Light-induced fluorescence changes in<Emphasis Type="Italic"> Phycomyces</Emphasis>: evidence for blue light-receptor associated flavo-semiquinones

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH^·), and the flavo-hydroquinone (FlH₂) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH₂, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.Abbreviations FAD flavin adenine dinucleotide - Fl oxidized flavin - FlH flavo-semiquinone radical - FlH₂ flavo-hydroquinone - LIAC light-induced absorbance change - LIFC light-induced fluorescence change - Pt oxidized pterin - PtH₂ dihydro-pterin - PtH₄ tetrahydro-pterin     

Pigments associated with the flagellum ofEuglena gracilis

The pigments associated with the flagellum of the phytoflagellateEuglena gracllis were characterized by HPLC. The pigment pattern of the wild-type strain was compared with a set of white mutants which did not display phototaxis and photoaccumulation in response to blue light. Flagella of the wild type contained FMN and FAD. Two mutants which lacked the stigma but retained a small paraxonemal body (PAB) contained less flavins. The whiteEuglena mutant FB, which retained a residual stigma and also a PAB, and the white phytoflagellateAstasia longa, a close relative ofEuglena, had normal amounts of flagellar flavins. Cells and flagella ofEuglena wild type contained an unldentified pterin-like pigment, called Pt₁₆, which was substantially reduced inAstasia and theEuglena mutants. A third pigment, designated P₅₂₈ with major absorption at 528 nm and fluorescence emission at 550 nm was present mainly in flagella. The association of the three pigment types with flagella and their respective alterations in the white strains indicates their possible role in photoreception. Dedicated to Pill-Soon Song on the occasion of his 60th birthday.     

Altered Pterin Patterns in Photoreceptor Mutants of Phycomyces blakesleeanus with Defective madl Gene

Action spectra of photogravitropic equilibrium were measured for the wild type of the lower fungus Phycomyces blakesleeanus and three photobehavioral mutants with defects in the madl gene. The action spectrum for the wild type NRRL1555 had major peaks at 383 and near 460 nm and subsidiary peaks at 365 and 422 nm. The action spectra of the mutants, L1 49 mad1712, L151 mad1714 and L153 mad1716 differed significantly from that of the wild type. One prominent feature of the three mutants was hat the near-UV peaks at 365 and 383 nm, which were not well resolved in the wild type, were of approximately equal height in the mutants and were separated by an extremely sharp valley at 378 nm. The steepness of this valley suggests interaction of multiple receptors. The second prominent feature of the mutants was their enhanced 422 nm peak. The gross changes of the photogravitropic action spectra associated with the madl genotype indicate that the respective gene product acts early in the photosensory transduction chain, very likely at the level of a complex photoreceptor system. Flavins and pterins, two pigment classes which were expected to function as chromophores of the near-UV/blue light photoreceptor system, were analyzed for stage I sporangiophores of the wild-type and the mutant strains by HPLC with fluorescence detection. In the wild-type strain NRRL1555, and also in the three madl mutants, flavins were found to be present at the following concentrations: riboflavin (2.9 × 10^?6 M), FMN (3.8 × 10^?6M) and FAD (1.3 × 10^?6 M). No significant effect of the madl mutations on the flavin content could be discerned. Among the pterins found in the wild type and the madl mutants were biopterin, 6,7-dimethylpterin, neopterin, pterin and xanthopterin. These pterins occurred in all strains in the micromolar range and none of them was significantly reduced in the mutants. However, biopterin, 6,7-dimethylpterin and xanthopterin occurred in some excess in one of the madl mutants. The most significant feature of the madl mutants was that they had almost completely lost one unidentified pterin with a retention time of 18 ? 20 min. Another two unidentified pterins were reduced about twofold in the mutants compared to the wild type. The results suggest an involvement of pterins in the photoreception of near-UV and blue light in Phycomyces.     

Microspectrofluorometry of the autofluorescent flagellum in phototactic brown algal zoids

Summary Posterior flagella of zoids ofScytosiphon lomemaria andChorda filum (Phaeophyceae, Chromophyta) were isolated and subjected to microspectrofluorometry using a high sensitivity microspectrofluorometer in order to characterize the flagellar autofluorescent substances. Vigorous agitation in a Hypertonic medium yielded preparations of largely intact flagella retaining detectable green flagellar autofluorescence. Under 380–425 nm excitation light, maximum emission of flagellar autofluorescence was observed at 495 nm, whereas under 400–440 nm excitation light fluorescence shifted to around 510 nm. Comparison of these spectra with the fluorescence spectra of 4,5-cyclic FMN isolated from fertile thalli ofS. lomentaria, and of 6-carboxypterin suggested that two (or more) different fluorescent substances (presumably a flavin and a pterin) are present in the flagella.Abbreviations DTT dithiothreitol - FMN flavin mononucleotide - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) - PEG polyethylene glycol - PFB paraflagellar body - Tris tris(hydroxymethyl) aminomethane. Dedicated to Professors Masakazu Tatewaki and Tadao Yoshida on the occasion of their academic retirement     

Partial purification of a plasma membrane flavoprotein and NAD(P)H-oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H-dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two-phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg^?1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non-covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H-oxidoreductase activity and suggests the presence of plasma membrane bound pterins.     

On the properties of fluorescing compounds in guard and epidermal cells of Allium cepa L.

Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (_max 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (_max 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.Abbreviations GCP guard-cell protoplast - HPLC high-performance liquid chromatography - TLC thin-layer chromatography     

Light dependence of sexual agglutinability in Chlamydomonas eugametos

The mating activity of mating-type plus gametes of Chlamydomonas eugametos depends on light. Cells lost their ability to agglutinate with mating-type minus gametes after a dark period of 30 min. They regained their agglutinability after 10 min exposure to light. Other mating reactions, such as tipping and flagellar tip activation, were not dependent upon light. Since cycloheximide and tunicamycin did not affect the light-induced activation of flagellar agglutinability, no protein synthesis or glycosylation is involved in this process. Equal amounts of biologically active agglutination factor could be extracted from cells placed either in light or in darkness. A minor portion of the active material was found to be located on the flagellar surface of illuminated cells. No active material was found on the flagellar surface of dark-exposed cells, whereas their cell bodies contained the same amount of active material as the cell bodies of illuminated cells. Since a light-induced flow of agglutination factors from the cell body to the flagella could not be detected and dark-exposed cells could be slightly activated by amputation or fixation by glutaraldehyde, we propose that light affects flagellar agglutinability by an in-situ modification of the agglutination factor on the flagella. When mt ⁺ and mt ^- strains were crossed and the progeny examined for light-sensitivity, it was apparent that this phenomenon is not mating type-linked.Abbreviations and symbols FTA flagellar tip activation - mt ^+/- mating type plus or minus - WGA wheat-germ agglutinin     

Green flagellar autofluorescence in brown algal swarmers and their phototactic responses

A flavin-like green autofluorescent substance is noticed to occur in one of the flagella of flagellated cells in the Phaeophyceae, Chrysophyceae, Synurophyceae, Xanthophyceae and Prymnesiophyceae. In the phaeophycean swarmers the autofluorescence occurs in the posterior flagellum throughout its length. It is considered to be involved in the photoreception of phototaxis, since it almost always occurs in the swarmers which have a flagellar swelling and stigma and show phototaxis. In the phaeophycean swarmers, the stigma is shown to act as a concave reflector mirror focusing the reflection light onto the flagellar swelling. In the action spectrum studies, phaeophycean swarmers showed phototaxis between 370 and 520 nm, having two major peaks at 420 or 430 nm and 450 or 460 nm. Their responses were true phototactic and not photophobic. Rotation of the swarmer was shown to be essential in the photoreception ofEctocarpus gametes. Recipient of the Botanical Society Award for Young Scientists, 1991.     

Nonpigment components of the photochlorophyllide photoactive complex: studies of low-temperature blue-green fluorescence spectra

Fluorescence spectra in the blue-green region and excitation fluorescence spectra of green wheat leaves, etiolated wheat leaves and isolated inner etioplast membranes (prolamellar bodies and prothylakoids) were compared to specify the structure of the active protochlorophyllide pigment-protein complex of inner etioplast membranes. Three bands in the blue region at 420, 443 and 470 nm and a broader green band at 525 nm were found. Comparison of the emission and excitation spectra suggests that the main components responsible for the blue fluorescence of etioplast inner membranes are pyridine nucleotides and pterins. The green fluorescence (525 nm) excitation spectra of etiolated samples were identical to the excitation spectrum of flavin fluorescence. The fact confirms the suggestion that flavins are the constituents of the active protochlorophyllide-protein complex.     

Swimming behavior and ultrastructure of sperm of<Emphasis Type="Italic"> Lygodium japonicum</Emphasis> (Pteridophyta)

Swimming behavior of the sperm of Lygodium japonicum (Pteridophyta) and the associated ultrastructure of the flagellar apparatus were studied by video microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The sperm has approximately 70 flagella that emerge from a sinistrally-coiled flagellar apparatus, and swims forward by ciliary beat of these flagella. Backward swimming was not observed even after sperm collided with obstacles. Video microscopy showed that the flagella of the swimming sperm are oriented laterally and oblique-anteriorly. TEM and SEM observations revealed that the basal bodies of these flagella are arranged in at least two rows and oriented in the same directions as observed by video microscopy. These basal bodies (flagella) are categorized into two types according to their orientation: group I (laterally directed) and group II (oblique-anteriorly directed). The directionality of the basal bodies appears to be fixed by electron-dense material around their base. The outer dynein arms of the flagellar axoneme are entirely absent. These morphological characteristics of basal bodies (flagella) may relate to the lack of backward swimming behavior of the sperm. Based on these results, the evolution of swimming behavior in the archegoniates is discussed in connection with lack of backward swimming in a distantly related green alga, Mesostigma viride, and the Streptophyta.     

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt⁺ or mt^-). It is shown that before mt⁺ and mt^- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt^- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt⁺ gametes constituting the outer surface of the clumps with the mt^- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt⁺ flagellar tips then deactivate and swimming is resumed. The original mt^- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt^- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonas eugametos gametes

Evidence is presented which supports the concept of a functional membrane barrier in the transition zone at the base of each flagellum of Chlamydomonas eugametos gametes. This makes it unlikely that agglutination factors present on the surface of the cell body can diffuse or be transported to the flagellar membrane. The evidence is as follows: 1) The glycoprotein composition of the flagellar membrane is very different to that of the cell-body plasma membrane. 2) The flagella of gametes treated with cycloheximide, tunicamycin or , -dipyridyl become non-agglutinable but the source of agglutination factors on the cell body is not affected. 3) Even under natural conditions when the flagella are non-agglutinable, for example in vis-à-vis pairs or in appropriate cell strains that are non-agglutinable in the dark, the cell bodies maintain the normal complement of active agglutinins. 4) When flagella of living cells are labeled with antibodies bound to fluorescein, the label does not diffuse onto the cell-body surface. 5) When gametes fuse to form vis-à-vis pairs, the original mating-type-specific antigenicity of each cell body is slowly lost (probably due to the antigens diffusing over both cell bodies), while the specific antigenicity of the flagellar surface is maintained. Even when the flagella of vis-à-vis pairs are regenerated from cell bodies with mixed antigenicity, the antigenicity of the flagella remains matingtype-specific. 6) Evidence is presented for the existence of a pool of agglutination factors within the cell bodies but not on the outer surface of the cells.Abbreviations and symbols CHI cycloheximide - GTC guaniline thiocyanate - mt ⁺/mt ^- mating type plus or minus - PAS Periodic-acid-Schiff reagent - SDS sodium dodecyl sulphate     

Morphology and ultrastructure ofOlisthodiscus luteus (Raphidophyceae) with special reference to the taxonomy

A flattened discoid flagellate collected from the Seto Inland Sea, Japan, has been examined by light and electron microscopy. This alga agrees well withClisthodiscus luteus Carter. It has two heterodynamic flagella emerging from a furrow on the upward side of the cell that contains six to 13 yellow-green parietal chloroplasts. It does not rotate but smoothly glide while swimming. The cell has a thin periplast lying between the plasmalemma and chloroplasts. Neither lipid bodies nor mucocysts are seen in the periplast. The pyrenoid matrix being free from thylakoids is penetrated by several cytoplasmic canals from various directions. There are no vesicles of periplastidal network in the narrow space between chloroplast envelope and chloroplast ER. The ultrastructural features ofO. luteus are unique, sharing certain characters with the raphidophycean algae but others withPseudopedinella pyriformis, a unique member of the Chrysophyceae.     

慢病毒直接诱导形成猪iPS细胞无需限定因子

为了证实慢病毒对细胞具有遗传修饰和重编程作用,在本实验中使用慢病毒感染猪胎儿成纤维细胞.结果显示:慢病毒介导的EGFP在猪胎儿成纤维细胞中稳定和高效表达,使用添加LIF和bFGF的细胞培养液,部分猪的胎儿成纤维细胞逐渐改变原有的纤维状形态,形成圆形的细胞,细胞逐步增殖形成细胞集落,细胞集落边界清晰,在饲养层上细胞集落生长迅速,具有稳定的生长性能和正常核型,细胞碱性磷酸酶染色为阳性,表达干细胞特有的标记Oct4、Nanog和SSEA1,在体外能够形成拟胚体,在体内分化形成包含三个生殖层的畸胎瘤.作为核移植的供体细胞,克隆胚的卵裂率为53.33%、桑椹胚率为9.03%、囊胚率为2.07%、孵化囊胚的总细胞数为26.5,在桑椹胚率和囊胚率方面显著低于猪普通胎儿成纤维细胞核移植克隆胚的发育能力(P<0.05).结果证实慢病毒能够直接使猪的胎儿成纤维细胞转变成iPS细胞,因此慢病毒将成为一种理想的材料和工具用于细胞的遗传修饰和细胞重构等方面的研究.     

Characterization of Flavin Binding in Flagella of Chiamydomonas

Abstract: Using an experimental approach similar to that used for Euglena flagella, we found that flagella and flagellar membrane preparations (isoagglutinins) of the unicellular green algae Chlamydomonas moewusii and C. reinhardtii , but not cells without flagella, bind radiolabelled riboflavin with high affinity and specificity. In addition, flagella and isoagglutinins contain high amounts of methanol-extractable flavins. These results indicate an abundance of proteins with high affinity for riboflavin in the flagella. Since sexual adhesiveness of gametic flagella in C. moewusii is controlled by light, the possibility is discussed that flavoproteins in the flagella are involved in this reaction. Action spectra exhibit maxima at 450 and 600 nm, suggesting–at least for the 450 nm band–a typical blue-light receptor.     

水蕨精子发生过程中中心体蛋白和微管蛋白的免疫荧光定位

采用透射电镜技术和免疫荧光标记技术对水蕨精子发生的超微结构以及中心体蛋白和微管蛋白在精子发生过程中的动态表达进行了观察。研究发现：（1）生毛体分化早期周围有放射状微管分布,这与线粒体向生毛体的聚集有关。（2）免疫荧光观察表明,中心体蛋白仅定位于生毛体、基体和鞭毛带上,自生毛体至基体阶段呈现明亮的荧光标记,在核塑形、鞭毛形成至精子成熟阶段,中心体蛋白荧光标记随着鞭毛的发生而逐渐减弱,至游动精子阶段中心体蛋白荧光标记信号几乎消失。（3）微管蛋白早期荧光标记与中心体蛋白标记形相同,在生毛体、鞭毛带、基体等运动细胞器上呈现明亮荧光标记,但微管蛋白随着鞭毛的发生其荧光标记越来越强。从二者的时空表达特征可以推断,中心体蛋白主要是运动细胞器的组织者,而非这些运动细胞器的结构成分,其功能是参与或负责中心粒、基体和鞭毛的发生。     

Riboflavin-binding sites associated with flagella of Euglena: A candidate for blue-light photoreceptor?

The hypothesis was tested that reversible riboflavin (RF)-binding sites are part of the photoreceptor in Euglena gracilis. Published evidence shows that the phototactic stimulus — with a flavin-type action spectrum — is perceived at the paraflagellar body (PFB). Flagella with PFBs were isolated from Euglena gracilis by a combined cold and Ca²⁺ shock. Saturable binding of [¹⁴C]RF was demonstrated with such preparations, in the oxidized state as well as under reducing conditions in the presence of dithionite. Affinities for RF were high: K _D (oxidized)=0.08 M, and K _D (reduced)=0.7 M. Flavin mononucleotide and flavin adenine dinucleotide showed lower binding affinities. The in vitro RF binding per unit of protein was enriched approximately tenfold in the flagellar preparations when compared with homogenates of whole cells. The number of (reduced) binding sites per entire flagellum was determined to be in the order of 10⁶. This number is in line with published estimates of chromophores bound in or at the PFB.Abbreviations FAD flavin adenine dinucleotide - FMN flavin mononucleotide - PFB paraflagellar body - RF riboflavin This work was supported by the Deutsche Forscungsgemeinschaft.     

The flagellar apparatus structure inMicrospora (Chlorophyceae) confirms a close evolutionary relationship with unicellular green algae

The spatial configuration of the flagellar apparatus of the biflagellate zoospores of the green algal genusMicrospora is reconstructed by serial sectioning analysis using transmission electron microscopy. Along with the unequal length of the flagella, the most remarkable characteristics of the flagellar apparatus are: (1) the subapical emergence of the flagella (especially apparent with scanning electron microscopy); (2) the parallel orientation of the two basal bodies which are interconnected by a prominent one-piece distal connecting fiber; (3) the unique ultrastructure of the distal connecting fiber composed of a central tubular region which is bordered on both sides by a striated zone; (4) the different origin of the d-rootlets from their relative basal bodies; (5) the asymmetry of the papillar region which together with the subapical position of the basal bodies apparently cause the different paths of corresponding rootlets in the zoospore anterior; (6) the presence of single-membered d-rootlets and multi-membered s-rootlets resulting in a 7-1-7-1 cruciate microtubular root system which, through the different rootlet origin, does not exhibit a strict 180° rotational symmetry. It is speculated that the different basal body origin of the d-rootlets is correlated with the subapical implant of flagella. It is further hypothesized that in the course of evolution the ancestors ofMicrospora had a flagellar papilla that has migrated from a strictly apical position towards a subapical position. Simultaneously, ancestral shift of flagella along the apical cell body periphery has taken place as can be concluded from the presence of an upper flagellum overlying a lower flagellum in the flagellar apparatus ofMicrospora. The basic features of the flagellar apparatus of theMicrospora zoospore resemble those of the coccoid green algal generaDictyochloris andBracteacoccus and also those of the flagellate green algal genusHeterochlamydomonas. This strengthens the general supposition thatMicrospora is evolutionarily closely related to taxa which were formerly classified in the traditionalChlorococcales.     

Photoreception inEuglena gracilis: Fluence-rate and time dependence of photoaccumulation and photodispersal

The fluence-rate and time dependence for photoaccumulation and photodispersal ofEuglena gracilis was measured for the wild-type strain and three white mutants. For wavelengths of 453 or 463 nm the threshold for photoaccumulation was close to 6×10⁻²Wm⁻². Photoaccumulation increased steadily with time and reached a maximum after about 4 hr. Red light elicited substantial photoaccumulation in the wild type and photodispersal in the white, non-photosynthetic mutant 1224-5/9f. The chromophore mediating the red-light response needs to be a non-photosynthetic pigment which remains presently unidentified. A whiteEuglena mutant, FB, which had retained a reduced stigma and a paraflagellar body, showed weak photoaccumulation. Two white mutants, 1224-5/1f and 1224-5/9f, both of which lacked the stigma and positive phototaxis, displayed during the first 90 min of irradiation photodispersal; after longer irradiations they showed instead photoaccumulation. These results contradict a widely held belief that the presence of a stigma represents a stringent requirement for photoaccumulation. Our results imply that phototaxis is not a prerequisite for photoaccumulation. Exogenous flavins and 5,10-methenyl-tetrahydrofolate (MTHF) influenced in a wavelength-dependent manner photoaccumulation and photodispersal. In the wild type FAD and riboflavin (RB) caused at 453 nm an increase of the responsiveness for photoaccumulation. The photoaccumulation of the white mutant FB, was sensitized by FMN and FAD. In the white mutant 1224-5/9f exogenous flavins lowered the threshold for photodispersal. FMN, which absorbs only blue light, altered also the responsiveness to red light: in the wild type FMN reduced photoaccumulation and in the white mutant 1224-5/9f it reduced photodispersal.     

Contribution from coenzyme related molecules to evolution of photoreceptors

Coenzyme related substances, particularly flavins and pterins, were available from primordial chemical processes. When excited by near UV and short wave visible light, these compounds sensitise transfer of redox equivalents in homogenous solution and across membrane. Such reactions result in accumulation of free energy. The evolutionary age of some flavoproteins is close to the age of the Earth's biosphere. In modern organisms coenzyme related substances perform photosensor function for enzymes, such as DNA photolyase and nitrate reductase, and are chromophores for a photoreceptor regulating metabolism and development. Relevance of flavins and pterins both to prebiotic and current photobiological phenomena indicates their possible role in primitive photoreception.