Fluorescence resonance energy transfer between polyphenolic compounds and riboflavin indicates a possible accessory photoreceptor function for some polyphenolic compounds

The photoreceptive extreme tip of the wheat coleoptile exhibits intense green-yellow fluorescence under UV light, suggesting the presence of UV-absorbing materials. Fluorescence spectra of the intact coleoptile tip and tip homogenate showed the presence of the known photoreceptor pigments flavin and carotene, and a preponderance of phenolic compounds. Absorption spectra and fluorescence spectra of various phenolic compounds showed close overlap with the absorption and fluorescence spectra of the wheat coleoptile tip homogenate. Fluorescence spectra of several phenolic compounds showed close overlap with the absorption bands of flavin, carotene and pterine, suggesting possible energy transduction from phenols to these photoreceptors. Excitation of gentisic acid and ferulic acid with 340 nm light in the presence of flavin showed enhancement of flavin fluorescence in a concentration- and viscosity-dependent fashion, indicating fluorescence resonance energy transfer between them and riboflavin. Furthermore, several phenolic compounds tested generated superoxide anion on excitation at 340 nm, suggesting that superoxide-dependent signal cascades could operate in a polyphenol-mediated pathway. Phenolic compounds thus may act as accessory photoreceptors bringing about excitation energy transfer to the reactive photoreceptor molecules, or they may take over the function of the normal photoreceptor in genetic mutations lacking the system, or both processes may occur. The responses of plants to UV-B and UV-A light in mutants may be explained in terms of various phenolics acting as energy transducers in photoreceptor functioning.     

RED LIGHT-INDUCED REDUCTION OF A PARTICLE-ASSOCIATED b-TYPE CYTOCHROME FROM CORN IN THE PRESENCE OF METHYLENE BLUE*

Abstract— In the presence of methylene blue, red light causes the reduction of a h-type cytochrome in particulate fractions from corn coleoptiles. Two types of difference spectra for the cytochromes in these fractions are presented: (a) red light-minus-dark in the presence of methylene blue, and (b) dithionite-reduced-minus-oxidized. Comparison of these spectra shows that photoexcited methylene blue selectively reduces a b-type cytochrome which constitutes at most only 30% of the total dithionite-reducible cytochrome present in the most active fractions. The photoreducible cytochrome has an alpha band at room temperature near 557 nm. Bleaching of methylene blue precedes cytochrome reduction under appropriate conditions, suggesting that the photoreduced dye is donating an electron to the cytochrome. This electron transfer does not involve a flavin, at least as judged by the absence of light-induced spectral changes attributable to flavins. Preliminary kinetic studies suggest that EDTA provides the pool of electrons for the reaction. The cytochrome cannot be assigned exclusively either to mitochondria or to endoplasmic reticulum, as judged by its sedimentation properties. These results and the current literature are discussed in the context of the hypothesis that this b-type cytochrome may be involved in the photoreception mechanism for blue and uv light in vivo.     

Discovery of a Unique Flavonoid Biosynthesis Mechanism in Fungi by Genome Mining

Flavonoids are important plant natural products with variable structures and bioactivities. All known plant flavonoids are generated under the catalysis of a type III polyketide synthase (PKS) followed by a chalcone isomerase (CHI) and a flavone synthase (FNS). In this study, the biosynthetic gene cluster of chlorflavonin, a fungal flavonoid with acetolactate synthase inhibitory activity, was discovered using a self-resistance-gene-directed strategy. A novel flavonoid biosynthetic pathway in fungi was revealed. A core nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) is responsible for the generation of the key precursor chalcone. Then, a new type of CHI catalyzes the conversion of a chalcone into a flavanone by a histidine-mediated oxa-Michael addition mechanism. Finally, the desaturation of flavanone to flavone is catalyzed by a new type of FNS, a flavin mononucleotide (FMN)-dependent oxidoreductase.     

Light‐Induced Conformational Changes in the Plant Cryptochrome Photolyase Homology Region Resolved by Selective Isotope Labeling and Infrared Spectroscopy

Plant cryptochromes are photoreceptors that regulate flowering, circadian rhythm and photomorphogenesis in response to blue and UV‐A light. It has been demonstrated that the oxidized flavin cofactor is photoreduced to the neutral radical state via separate electron and proton transfer. Conformational changes have been found in the C‐terminal extension, but few studies have addressed the changes in secondary structure in the sensory photolyase homology region (PHR). Here, we investigated the PHR of the plant cryptochrome from the green alga Chlamydomonas reinhardtii by light‐induced infrared difference spectroscopy in combination with global ¹³C and ¹⁵N isotope labeling. Assignment of the signals is achieved by establishing a labeling strategy for cryptochromes that preserves the flavin at natural abundance. We demonstrate by UV/vis spectroscopy that the integrity of the sample is maintained and by mass spectrometry that the global labeling was highly efficient. As a result, difference bands are resolved at full intensity that at natural abundance are compensated by the overlap of flavin and protein signals. These bands are assigned to prominent conformational changes in the PHR by blue light illumination. We postulate that not only the partial unfolding of the C‐terminal extension but also changes in the PHR may mediate signaling events.     

Bacteriocin release protein-mediated secretory expression of recombinant chalcone synthase in Escherichia coli

Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD₆₀₀ induction time of 0.5 was found to be the best condition for chalcone synthase secretion.     

THE EFFECT OF POTASSIUM IODIDE ON PHOTOPHOBIC RESPONSES IN EUGLENA: EVIDENCE FOR TWO PHOTORECEPTOR PIGMENTS*

Abstract— Potassium iodide, a quencher of flavin fluorescence, inhibits the shock reaction which Euglena experiences upon a sudden decrease in light intensity (inverse photophobic response) completely at a concentration of 150 mM. The rate of swimming of the cells at the same concentration of KI is reduced to 30% of the control. The direct photophobic response, a shock reaction which appears identical but occurs upon an increase in light intensity, is unaffected by KI as is negative phototaxis of Euglena. It is concluded that a non-flavin pigment system mediates photoreception for the direct photophobic response and negative phototaxis.     

ROSEOFLAVIN INHIBITION OF PHOTOCONIDIATION IN A Trichoderma RIBOFLAVIN AUXOTROPH: INDIRECT EVIDENCE FOR FLAVIN REQUIREMENT for PHOTOREACTIONS

Roseoflavin, an analog known to compete with riboflavin in Lactobacillus casei , riboflavin-deficient rats, and fungi, was used to ascertain whether a flavin plays a role as a sensitizing pigment for photomorphogenesis in the fungus Trichoderma . Roseoflavin inhibited blue light induced conidiation of a riboflavin-requiring auxotroph of Trichoderma . Colonies pre-incubated with roseoflavin needed six times more light to saturate conidiation. Roseoflavin did not change the dark rate of conidial development indicating that it interfered only with the photoact. A revertant from the riboflavin auxotroph strain was not inhibited by roseoflavin. Simultaneous addition of 10 μ M riboflavin prevented inhibition by 6.4 μ M roseoflavin. Thus, roseoflavin inhibition is probably specific, possibly replacing riboflavin or one of its metabolites in the light reactions. There was no detectable shift in the action spectrum towards the green where roseoflavin absorbs; thus it did not replace a flavin as an efficient photoreceptor.     

REDUCED NEAR-UV SENSITIVITY IN Phycomyces MUTANTS AFFECTED IN THE BIOSYNTHESIS OF 6,7-DIMETHYL-8-RIBITYLLUMAZINE

Riboflavin-requiring mutants of Phycomyces blakesleeanus with defects in the genes ribA, ribB, ribC and ribD were analyzed with respect to their contents of flavins, 6,7-dimethyl-8-ribityllu-mazine (DMRL) and pterins as well as their phototropic sensitivity. Strains were grown on minimal medium enriched with 10^?6M riboflavin (RB), and the concentrations of the respective pigments in sporangiophores were determined by HPLC. In strains A607 ribC401 and A641 ribC402 madA7 a loss of DMRL correlated with a loss of near-UV sensitivity. In general terms, the results suggest the participation of DMRL in photoreception, which does not necessarily imply DMRL as a photoreceptor chromophore. In more specific terms, the result could be understood on the basis of a UV/blue-light photoreceptor, which includes besides a flavin also a lumazine-like chromophore. Mutants C318 ribA I and C323 ribA4 accumulated DMRL, the immediate precursor of RB, as well as biopterin and neopterin. Mutant C322 ribB contained normal amounts of DMRL and pterins. Mutant C324 ribD5 had reduced amounts of neopterin and biopterin. The fact that some of the RB-requiring mutants displayed abnormal amounts of pterins indicates a common regulation for the flavin and the pterin pathway.     

A FLAVOPROTEIN MAY MEDIATE THE BLUE LIGHT-ACTIVATED BINDING OF GUANOSINE 5'-TRIPHOSPHATE TO ISOLATED PLASMA MEMBRANES OF Pisum sativum L.*

Abstract— A blue light photoreceptor has not been identified in higher plants. Most proposals for a blue light-absorbing chromophore lack evidence for a direct connection between the putative chromophdre and a biological effect. Fluorescence data for the plasma membrane from etiolated buds of Pisum sativum L. suggest that we are measuring fluorescence emission of flavin species, and probably not pterin species. Fluorescence data indicate that a putative flavin exists associated with a protein or protein complex in the plasma membrane. Excitation of plasma membranes that were boiled in the presence of 0.1% sodium dodecyl sulfate and treated with blue light yields a fluorescence band with a maximum of approximately 552 nm. This fluorescence emission can be rapidly quenched by the flavin antagonists phenylacetic acid (PAA) and KI. Blue light-enhanced binding of guanosine 5'-[Γ-thio]triphosphate (GTPγS) to a protein in the plasma membrane is strongly inhibited by PAA, KI, and NaN₃, all quenchers of flavin excited states, indicating that a chromophore for this photoreaction may be a flavin associated with a plasma membrane protein. The above evidence is consistent with the participation of a flavin as the chromophore for the light-induced GTP-binding reaction in pea plasma membrane.     

A NOVEL EFFECT OF UV-B IN A HIGHER PLANT (SORGHUM VULGARE)

Abstract— Two non-photosynthetic photoreceptors (phytochrome and the usual blue/UV light photoreceptor) were previously found to be involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings (Drumm and Mohr, 1978). The decisive point is that phytochrome can act only after a blue/UV light effect has occurred. On the other hand, the expression of the blue/UV light effect is controlled by phytochrome ('obligatory sequential action'). A strong positive interaction between the blue/UV-A and the UV-B part of the spectrum was found, in addition to the above sequential action: an inductive effect of blue or UV-A light can only express itself fully if short wavelength UV (approximately 300–320nm. UV-B range) is also given, either after the blue/UV-A light or simultaneously. Since even small amounts of the UV-B are strongly effective it is probable that this effect plays a role under natural conditions and may not be considered as a mere laboratory artifact.     

RESPONSES OF LIGHT-GROWN WILD-TYPE and LONG-HYPOCOTYL MUTANT CUCUMBER SEEDLINGS TO NATURAL and SIMULATED SHADE LIGHT*

The photomorphogenic control of hypocotyl extension growth was characterized in wild type (WT) and long hypocotyl (Ih) mutant seedlings of cucumber (Cucumis sativus L.) grown under natural radiation in outdoor and glasshouse experiments. Hypocotyl extension growth of WT plants was promoted by supplementing sunlight with far-red light during the photoperiod, by reducing the amount of blue light reaching either the whole shoot or the hypocotyl, and by reducing the amount of UV reaching the whole shoot.The Ih seedlings only responded to a reduction in UV-B levels. Both WT and Ih seedlings showed phototropic responses to the direction of blue light. Increasing degrees of vegetational shade promoted hypocotyl growth of WT plants. The Ih mutant showed no hypocotyl growth promotion by natural shade in glasshouse experiments (no UV-B, low water demand) and a reduced response (10-23% of the WT response, according to pretreatment conditions) in outdoor experiments (UV-B, high water demand).     

INHIBITION OF LIGHT-DEPENDENT PHOTOMORPHOGENESIS OF SPORANGIOPHORES FROM Phycomyces blakesleeanus BY APPLICATION OF PTERIDINE BIOSYNTHESIS INHIBITORS*†

Abstract— In the zygomycete Phycomyces blakesleeanus the morphogenesis of both macrosporangiophores and microsporangiophores is under separate control of different blue-light photoreceptors. Blue light represses microphorogenesis and enhances macrophorogenesis. Both, flavins and pterins are discussed as possible components of these blue-light photoreceptors. 2,4-Diamino-6-hydroxypyrimidine (DAHP) was shown to be an inhibitor of GTP-cyclohydrolase I, the initial enzyme of pteridine biosynthesis, in vitro and in vivo in Phycomyces. When grown on agar plates containing 5 mM of DAHP, Phycomyces shows a 6.0-fold reduction of light-dependent enhancement of macrophorogenesis, and microphorogenesis was 3.1 times less repressed when compared with controls grown without DAHP. This implies a participation of pteridines in blue-light photoreception in both phenomena, with greater influence on macrophorogenesis enhancement than on microphorogenesis repression.     

PROTECTION OF THE D1 PHOTOSYSTEM II REACTION CENTER PROTEIN FROM DEGRADATION IN ULTRAVIOLET RADIATION FOLLOWING ADAPTATION OF Brassica napus L. TO GROWTH IN ULTRAVIOLET-B

As depletion of the stratospheric ozone layer continues, the biosphere will most likely be exposed to higher levels of ultraviolet-B (UV-B) irradiation (290–320nm). For plants, damage from UV-B can occur at several molecular targets with the photosynthetic apparatus being especially vulnerable. We are interested both in the mechanisms of UV-B-induced damage and identifying adaptation processes that can confer protection from UV-B. Toward this end, Brassica napus (oil seed rape) plants grown under visible light plus a low level of UV-B radiation (adapted plants) were compared to plants grown under visible light alone (control plants). Relative to the control plants, the adapted plants showed little evidence of damage at the levels of morphology or photosynthesis, indicating that B. napus has some tolerance of UV-B and that the plants may have protection mechanisms. Consistent with this, a strong UV-B adaptation process was observed in the plants-accumulation of flavonoids in the epidermis. These pigments seemed to screen a molecular target in the mesophyll. Namely, the D1 photosystem II reaction center protein, which is rapidly degraded in UV-B, was partially protected from degradation in UV-B in the adapted plants. Moreover, the extent that the half-life of the D1 protein increased in the adapted plants was on par with the elevation in total flavonoid concentrations. These experiments demonstrate that degradation of the D1 protein can be used as an in vivo assay of penetration of UV-B photons to the mesophyll.     

BLUE LIGHT PERCEPTION BY ENDOGENOUS REDOX COMPONENTS OF THE PLANT PLASMA MEMBRANE

b-Type cytochromes of the higher plant plasma membrane may be reduced by irradiation with actinic blue light (light-induced absorbance change). Although this reaction has been reported to depend on the presence of an exogenous oxygen-scavenging system, significant cytochrome reduction was obtained in bean hook (Phaseolus vulgaris L. cv. “Limburgse Vroege”) plasma membranes without any addition. An endogenous oxygen-consuming reaction is apparently sufficient to achieve a proper redox balance. A blue light-mediated absorbance change with absorbance minima at 450 and 475 nm precedes cytochrome b reduction and indicates the presence of a flavoprotein in the plasma membrane fraction. Cytochrome b reduction by blue light in the absence of an oxygen scavenger is highly sensitive to flavin photosensitizers. Glucose oxidase, which has previously been used to lower the oxygen concentration in membrane samples, was demonstrated to have a photosensitizing effect. Inhibitors of flavin photochemical reactions (KI and phenylacetic acid) were highly effective in preventing cytochrome b reduction. These results indicate that the blue light-mediated reaction probably involves an endogenous plasma membrane flavoprotein as the photoreceptor. As plasma membrane NADH-dependent oxidoreductases potentially are flavoproteins these experiments raise the question whether a plasma membrane cytochrome b and a flavin-enzyme may cooperate in blue light reactions. Evidence is also discussed, suggesting the possible involvement of oxygen radicals in the blue light-induced cytochrome b reduction.     

Solving Blue Light Riddles: New Lessons from Flavin‐binding LOV Photoreceptors

Detection of blue light (BL) via flavin‐binding photoreceptors (Fl‐Blues) has evolved throughout all three domains of life. Although the main BL players, that is light, oxygen and voltage (LOV), blue light sensing using flavins (BLUF) and Cry (cryptochrome) proteins, have been characterized in great detail with respect to structure and function, still several unresolved issues at different levels of complexity remain and novel unexpected findings were reported. Here, we review the most prevailing riddles of LOV‐based photoreceptors, for example: the relevance of water and/or small metabolites for the dynamics of the photocycle; molecular details of light‐to‐signal transduction events; the interplay of BL sensing by LOV domains with other environmental stimuli, such as BL plus oxygen‐mediating photodamage and its impact on microbial lifestyles; the importance of the cell or chromophore redox state in determining the fate of BL‐driven reactions; the evolutionary pathways of LOV‐based BL sensing and associated functions through the diverse phyla. We will discuss major novelties emerged during the last few years on these intriguing aspects of LOV proteins by presenting paradigmatic examples from prokaryotic photosensors that exhibit the largest complexity and richness in associated functions.     

FLAVIN COMPOUNDS AS AGENTS FOR THE OXIDATION OF PLASTOCYANIN IN BLUE LIGHT

Abstract—Flavins, flavin nucleotides and selected flavoproteins have been compared in a reaction using blue light. in which plastocyanin is oxidized as the flavin is photoreduced. Per unit of light absorbed. flavin mononucleotide is more effective than flavin adenine dinucleotide, ribotlavin or lumiflavin. Of the flavoproteins tested, diaphorase from Clostridium kluyveri was most effective, but was less active than free flavin mononucleotide. The oxidation of plastocyanin requires aerobic conditions. and appears to be mediated by the production of singlet oxygen when the flavin is irradiated.     

A PHOTORECEPTOR SYSTEM REGULATING in vivo AND in vitro PHOSPHORYLATION OF A PEA PLASMA MEMBRANE PROTEIN*

Abstract— We have continued to characterize the blue light-regulated phosphorylation of a 120 kDa pea plasma membrane protein thought to be involved in sensory transduction for phototropism (Short and Briggs, 1990, Plant Physiol. 92 , 179–185). By incubating pea stem sections in ³²P-phosphate, we show that the 120 kDa protein is phosphorylated in vivo only after blue light irradiation and that the photosensitive fluence range matches that for phototropism. Blue light induces phosphorylation of the protein in vitro as well, but the fluences required to elicit the response are at least 30-fold higher. Triton solubilization of the plasma membrane vesicles does not further alter the fluence-response relationship. Very little turnover was detected over 20 min phosphorylation time courses or by pulse-chase experiments on unirradiated, blue light pulse-irradiated, or continuously irradiated membranes. Experiments with a dark period intervening between irradiation and addition of adenosine triphosphate show the light-induced change to persist for several minutes at 30°c. Agents that disrupt the normal photochemistry of flavins preferentially inhibit the light-induced enhancement of phosphorylation, suggesting a flavin chromophore. However, exogenous free flavins do not affect the sensitivity of the response. Staphylococcus aureus V-8 proteolysis of the phosphorylated protein from membranes subjected to a range of fluences before phosphorylation shows that the radiolabel on each of three peptides increases in proportion to the phosphorylation level of the undigested polypeptide. These studies may be valuable for assessing the nature of the photoreceptor and for unravelling the early sensory transduction steps in phototropism.     

Flavin Redox Bifurcation as a Mechanism for Controlling the Direction of Electron Flow during Extracellular Electron Transfer

The iron‐reducing bacterium Shewanella oneidensis MR‐1 has a dual directional electronic conduit involving 40 heme redox centers in flavin‐binding outer‐membrane c‐type cytochromes (OM c‐Cyts). While the mechanism for electron export from the OM c‐Cyts to an anode is well understood, how the redox centers in OM c‐Cyts take electrons from a cathode has not been elucidated at the molecular level. Electrochemical analysis of live cells during switching from anodic to cathodic conditions showed that altering the direction of electron flow does not require gene expression or protein synthesis, but simply redox potential shift about 300 mV for a flavin cofactor interacting with the OM c‐Cyts. That is, the redox bifurcation of the riboflavin cofactor in OM c‐Cyts switches the direction of electron conduction in the biological conduit at the cell–electrode interface to drive bacterial metabolism as either anode or cathode catalysts.