Partial Reversal by Cytokinin and (2-Chloroethyl)-Trimethylammonium Chloride of Near-Ultraviolet Inhibited Growth and Morphogenesis in Callus Cultures

Callus cultures of Haplopappus gracilis, Nicotiana tabacum and Allium cepa var. proliferum were in varying degrees inhibited by blue to near-UV light obtained from fluorescent tubes. The inhibition was considerably reduced for Haplopappus cultures by 6-(3-methyl-2-buten-1-ylamino)-purine (2 iP) and (2-chloro-ethyl)-trimethylammonium chloride (CCC) in combination. Even separately these compounds stimulated growth in blue but not in white light. A high concentration of 2 iP reduced the inhibiting effects of near-UV on tobacco tissue cultures, and a synergism was observed between 2 iP and CCC in respect to shoot formation in blue light. Allium callus was not significantly affected by CCC. It was also observed that the concentration of indol-3-acetic acid (IAA) was more important for the growth of tobacco cultures in blue than in white light. It is believed that the light-inhibition of growth is partly due to a photoinactivation of IAA and that 2 iP and CCC might be active through processes controlling the levels of IAA and other growth hormones.     

Kinetics of photoinactivation and photooxidation of mitomycin C in the presence of riboflavin

The kinetic relation between the photoinactivation and photooxidation of mitomycin C in the presence of riboflavin was investigated. The photoinactivation was tested for lambda-phage induction in Escherichia coli K-12 (lambda) cells and colony formation of E. coli Bs-1 cells. Mitomycin C lost its phage-inducing and antibiotic activities when the antibiotic was irradiated in vitro with visible light in the presence of riboflavin. The loss of phage-inducing activity followed a Stern-Volmer type equation with respect to the dose of irradiation, and the inactivation constant was evaluated to be 0.96X10(-4)m2/J. The initial rate of decay of mitomycin C by photooxidation in the presence of riboflavin obeyed first order kinetics, and its cross section was estimated to be 2.51X10(-6)m/J independent of the intensity of incident light. The cross section for photooxidation was found to be proportional to the inactivation constant. These results suggest that the photoinactivation of mitomycin C is caused by its photooxidation. In order to rationalize this conclusion, a mechanism of photooxidation was proposed and reactions in vivo of the photoproduct were discussed in relation to the inactivation.     

INVESTIGATIONS ON AUXIN-GIBBERELLIN INTERACTION ON THE PHOTOTROPISM OF HELIANTHUS ANNUUS HYPOCOTYL

On applying IAA-GB and IAA-NAA mixtures to the hypocotyls ofHelianthus annuus, the plants showed an increase and a decrease,respectively, in their phototropic response compared with thecontrol treated with IAA-H₂O only. In the in vitro experimentsit was observed that the illumination (with 1,500 lux light,lasting for 24 hr) caused photoinactivation of IAA in the mixturesof IAA-H₂O, IAA-GB and IAA-NAA by 50%, 24% and 0%, respectively.In the presence of 0.01% riboflavin as a photosensitizer inthe same mixtures, the degree of photodestruction of IAA causedby the same illumination was 92%, 97% and 86%, respectively. The stronger positive phototropism in the IAA-GB treated hypocotylwas accounted for by the situation that on the illuminated sidethe photo-inactivation of IAA was accelerated by GB, while onthe shaded side IAA and GB acted synergistically. On the otherhand, the weaker phototropism in the IAA-NAA treated hypocotylwas explained as due to a partial inhibition of photoinactivationof IAA by NAA. (Received October 6, 1962; )     

Effect of Auxin and Anti-auxin (4-Phenyl-3-carbostyriloxyacetic Acid) on the Growth of Escherichia coli

Indole-3-acetic acid (IAA) inhibited specifically the growth of a wild strain of Escherichia coli IFO 3545 in a glucose-free polypeptone medium adjusted to pH below 6.3. When 50 ppm of IAA was combined with 10 ppm of 4-phenyl-3-carbostyriloxyacetic acid (V-OCH₂COOH), an anti-auxin, inhibitory effect of IAA on the bacterial growth was markedly increased though V-OCH₂COOH alone had no effect. When 30 ppm of IAA was combined with 10 ppm of V-OCH₂COOH, inhibition increased initially, but soon decreased and disappeared. Riboflavin also increased the inhibitory effect of IAA under fluorescent light. Cysteine restored not only the effect of IAA alone but also the combination effect of IAA with riboflavin or V-OCH₂COOH. An intermediary metabolite of IAA in bacteria that appeared to be identical with a photooxidation product from IAA may actually inhibit the bacterial growth. It was suggested that V-OCH₂COOH stimulated the induction of IAA-metabolizing enzymes in bacteria, as in the case of plants.     

THE KINETICS OF INACTIVATION OF COMPLEMENT BY LIGHT

The photoinactivation of complement has been studied with a view to determining if possible how many kinds of molecules disappeared during the reaction. It was found that: 1. The apparent course of photoinactivation is that of a monomolecular reaction. 2. Diffusion is not the limiting factor responsible for this fact, because the temperature coefficient of diffusion is much higher than that of photoinactivation (Q ₁₀ = 1.22 to 1.28, and Q ₁₀ = 1.10 respectively). 3. There is no change in the transparency of serum solutions during photoinactivation, at least for light of the effective wave-length, which is in the ultra-violet region probably at about 2530 Ångström units. It is pointed out that under these conditions only one interpretation is possible; namely, that during photoinactivation a single disappearing molecular species governs the rate of reaction. This substance must be primarily responsible for the hemolytic power of serum when it is used as complement.     

Light-induced binding of riboflavin to lysozyme

Summary The photodynamic inactivation of lysozyme in air saturated H₂O and D₂O (phosphate buffer 0.05 M, pH 7.0) in the presence of methylene blue and riboflavin has been studied. When H₂O was replaced by D₂O a great increase in the rate of photoinactivation of lysozyme was observed. This finding, together with the fact that photooxidation is inhibited by singlet oxygen quenchers like NaN₃, suggests that these reactions occur via a singlet oxygen mechanism.During the course of the studies of the riboflavin sensitized photoinactivation of lysozyme, it was found that riboflavin is strongly bound to the enzyme as a result of illumination. This finding would explain the higher quantum yield observed when riboflavin is used, although this dye is bleached during irradiation.     

Photoinactivation of photophosphorylation and dark ATPase in Rhodospirillum rubrum chromatophores.

Preillumination of Rhodospirillum rubrum chromatophores with strong, far-red light in the presence of phenazine methosulfate under non-phosphorylation conditions results in a selective, irreversible inactivation (typically about 70%) of photophosphorylation and of uncoupler-stimulated dark ATPase. The time course of the photoinactivation is similar to the light-on kinetics of the light-induced proton uptake in the absence of ADP. Only little photoinactivation occurs when the uncoupler carbonyl cyanide m-chlorophenyl hydrazone is present or when phenazine methosulfate is absent during the preillumination, indicating that the reaction occurs only when the membrane is energized. Phosphorylation conditions offer a practically complete protection against the photoinactivation. Inorganic phosphate, Mg2+ or ADP do not provide a significant protection against the photoinactivation, nor does ATP. The pH-dependence of the reaction(s) leading to photoinactivation may indicate that a partial reaction of the photophosphorylation process (perhaps only a conformational change of the coupling factor) precedes the photoinactivation.     

Photoinactivation of photophosphorylation and dark ATPase in Rhodospirillum rubrum chromatophores

Preillumination of Rhodospirillum rubrum chromatophores with strong, far-red light in the presence of phenazine methosulfate under non-phosphorylation conditions results in a selective, irreversible inactivation (typically about 70%) of photophosphorylation and of uncoupler-stimulated dark ATPase. The time course of the photoinactivation is similar to the light-on kinetics of the light-induced proton uptake in the absence of ADP. Only little photoinactivation occurs when the uncoupler carbonyl cyanide m-chlorophenyl hydrazone is present or when phenazine methosulfate is absent during the preillumination, indicating that the reaction occurs only when the membrane is energized.

Phosphorylation conditions offer a practically complete protection against the photoinactivation. Inorganic phosphate, Mg²⁺ or ADP do not provide a significant protection against the photoinactivation, nor does ATP. The pH-dependence of the reaction(s) leading to photoinactivation may indicate that a partial reaction of the photophosphorylation process (perhaps only a conformational change of the coupling factor) precedes the photoinactivation.     

Very strong UV-A light temporally separates the photoinhibition of photosystem II into light-induced inactivation and repair

When organisms that perform oxygenic photosynthesis are exposed to strong visible or UV light, inactivation of photosystem II (PSII) occurs. However, such organisms are able rapidly to repair the photoinactivated PSII. The phenomenon of photoinactivation and repair is known as photoinhibition. Under normal laboratory conditions, the rate of repair is similar to or faster than the rate of photoinactivation, preventing the detailed analysis of photoinactivation and repair as separate processes. We report here that, using strong UV-A light from a laser, we were able to analyze separately the photoinactivation and repair of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803. Very strong UV-A light at 364 nm and a photon flux density of 2600 micromol photons m(-2) s(-1) inactivated the oxygen-evolving machinery and the photochemical reaction center of PSII within 1 or 2 min before the first step in the repair process, namely, the degradation of the D1 protein, occurred. During subsequent incubation of cells in weak visible light, the activity of PSII recovered fully within 30 min and this process depended on protein synthesis. During subsequent incubation of cells in darkness for 60 min, the D1 protein of the photoinactivated PSII was degraded. Further incubation in weak visible light resulted in the rapid restoration of the activity of PSII. These observations suggest that very strong UV-A light is a useful tool for the analysis of the repair of PSII after photoinactivation.     

Very strong UV-A light temporally separates the photoinhibition of photosystem II into light-induced inactivation and repair

When organisms that perform oxygenic photosynthesis are exposed to strong visible or UV light, inactivation of photosystem II (PSII) occurs. However, such organisms are able rapidly to repair the photoinactivated PSII. The phenomenon of photoinactivation and repair is known as photoinhibition. Under normal laboratory conditions, the rate of repair is similar to or faster than the rate of photoinactivation, preventing the detailed analysis of photoinactivation and repair as separate processes. We report here that, using strong UV-A light from a laser, we were able to analyze separately the photoinactivation and repair of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803. Very strong UV-A light at 364 nm and a photon flux density of 2600 μmol photons m⁻² s⁻¹ inactivated the oxygen-evolving machinery and the photochemical reaction center of PSII within 1 or 2 min before the first step in the repair process, namely, the degradation of the D1 protein, occurred. During subsequent incubation of cells in weak visible light, the activity of PSII recovered fully within 30 min and this process depended on protein synthesis. During subsequent incubation of cells in darkness for 60 min, the D1 protein of the photoinactivated PSII was degraded. Further incubation in weak visible light resulted in the rapid restoration of the activity of PSII. These observations suggest that very strong UV-A light is a useful tool for the analysis of the repair of PSII after photoinactivation.     

Levels of Indol-3yl-acetic Acid and Acid Inhibitors in Green and Etiolated Bean Seedlings (Phaseolus vulgaris)

Indol-3yl-acetic acid (IAA) was identified in Phaseolus vulgaris L. Shoot tissue of seedlings, exposed to light for 5 days, had a higher level of IAA than etiolated seedlings of the same age. The content of IAA increased in green seedlings during light treatment for 5–12 days. No increase could be measured in dark-grown seedlings. Inhibitory substances appeared at different R_f-values. The main part was identical to the inhibitor-β complex and occurred in a higher amount in light-grown seedlings than in etiolated taller ones. One part of the inhibitor-complex appeared to be abscisic acid (ABA). It is suggested that both IAA and acid inhibitors may play an important role in the control of stem growth and differentiation, although light effects on other hormones and regulatory systems cannot be ignored.     

Reversal of cytokinin action by riboflavin during stage II micropropagation: The role of 3-methyleneoxindole

Summary Nicotiana tabacum explants were grown on stage II micropropagation medium containing 6-[γ,γ dimethylallyl amino]-purine (2iP), a cytokinin, and indole-3-acetic acid (IAA), an auxin, under a photon fluence rate of ≈60 μmol m⁻² s⁻¹. In the presence of riboflavin the explants showed growth patterns suggesting that IAA had assumed a dominant regulatory role, even though the medium contained a high 2iP:IAA molar ratio. It was determined that this effect was produced by 3-methyleneoxindole, a product of the riboflavin-catalyzed photooxidation of IAA. Methyleneoxindole was recovered from the riboflavin-treated agar medium and found to be much more active than IAA in supporting stage II and stage III micropropagation of tobacco explants. Reversal of cytokinin action by riboflavin did not result from the inhibition of any cytokinin-specified growth function because, in the presence of riboflavin, normal stage II growth was obtained if naphthaleneacetic acid was used as the auxin.     

Identification of possible light emitters in the gills of a bioluminescent fungus Mycena chlorophos

The pileus of Mycena chlorophos actively, spontaneously, and continuously emits green light. Molecular mechanisms underlying this bioluminescence remain unclear. We investigated light emitters in the pileus of M. chlorophos to determine the underlying mechanisms. High‐performance liquid chromatography–fluorescence–photodiode array–mass detection analyses showed that actively luminescent gills in the pileus exclusively and abundantly possessed riboflavin, riboflavin 5′‐monophosphate, and flavin adenine dinucleotide as green‐fluorescent components. These components were localized in the bioluminescent region of the gills at the microscopic level. Fluorescence spectra of these green‐fluorescent components and the gills were identical with the spectrum of gill bioluminescence (maximum emission wavelength, 525 nm). Thus, our results indicated that the possible light emitters in the pileus of M. chlorophos were riboflavin, riboflavin 5′‐monophosphate, and/or flavin adenine dinucleotide. Copyright © 2016 John Wiley & Sons, Ltd.     

Photodegradation of DNA with fluorescent light in the presence of riboflavin, and photoprotection by flavin triplet-state quenchers

Superoxide anion was photogenerated upon illumination of nucleic acids with fluorescent light in a solution containing phosphate buffer, pH 7.8 and riboflavin. DNA was a better reducing substrate for this reaction than was RNA. A similar riboflavin-sensitized photoreaction caused single- and double-strand scissions of supercoiled PM2 DNA as detected by electrophoresis in agarose gels. None of specific scavengers or quenchers for superoxide anion and other active oxygen species prevented the DNA strand breaks. However, among the flavin triplet-state quenchers, potassium iodide, butylated hydroxyanisole, and ferricytochrome c protected the supercoiled DNA from photodegradation; butylated hydroxytoluene, alpha-tocopherol, tyrosine and hemoglobin did not have any protective effect. These results indicate that triplet-state riboflavin or a derivative formed from it participate directly in the observed riboflavin-sensitized DNA photodegradation and that active oxygen species are not directly involved.     

Photodynamic antimicrobial therapy

Photodynamic antimicrobial therapy (PACT) involves the utilisation of photosensitizers activated by exposure to visible light in order to eradicate microbes (this method has already been applied in photodynamic therapy of tumours). Photodynamic effect of the particular photosensitive substance (PS) is attributed to its ability to penetrate susceptible microorganisms, to absorb the light of certain wavelength, and to generate reactive cytotoxic oxygen products. The target microorganisms for photoinactivation are bacteria, fungi, viruses and protozoa. Photodynamic antimicrobial therapy is proposed as a potentially topical, non-invasive approach suitable for treatment of locally occurring infection. The fact that bacteria are becoming increasingly resistant to antibiotics and antiseptics has lead to an increased interest in the development of new alternative eradication methods, such as PACT. Research and development of photosensitive substances are aimed at finding effective antimicrobial substances, which would have a broad-spectrum potency.     

Degradation of exogenous indole-3-butyric acid and riboflavin and their influence on rooting response of papaya in vitro

Varying concentrations of riboflavin were added to a De Fossard et al. (1974) basal medium containing 10 µM IBA and the effect on adventitious root initiation on shoots of Carica papaya L. was studied. Ninety percent root initiation occurred in 11 days when 1 µM riboflavin was added to the culture medium. Smaller rooting percentages were observed and roots emerged more slowly with riboflavin concentrations greater and less than 1 µM. Tissue culture media were maintained at 27°±1°C in either darkness or 12-h photoperiods for 28 days, and concentrations of riboflavin and IBA were measured at regular intervals using HPLC analysis. In a De Fossard et al. (1974) basal medium, riboflavin concentrations (0.1, 1.0, 10.0 µM) decreased rapidly in light and were independent of the presence of IBA. IBA concentration steadily decreased when media was placed in light, and increasing riboflavin concentrations accelerated the reduction of IBA levels. Concentrations of IBA and riboflavin were stable with dark incubation.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Regularities in the kinetic of photoactivation of lactate dehydrogenase by the action of UV light in different microenvironment

The kinetics of photoinactivation of cardiac (H4) and muscular (M4) isoforms of lactate dehydrogenase irradiated by UV light (240-390 nm) in the free form and in the presence of sodium azide, D-mannitol, and serotonin was studied. It was shown that the decrease in the catalytic activity of both enzymes can be described by the kinetics of the first-order monomolecular reaction. The inactivation rate constant of lactate dehydrogenase M4 is considerably higher than that of lactate dehydrogenase H4, indicating a greater photochemical lability of the isoform M4. It was shown that sodium azide has a different protective action on the proteins studied. The irradiation of the muscular isoform in the presence of serotonin and D-mannitol did not change the character of the "dose-effect" curve and only led to a decrease in the photoinactivation rate constant of the protein.     

Degradation of Indol-3yl-acetic Acid in Homogenates and Segments of Cabbage Roots

Plots of reaction rate versus substrate concentration of the enzymatic decarboxylation of IAA yield sigmoid, rather than the usual, hyperbolic curves, suggesting that the IAA oxidase of cabbage roots is an allosteric enzyme. The quantity of this enzyme in roots is so high that the IAA concentration is likely to limit IAA degradation in intact cells. Thus, variations in the level of this enzyme seem not to be essential for the regulation of the endogenous IAA concentration. Cabbage roots contain substances that can inhibit IAA oxidase. These substances are spatially separated from IAA oxidase in intact cells, but the same inhibitors are able to reach the enzyme when added exogenously to tissue segments. The possibility that added IAA is treated by tissue segments in another manner than endogenous IAA is discussed.     

EFFECT OF LIGHT ON AUXIN TRANSPORT AND ELONGATION OF AVENA MESOCOTYL

The present work was undertaken to find if there are relations between light and auxin action on elongation of coleoptilar node and mesocotyl with Avena seedlings. Red light inhibited the elongation of mesocotyl and simultaneously decreased the rate of transport of diffusible auxin through the node. Red light also inhibited the transport of exogenously given IAA through the nodal region. The light inhibition of IAA transport was closely related to the increase of IAA immobilization. As the age proceeds, the ability of IAA immobilization increased with the decrease in the rate of mesocotyl elongation, even if the seedling was grown in complete darkness. The nature of radioactive substances found in the IAA-C¹⁴ treated tissue was examined by paper chromatography. The above results strongly suggested that the increase of IAA immobilization might result in the inhibition of mesocotyl elongation.