eytnkinin concentration in the sap with increase in flooding time up to 72 hours. Flooding for 96 hours drastically reduce all four parameters of root activity. After flooding for this period there was a large increase in the number of blackened and tetrazolium-negative root apices which were in all probability dead. The correlation between the metabolic activity of the root apices and the total cytokinin content of the sap supports tbe view that root apices may be sites of cytokinin synthesis.

Cytokinin from soluble RNA of Escherichia coli: 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-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-beta-D-ribofuranosylpurine, C(16)H(23)N(5)0(4)S, 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.

Studies on leaflet abscission of blue lupin leaves : I. Interaction of leaf age, kinetin and light.

In blue lupin leaves, each leaflet abscises at an abscission zone situated in the pulvinus at its base. The time to abscission of leaflets of detached leaves is proportional to leaf age. Light accelerates abscission; within certain limits the acceleration is the greater the younger the leaf. At a given concentration, kinetin applied to a single leaflet accelerates leaflet abscission in young leaves kept in darkness, delays it in older ones. There is an interaction between kinetin and light which is dependent also on leaf age and kinetin concentration. The leaf can be considered as consisting of three regions, the petiole, the pulvinar region and the leaflets. The effects of kinetin and of light as well as their interactions depent on the regions of the leaf treated with these agents. Kinetin applied to a leaflet of a young leaf kept in darkness accelerates abscission, but kinetin applied to the pulvinar region of a similar leaf kept in darkness delays abscission. When any part of a leaf is illuminated, abscission is accelerated. The most light-sensitive region of the leaf is the pulvinar region, despite its relatively small area. Acceleration of abscission by light is greatest when illumination of the pulvinar region is combined with illumination of either the leaflets or the petiole. The interaction of light with kinetin is complex. Where the illuminated area includes the pulvinar region, kinetin delays abscission. This effect is most marked in the case where the pulvinar region alone is illuminated and kinetin is applied to a leaflet.Intrafoliar abscission as found in lupin leaves permits study of complex interactions of both distal and proximal stimuli involved in abscission.

Studies on the abscission of blue lupin leaves : II. effects of kinetin and light on pulvinar and other types of explants.

The responses of three types of explants of blue lupin leaves are considered: pulvinar explants, consisting of the pulvinar region alone, petiolar explants, consisting of the pulvinar region plus petiole and laminar explants consisting of the pulvinar region plus leaflets. Abscission is accelerated by removal of the leaflets; removal of the petiole has much less effect. Pulvinar explants fail to abscise in darkness but are the first to abscise in the light. This is in accordance with previous evidence of high light sensitivity of the pulvinar region. Kinetin applied directly to the pulvinar region delays abscission, as does kinetin supplied via the transpiration stream. As shown by experiment, this is probably due to transported kinetin reaching the abscission zones of the pulvinar region. The effects of photoperiodic treatments on explants or whole leaves are described. Abscission in the whole leaf is delayed by short daily photoperiods; the delay reaches a maximum with 8 hours light per day. However, abscission is more rapid in continuous light than in darkness. Removal of the leaflets greatly accelerates abscission even in darkness. The pulvinar explant fails to abscise with photoperiods of 4 hours or less; although it appears to have a long day response, preliminary attempts failed to demonstrate that this is a true photoperiodic response (replacement of a long day by a short day together with a light break). The complex responses of leaves and explants to day length lend further support to the hypothesis that light has effects on abscission other than in photosynthesis.