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Effect of the kinetin-naphthaleneacetic acid interaction upon total RNA and protein in senescing detached leaves.

The interaction between kinetin and naphthaleneacetic acid in the regulation of senescence of excised tissue of mature broccoli leaves has been used to examine the extent of synchrony between changes in chlorophyll, RNA, and protein. Kinetin increased the net uptake of (14)C-labeled orotic acid and leucine. Naphthaleneacetic acid decreased the effect of kinetin on net uptake after long treatment, but in short-time treatments the auxin increased the effect of kinetin on net uptake. Results of long (24 hr) treatments indicated a general synchrony between the loss of RNA, protein, and chlorophyll. Naphthaleneacetic acid reduced the stabilizing effect of kinetin upon chlorophyll content and upon the content and synthesis of RNA. In short-time experiments, however, RNA content and synthesis were transiently increased by kinetin, and further increased by kinetin plus naphthaleneacetic acid, while chlorophyll content decreased in the presence of kinetin and decreased further in the presence of kinetin plus naphthaleneacetic acid. Actinomycin-D accelerated the loss of chlorophyll, RNA and protein and strongly depressed the rate of RNA synthesis. In the presence of actinomycin-D the stabilizing effect of kinetin upon RNA was substantially reduced. In contrast, the chlorophyll and protein contents remained higher than in the control. Actinomycin-D did not nullify the basal incorporation of orotic acid into RNA, nor did it negate the effect of kinetin upon incorporation. The failure of synchrony between changes in chlorophyll and RNA does not substantiate the proposal that kinetin regulates senescence by a direct effect upon DNA-dependent RNA synthesis.

Naphthaleneacetic Acids

Effect of kinetin on the formation of chromosome aberrations during irradiation of Crepis capillaris L. seeds and its radioprotective effect.

The object of this investigation was the mutagenic effect of kinetin (6-phurphuryl-aminopurine) on chromosomes of Crepis capillaris L. Dry seeds were soaked in the 0.05% kinetin solution for 4, 6 and 8 hours before the irradiation with X-rays. It is observed that soaking of dry seeds in kinetin solution stimulates the appearance of chromatid aberrations for almost two hours earlier, than in the control (soaking seeds in water). A regular increase of the chromatid-type rearrangements with increasing the duration of soaking in the kinetin solution was observed (3.3%, 16-20% and almost 50% at 4 hours; 6 hours' and 8 hours' soaking respectively). This increase is indicative for the effect of kinetin on the processes taking place in the cell before the irradiation. Apparently kinetin stimulates the onset of the stage S for a certain proportion of cells, and thus at the moment of irradiation the cell population is at the asynchronous state. A considerable radioprotective effect of kinetin was observed in all the variants of the experiment, which is not associated with the capacity of stimulating the initiation of chromatid aberrations inherent in kinetin. Kinetin alone (without irradiation) exerts no mutagenic effect on the cells of Crepis capillaris.

Chromosome Aberrations

A mechanism for the in vivo formation of N6-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA.

Recently, we have reported the presence of kinetin (N6-furfuryladenine) in commercially available DNA, in freshly extracted cellular DNA and in plant cell extracts. We have also found that kinetin has electrochemical properties which can be used for monitoring the level of this modified base in DNA. Here, for the first time, we propose a mechanism for the formation of kinetin in DNA in vivo, based on the analyses of its mass spectra. Since hydroxy radical oxidation at the carbon 5' of the deoxyribose residue yields furfural, we propose that this aldehyde reacts with the amino group of adenine and, after intramolecular rearrangement, kinetin is formed in vivo. Thus kinetin is the first stable secondary DNA damage product known to date with very well defined cytokinin and anti-aging properties, linked to oxidative processes in the cell. These results also indicate that N6-furfuryladenine or kinetin is an important component of a new salvage pathway of hydroxy radicals constituting a 'free radical sink'. In this way, the cells can neutralize the harmful properties of hydroxyl radical reaction products, such as furfural, and respond to oxidative stress by inducing defence mechanisms of maintenance and repair.

Adenine

Alcohol dehydrogenase (EC 1.1.1.1) isozymes as markers at 2,4-dichlorophenoxyacetic acid x kinetin combinations in callus cultures of Cereus peruvianus (Cactaceae).

Alcohol dehydrogenase (ADH; EC 1.1.1.1) isozymes were investigated in tissue of Cereus peruvianus cultured in different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Five ADH isozymes were detected in starch gel and showed different patterns in seeds, seedlings, calli cultured at 32 and 22 degrees C, and plants regenerated from calli cultured in three 2,4-D and kinetin combinations. Four phenotypes formed by different combinations of ADH-2, ADH-3, ADH-4, and ADH-5 were detected in calli cultured at 32 degrees C and in plants regenerated from calli. ADH-1 isozyme was detected only in calli subcultured for 1 or 2 weeks at 22 degrees C and was indicated as a marker of stress conditions that affect the growth of C. peruvianus callus tissues in culture. ADH phenotypes with either a higher or a lower number of isozymes were detected in different proportions in the callus tissues cultured in media containing different 2,4-D and kinetin ratios. ADH isozyme patterns were found to be sensitive markers at the highest kinetin concentration or at high kinetin/2,4-D ratios. The results indicate a high correlation between the ADH isozyme patterns and the capacity for regeneration. Thus, ADH isozymes are indicated as good biochemical markers and as a powerful tool for monitoring studies of C. peruvianus callus cultures.

2,4-Dichlorophenoxyacetic Acid

Hill-acitivity and P700 concentration of chloroplasts isolated from radish seedlings treated with-indoleacetic acid, kinetin of gibberellic acid.

The Hill-activity (reduction of DCPIP or methylviolgen) and the concentration of P700 were studied in chloroplasts isolated from cotyledons of radish seedlings (Raphanus sativus L. saxa Treib), which had been grown with the addition of beta-indoleacetic acid (IAA), kinetin, or gibberellic acid. 1) The photosynthetic activity of young chloroplasts from 3 day old Raphanus seedlings is very high (c. 180 micron mol O2/mol chlorophyll X h) and decreases continuously thereafter with increasing age. The steady state Hill-activity is reached after 8 to 10 days (values of 55 to 50 micron mol O2/mg chlorophyll X h). 2) Chloroplasts from plants treated with IAA or kinetin not only exhibit higher plastoquinone levels 1,2, but also a higher P700-content and a higher Hill-activity. The promotion effect is more pronounced with kinetin (+36 tb 40%) than with IAA (+12 to 17%). 3) Gibberellic acid has a different effect on composition and activity of chloroplasts. In younger seedlings the Hill-activity appears to be somewhat stimulated, without promotion effect on plastoquinone 2 or P700 concentration. After 10 days GA3-treated plants show signs of chlorosis combined with a strong decrease in photosynthetic activity. 4) The data clearly demonstrate that the composition and activity of the photosynthetic apparatus are under phytohormone control. IAA and even better kinetin promote the light induced formation of pigment systems and electrontransport chains. GA3 seems to block the rebuilding of the photosynthetic apparatus under steady state conditions.

2,6-Dichloroindophenol

Influence of kinetin (6-furfurylo-amino-purine) on human fibroblasts in the cell culture.

Influence of kinetin on human fibroblasts cultured in vitro was studied. Investigations were carried out on I passage fibroblasts, taken from the human skin. Feulgen's method and radioautography were used. Quantitative estimations of DNA in nuclei of fibroblasts were done on cytophotometer. An increase in amount of DNA in nuclei of fibroblasts cytophotometer. An increase in amount of DNA in nuclei of fibroblasts cultured in the presence of kinetin was found. Stronger 3H uridine incorporation was recorded in cultures after administration of the lowest dosis of kinetin. Kinetin, derivative of adenine (6-furfurylo-amino-purine) belongs to cytokinins and it regulates the growth of plants.

Adenine

Stomatal guard cell responses to kinetin and natriuretic peptides are cGMP-dependent.

Immunological evidence suggest that plants contain natriuretic peptides (NPs) and furthermore (3-[I]iodotyrosol) rat atrial NP (rANP) binds specifically to plant membranes. rANP and immunoaffinity-purified plant NP analogues also promote concentration-dependent stomatal opening. Here we report that kinetin, a synthetic cytokinin, and rANP induce stomatal opening in Tradescantia albiflora and that the effect of rANP is critically dependent on the secondary structure of the peptide hormone. The native circular molecule is active, whereas the linearized molecule shows no biological activity. Furthermore, kinetin- and rANP-induced stomatal opening is reversibly inhibited by two inhibitors of guanylate cyclase, LY 83583 and methylene blue. Stomatal opening is also induced in a concentration-dependent manner by the cell-permeant cyclic guanosine-3',5'-monophosphate (cGMP) analogue 8-Br-cGMP, and this effect is prevented by the stomatal closure promoting plant hormone abscisic acid (ABA). We conclude that in guard cells kinetin and rANP pathways operate via guanylate cyclase upregulation, and we propose that ABA-induced closure is not cGMP-dependent.

Adenine

A transient increase of phenylalanine ammonia-lyase transcript in kinetin-treated tobacco callus.

In tobacco cell culture (Nicotiana tabacum L. "Bright Yellow" T-13), phenylalanine ammonia-lyase (PAL) activity was induced in response to an exogenously added kinetin. RNA blot hybridization analysis showed that a single species of PAL transcript 2.9-kb in size was detected using the PAL cDNA cloned from kinetin-treated cells. The cellular content of this transcript increased transiently (2.5-fold) 4 h after the addition of kinetin followed by an increase in PAL enzyme activity at 16 h.

Adenine

Specific incorporation of kinetin into eukaryotic and prokaryotic transfer ribonucleic acid molecules.

We show that kinetin, a non-natural product with strong cytokinin activity, is incorporated into prokaryotic and eukaryotic tRNAs in the exchange reaction catalysed by a putative tRNA-kinetin transglycosylase. We also show that kinetin is specifically incorporated into E. coli tRNA(Tyr) and most probably at position 37. To our knowledge, this is the first report of a nucleic acid base exchange reaction occurring at this position.

Adenine

[Antagonism in the action of kinetin and actinomycin D on the cytogenetic effect of radiation].

A study was made of the effect of kinetin when administered separately or in combination with actinomycin D on the chromosomal aberrations in Crepis capillaris L. irradiated by X-rays. Dry seeds were treated after irradiation or seeds soaked, prior to irradiation. Significant decrease in the frequency of chromosomal aberrations was shown when actinomycin D was administered separately and prior to kinetin. The latter used separately and prior to actinomycin D slightly increased the level of the aberrations, as compared with the control. Noticeable radioprotective effect was observed after treatment of seeds with kinetin, prior to irradiation.

Adenine

Furfural, a precursor of the cytokinin hormone kinetin, and base propenals are formed by hydroxyl radical damage of DNA.

Recently, we have detected kinetin (N6-furfuryladenine), a well known cytokinin plant hormone, in commercially available DNA, in freshly extracted cellular DNA and in plant cell extracts. We had suggested that the furfuryl moiety of kinetin originates from furfural which is one of the primary oxidation products of deoxyribose in DNA. Here we show that the human cell extracts treated with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) give rise to oxime derivatives of various aldehydes present in the cell. Mass spectrometric analysis of silylated oximes showed several mass signals of different species, one of which was identified as furfural. Furthermore, detailed inspection of the mass spectra of DNA showed the mass signals of 165, 180, 189 and 206 m/z which correspond to cytosine-propenal, thymine-propenal, adenine-propenal and guanine-propenal, respectively. The presence of furfural, along with four base-propenals in the cell extract, as the primary oxidation products of deoxyribose, suggests that degradation of sugar residues in DNA is one of the major routes of cellular damage in addition to the modification of nucleic acid bases.

Adenine

Evidence for the presence of kinetin in DNA and cell extracts.

In contrast to the current view that kinetin (N6-furfuryladenine) is an unnatural and synthetic compound, we have detected it in commercially available DNA, in freshly extracted cellular DNA from human cells and in plant cell extracts by two independent methods. First, we discovered that N6-furfuryladenine has electrochemical properties that can be applied for monitoring this modified base by a HPLC/UV/EC method. Second, we have confirmed electrochemical assignments by mass-spectrometric analysis. A pathway of kinetin formation is proposed in which the formation of furfural by oxidative damage of the deoxyribose moiety of DNA is followed by its reaction with adenine residues to form N6-furfuryladenine. Since this modification can lead to mutations, the odd DNA base has to be removed by repair enzymes.

Adenine

Effect of abscisic acid, gibberellic acid, indoleacetic acid, and kinetin on selective ribosomal cistron regulation in quiescent and senescent onion leaf base tissue.

Small pieces of tissue from the basal, equatorial, near-apical, and apical regions of the third turgid onion leaf base were treated (3 and 6 h in the dark) with abscisic acid (ABA), gibberellic acid (GA3), indoleacetic acid (IAA), and kinetin (K) and compared with responses in water controls. ABA inhibited the activation (increase in size and changes in morphologies from round or oval to elongated-oval and dumbbell) of major nucleolar organizer regions (NORs) in basal, equatorial, and near-apical tissue. GA3 and K activated the major NORs in the basal, equatorial, and near-apical tissue. IAA stimulated the activation of major NORs in basal tissue but inhibited their activation in equatorial and near-apical tissue. No major nucleoli were activated in control or plant growth regulator-treated apical tissue. Minor NORs were not expressed in the control and plant growth regulator-treated tissue in these four locations. Actinomycin D and cycloheximide inhibited major NOR activation in equatorial control and kinetin-treated tissue. We propose that ABA, GA3, IAA, and K are major NOR regulators. We infer that the basal through near-apical cells were quiescent during post-harvest storage and that the cells in the apical tissue had senesced beyond the point of no return (degeneration of the karyoskeleton) in the cellular senescence pathway.

Abscisic Acid

Effects of kinetin on phosphorylation of leaf membrane proteins.

Isolated Chinese cabbage leaf membranes were phosphorylated by membrane-associated protein kinase(s) in the presence or [gamma-32P]ATP. Membrane-associated 32P radioactivity appeared to be bound to membrane proteins. Both smooth cell membranes and chloroplast lamellae reacted with ATP. Phosphorylation of the membranes was inhibited by Ca2+ and partially inhibited by kinetin or 6-benzyladenine. The possibility that cytokinin effects on membrane phosphorylation might increase ion availability was investigated in vivo. It was found that Ca2+ could substitute for kinetin in the leaf disc expansion assay.

Cell Membrane

[Kinetin and the germinating capacity of Lupinus multilupa seeds].

The effect of kinetin (10(-6) M and 10(-4) M) on the germinating capacity and incorporation of 8-C14 adenine into DNA, RNA and RNA Poly A+ of embryos and cotyledons from Lupinus multilupa L. seeds have been studied. Kinetin enhanced the germinative capacity of the seeds and the incorporation of 8-C14 adenine into DNA, RNA and Poly A+ of embryos and cotyledons. However, there seems to be no close relationship between the DNA and RNA biosynthesis of embryos and cotyledons and the ability of the seeds to germinate and their embryos to continue growing.

DNA

Effect of second-leaf removal or kinetin treatment on the nucleic acid metabolism of senescing first seedling leaf of barley.

1. Changes in nucleic acid metabolism in first seedling leaves of barley plants during aging (from 7 to 27 days) were followed, and the effect of continual removal of the second leaf and basal meristem or of treating the first leaf with 20p.p.m. kinetin on these changes was examined. During aging of the first seedling leaves the ribosomal RNA, DNA and soluble RNA declined, with ribosomal RNA showing the most rapid fall. This was, however, accompanied by increased incorporation of (32)P into RNA, which reached its peak on the fifteenth day. 2. Second-leaf removal partially suppressed first-leaf senescence as judged by retarded chlorophyll and nucleic acid decline and by a decreased extent of RNA labelling. Treatment with kinetin, however, did not prove effective. 3. No significant differences in the sucrose-gradient pattern of (32)P-labelled nucleic acids or in the (32)P-labelled nucleotide composition of RNA fractions during aging or during the two treatments were noted, except for a decrease in CMP content of soluble RNA during aging. 4. The results demonstrate that important changes in RNA metabolism are associated with leaf senescence.

Aging

The binding of kinetin to plant ribosomes.

The synthetic cytokinins kinetin and 6-benzylaminopurine exhibit equilibrium-type binding to purified chinese-cabbage leaf ribosomes. At 23mum and 4 degrees C one molecule of kinetin and 1.34 molecules of 6-benzylaminopurine are bound per ribosome. Adenine and adenine derivatives that are inactive as cytokinins showed much less affinity for ribosomes. Pretreatment of ribosomes with 0.5m-ammonium chloride or Triton X-100 did not decrease the extent of cytokinin binding. Binding appeared to be to the 83S ribosome species. A positive correlation between the extent of binding and the biological effect of various cytokinin analogues was demonstrated. These results are discussed in terms of cytokinin control of growth processes at the ribosomal level.

Adenine

Kinetin-induced chloroplast maturation in cultures of tobacco tissue.

Cultured tobacco tissue possesses proplastids capable of differentiating into mature chloroplasts. Kinetin (6-furfurylaminopurine) is a specific requirement for this differentiation; the absence of this compound results in a blockage of the formation of grana. The possibility that kinetin exerts a direct effect upon chloroplast differentiation is considered.

Cell Differentiation