Effects of Some Metabolic Inhibitors on the Pigments of Euglena gracilis in an Acidic Medium

SYNOPSIS. Darkness and O₂-lack promote formation of a pheophytin-like pigment from chlorophyll in Euglena gracilis in an acidic medium. Dinitrophenol (DNP) produces similar but more drastic pigment alterations in aerated and illuminated cells. The extent of pigment decay was dependent on DNP concentration and external pH. Since volume expansion and inhibition of the contractile vacuole were also noted, it is suggested that pheophytin formation is secondary to hydrogen ion influx from the external medium. Major structural alterations of the chloroplast accompanied the pigment changes.
Pentachlorophenol, iodoacetamide, Na fluoride, and Na azide produced similar pigment changes. Malonic acid and fluoroacetate were ineffective under the conditions described. However, in the dark, cells treated with fluoroacetate formed pheophytin rapidly. These effects are discussed in relation to the maintenance of intracellular [H⁺].     

The Physical Mechanism for Retinal Discrete Dark Noise: Thermal Activation or Cellular Ultraweak Photon Emission?

For several decades the physical mechanism underlying discrete dark noise of photoreceptors in the eye has remained highly controversial and poorly understood. It is known that the Arrhenius equation, which is based on the Boltzmann distribution for thermal activation, can model only a part (e.g. half of the activation energy) of the retinal dark noise experimentally observed for vertebrate rod and cone pigments. Using the Hinshelwood distribution instead of the Boltzmann distribution in the Arrhenius equation has been proposed as a solution to the problem. Here, we show that the using the Hinshelwood distribution does not solve the problem completely. As the discrete components of noise are indistinguishable in shape and duration from those produced by real photon induced photo-isomerization, the retinal discrete dark noise is most likely due to ‘internal photons’ inside cells and not due to thermal activation of visual pigments. Indeed, all living cells exhibit spontaneous ultraweak photon emission (UPE), mainly in the optical wavelength range, i.e., 350–700 nm. We show here that the retinal discrete dark noise has a similar rate as UPE and therefore dark noise is most likely due to spontaneous cellular UPE and not due to thermal activation.     

Physiological and ultrastructural changes in “green islands” on Avena sterilis leaves caused by (8R,16R)-(–)-pyrenophorin

The biochemical and ultrastructural changes in "green islands" (GIs) on detached Avena sterilis leaves caused by the macrodiolide (8R,16R)-(-)-pyrenophorin in the dark were examined. In the absence of light, leaf segments retained their photosynthetic pigments for 96 h after treatment with (8R,16R)-(-)-pyrenophorin (70 muM), whereas in the untreated leaves complete senescence, loss of photosynthetic pigments and cell disorganization were observed 72 h after detachment. Proteolytic enzyme activity in treated tissues with pyrenophorin remained at low levels for 96 h after treatment and protein dissipation was lower in the treated than in the untreated. Although tissues in "GIs" seem macroscopically healthy, electron microscopy observations revealed structurally disorganized cells filled with granular, electron-dense material. Chloroplasts were severely damaged and contained a large number of plastoglobuli. Similar ultrastructural changes were also observed in A. sterilis tissues treated with the phytotoxin under illumination, indicating a mechanism operating both under illumination and in the dark.     

Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+. II. Cell-cycle variations in the synthesis and assembly of pigment

Synthesis of the chlorophyll and the major carotenoid pigments and their assembly into thylakoid membrane have been studied throughout the 12-h light/12-h dark vegetative cell cycle of synchronous Chlamydomonas reinhardtii 137+ (wild-type). Pulse exposure of cells to radioactive acetate under conditions in which labeling accurately reflects lipogenesis, followed by cellular fractionation to purify thylakoid membrane, allowed direct analysis of the pigment synthesis and assembly attendant to thylakoid biogenesis. All pigments are synthesized and assembled into thylakoids continuously, but differentially, with respect to cell-cycle time. Highest synthesis and assembly rates are confined to the photoperiod (mid-to-late G1) and support chlorophyll and carotenoid accretion before M-phase. The lower levels at which these processes take place during the dark period (S, M, and early-to- mid G1) have been ascribed to pigment turnover. Within this general periodic pattern, pigment synthesis and assembly occur in a "multi- step" manner, i.e., by a temporally-ordered, stepwise integration of the various pigments into the thylakoid membrane matrix. The cell-cycle kinetics of pigment assembly at the subcellular level mirror the kinetics of pigment synthesis at the cellular level, indicating that pigment synthesis not only provides chlorophyll and carotenoid for thylakoid biogenesis but may also serve as a critical rate-determinant to pigment assembly.     

EFFECT OF CHROMATIC LIGHTS ON PHYCOBILIN FORMATION IN A BLUE-GREEN ALGA, TOLYPOTHRIX TENUIS

1. The formation of phycobilin pigments in a blue-green alga Tolypothrixtenuis was investigated with special reference to the effectsof preillumination with colored lights.
2. It was discoveredthat the algal cells are capable of formingphycobilin pigmentsin the dark, if they have been previouslyilluminated for severalhours in the presence of CO₂.
3. The color of light applied inthe later period of preillumination(chromatic illumination)was found to affect the ratio of phycoerythrinto phycocyaninformed in the subsequent dark period. A greenlight acceleratesthe dark-formation of phycoerythrin, a redlight that of phycocyanin,and the two lights counteractingwith each other in their effects.
4. These directive effects of the "chromatic illumination" canbe accomplished within a very short period, for instance, in3 minutes if it is preceded by sufficient "preillumination"with an incandescent or day light fluorescent light. The reactionsoccurring during the period of chromatic illumination does notrequire the presence of CO₂ and the aerobic condition.
5. Thealga can be grown heterotrophically when supplied with casaminoacids and glucose. Under such a condition the alga forms phycocyanintogether with chlorophyll and carotenoids, but not phycoerythrin.
6. On the basis of the results obtained, a tentative scheme forthe biosynthesis of phycobilin pigments in the alga was proposed,assuming the light-induced formation of unknown precursors whichare converted into phycocyanin and phycoerythrin in the subsequentdark period.

(Received July 4, 1960; )     

Incompetence of dark-synthesized phycocyanin in excitation transfer to photosystem II chlorophyll

Summary When cells of Anabaena variabilis, all the phycobilin pigments of which had been newly synthesized in the dark, were excited by light absorbed in phycocyanin, the fluorescence emission spectrum showed a peak corresponding to the emission from allophycocyanin, but no emission from chlorophyll. These cells were active in photosynthesis and, when excited by light absorbed by chlorophyll, the emitted fluorescence was characteristic of photosystem II chlorophyll. This indicates that dark synthesized phycocyanin is capable of excitation transfer to allophycocyanin but not to photosystem II chlorophyll.Abbreviation CMU 3-(p-chlorophenyl)-1,1-dimethylurea     

β-arrestin functionally regulates the non-bleaching pigment parapinopsin in lamprey pineal

The light response of vertebrate visual cells is achieved by light-sensing proteins such as opsin-based pigments as well as signal transduction proteins, including visual arrestin. Previous studies have indicated that the pineal pigment parapinopsin has evolutionally and physiologically important characteristics. Parapinopsin is phylogenetically related to vertebrate visual pigments. However, unlike the photoproduct of the visual pigment rhodopsin, which is unstable, dissociating from its chromophore and bleaching, the parapinopsin photoproduct is stable and does not release its chromophore. Here, we investigated arrestin, which regulates parapinopsin signaling, in the lamprey pineal organ, where parapinopsin and rhodopsin are localized to distinct photoreceptor cells. We found that beta-arrestin, which binds to stimulated G protein-coupled receptors (GPCRs) other than opsin-based pigments, was localized to parapinopsin-containing cells. This result stands in contrast to the localization of visual arrestin in rhodopsin-containing cells. Beta-arrestin bound to cultured cell membranes containing parapinopsin light-dependently and translocated to the outer segments of pineal parapinopsin-containing cells, suggesting that beta-arrestin binds to parapinopsin to arrest parapinopsin signaling. Interestingly, beta-arrestin colocalized with parapinopsin in the granules of the parapinopsin-expressing cell bodies under light illumination. Because beta-arrestin, which is a mediator of clathrin-mediated GPCR internalization, also served as a mediator of parapinopsin internalization in cultured cells, these results suggest that the granules were generated light-dependently by beta-arrestin-mediated internalization of parapinopsins from the outer segments. Therefore, our findings imply that beta-arrestin-mediated internalization is responsible for eliminating the stable photoproduct and restoring cell conditions to the original dark state. Taken together with a previous finding that the bleaching pigment evolved from a non-bleaching pigment, vertebrate visual arrestin may have evolved from a "beta-like" arrestin by losing its clathrin-binding domain and its function as an internalization mediator. Such changes would have followed the evolution of vertebrate visual pigments, which generate unstable photoproducts that independently decay by chromophore dissociation.     

CAROTENOID INTERCONVERSION IN LIGHT-DARK CULTURES OF THE DINOFLAGELLATE AMPHIDINIUM KLEBSII1

The carotenoid pigments of Amphidinium klebsii cultures grown in LD 12:12 light regimes were determined in cells harvested during the log phase growth in the dark and light photoperiods. The analysis of the individual pigments revealed the presence of an endogenous redox carotenoid system involving the epoxide carotenoid diadinoxanthin and an unidentified carotenoid with the properties of a dihydroxy xanthophyll.     

Visual pigments in the individual rods of deep-sea fishes

Summary The visual pigments in the rods of 15 species of deep-sea fish were examined by microspectrophotometry. In 13 species a single visual pigment was found. The max of these pigments, which ranged from 475 nm to 488 nm, suggest they give the fish maximum sensitivity to the ambient light in the deep, blue ocean waters where they live. In two species two visual pigments were found in separate rods.Bathylagus bericoides had rhodopsins of max 466 nm and 500 nm andMalacocephalus laevis had two rhodopsins of max 478 nm and 485 nm. It is noted that the species with two visual pigments tend to be dark in colour and live in deeper, darker, water.     

Ultrastructure and migration of screening pigments in the retina of Pieris rapae L. (Lepidoptera,Pieridae)

Summary The retinal morphology of the butterfly, Pieris rapae L., was investigated using light and electron microscopy with special emphasis on the morphology and distribution of its screening pigments. Pigment migration in pigment and retinula cells was analysed after light-dark adaptation and after different selective chromatic adaptations. The primary pigment cells with white to yellow-green pigments symmetrically surround the cone process and the distal half of the crystalline cone, whilst the six secondary pigment cells, around each ommatidium, contain dark brown pigment granules. The nine retinula cells in one ommatidium can be categorised into four types. Receptor cells 1–4, which have microvilli in the distal half of the ommatidium only, contain numerous dark brown pigment granules. On the basis of the pigment content and morphology of their pigment granules, two distal groups of cells, cells 1, 2 and cells 3, 4 can be distinguished. The four diagonally arranged cells (5–8), with rhabdomeric structures and pigments in the proximal half of the cells, contain small red pigment granules of irregular shape. The ninth cell, which has only a small number of microvilli, lacks pigment. Chromatic adaptation experiments in which the location of retinula cell pigment granules was used as a criterium reveal two UV-receptors (cells 1 and 2), two green receptors (cells 3 and 4) and four cells (5–8) containing the red screening pigment, with a yellow-green sensitivity.     

Chloroplast ultrastructure,photosynthetic apparatus activities and production of steviol glycosides in <Emphasis Type="Italic">Stevia rebaudiana in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2–3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O₂ evolution rate. However, photosynthetic O₂ evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.     

Light-induced changes of carotenoid pigments in anaerobic cells of the aerobic photosynthetic bacterium,Roseobacter denitrificans (Erythrobacter species OCh 114): reduction of spheroidenone to 3,4-dihydrospheroidenone

Roseobacter denitrificans, previously named Erythrobacter species OCh 114, synthesized spheroidenone as a major carotenoid under aerobic dark conditions. When the dark-grown cells were subjected to illumination under anacrobic conditions, many unknown yellow pigments appeared and a considerable amount of spheroidenone disappeared. Absorption maxima of these pigments were blue-shifted from those of spheroidenone. The most abundant of the pigments was isolated, and its chemical structure was determined as 3,4-dihydrospheroidenone on spectroscopic and chemical evidence. Presumably, over-reduction of the photosynthetic apparatus interfered with normal photosynthetic electron transfer and resulted in photoreduction of C=C double bond at the 3,4-position of spheroidenone.     

Physiological colour changes in Odonata eyes. A comparison between eye and epidermal chromatophore pigment migrations

Pigment migration in the eyes of Austrolestes annulosus and Ischnura heterosticta cause pronounced colour changes which superficially resemble those of Odonata epidermal chromatophores. In both species, the migratory pigment is confined to the distal pigment cells of dorsal ommatidia. When the pigment is concentrated around the base of the crystalline cones, a dense layer of Tyndall blue bodies produce bright ‘blue phase’ colours. Distal migration of the pigment disrupts the Tyndall effect and produces ‘dark phase’ (grey-brown) colours. As in chromatophores, eye pigments consist of a mixture of xanthommatin and dihydroxanthommatin together with an additional pigment, possibly ommin A, not found in chromatophores.As with chromatophores, eye pigments respond to change in temperature only, change in light intensity having no effect. The change from blue to dark phase (at 8°C) occurs at the same rate as in chromatophores, whereas the reverse change (at 20°C) is significantly slower. Equilibrium colours at constant temperature are variable but significantly different from those of chromatophores at 12°C and above. There is no diurnal variation in responsiveness as is found in chromatophores.Isolated dark phase eyes or undamaged pieces of eye are able to change to blue phase after temperature increase. Isolated blue phase eyes show little response to temperature decrease, isolated undamaged pieces show no response. A temperature difference between the eyes of the same intact insect may result in minor colour differences. Ablation of the optic tract or of tissue posterior to the optic tract prevents normal colour change from blue to dark phase. The above results indicate that eye pigment cells are structurally similar to Odonata chromatophores and are under similar environmental and physiological control.     

Freezing tolerance of sea urchin embryo pigment cells

Various stresses, including exposure to cold or heat, can result in a sharp increase in pigmentation of sea urchin embryos and larvae. The differentiation of pigment cells is accompanied by active expression of genes involved in the biosynthesis of naphthoquinone pigments and appears to be a part of the defense system protecting sea urchins against harmful factors. To clarify numerous issues occurring at various time points after the cold injury, we studied the effect of shikimic acid, a precursor of naphthoquinone pigments, on cell viability and expression of some pigment genes such as the pks and sult before and after freezing the cultures of sea urchin embryo cells. The maximum level of the pks gene expression after a freezing–thawing cycle was found when sea urchin cells were frozen in the presence of trehalose alone. Despite naphthoquinone pigments have been reported to possess antioxidant and cryoprotectant properties, our data suggest that shikimic acid does not have any additional cryoprotective effect on freezing tolerance of sea urchin embryo pigment cells.     

Physiology of dark fermentative growth of Rhodopseudomonas capsulata

The photosynthetic bacterium Rhodopseudomonas capsulata can grow under anaerobic conditions with light as the energy source or, alternatively, in darkness with D-fructose or certain other sugars as the sole source of carbon and energy. Growth in the latter mode requires an "accessory oxidant" such as trimethylamine-N-oxide, and the resulting cells contain the photosynthetic pigments characteristic of R. capsulata (associated with intracytoplasmic membranes) and substantial deposits of poly-beta-hydroxybutyrate. In dark anaerobic batch cultures in fructose plus trimethylamine-N-oxide medium, trimethylamine formation parallels growth, and typical fermentation products accumulate, namely, CO2 and formic, acetic, and lactic acids. These products are also found in dark anaerobic continuous cultures of R. capsulata; acetic acid and CO2 predominate when fructose is limiting, whereas formic and lactic acids are observed at elevated concentrations when trimethylamine-N-oxide is the limiting nutrient. Evidence is presented to support the conclusions that ATP generation during anaerobic dark growth of R. capsulata on fructose plus trimethylamine-N-oxide occurs by substrate level phosphorylations associated with classical glycolysis and pyruvate dissimilation, and that the required accessory oxidant functions as an electron sink to permit the management of fermentative redox balance, rather than as a terminal electron acceptor necessary for electron transport-driven phosphorylation.     

Studies on red pigments excreted by cells of Chlorella protothecoides during the process of bleaching induced by glucose or acetate II. Mode of formation of the red pigments

In parallel with the studies reported in the preceding paper(I), the modes of production of characteristic red pigmentsby Chlorella protothecoides cells were investigated under variousculture conditions, (i) During the course of "acetate-bleaching"of algal cells, excretion of red pigments in the medium proceededwith simultaneous disappearance of chlorophyll from algal cells.The total amount (weight) of the red pigments excreted intomedium was slightly less than that of the chlorophyll lost.No red pigment was detectable within the bleaching algal cells.Carotenoids were found to increase or remain nearly constantin their quantities per culture during the process of bleaching,(ii) In a later phase of "glucose-bleaching" some red pigmentswere found to be present inside as well as outside the algalcells, and the excreted pigments underwent further changes turningcolourless, (iii) Both the production of red pigments and disappearanceof chlorophyll were suppressed by light and this light effectwas insensitive to CMU. (iv) During the process of "regreening"of "glucose-bleached" algal cells, no production of red pigmentswas observed either in or outside the algal cells. Based on these results we concluded that the red pigments areproduced from chlorophyll during the bleaching process of algalcells induced by an organic carbon source. (Received July 23, 1968; )     

Light acclimation potential and xanthophyll cycle pigments in photoautotrophic suspension cells of Chenopodium rubrum

The present study investigates the light acclimation potential of photoautotrophic suspension culture cells of Chenopodium rubrum L. grown in 16 h light/8 h dark cycles. Typical features of sun/shade acclimation could be demonstrated in cultures grown at photon flux densities of 30 and 150 μmol m⁻² s⁻¹. Low light grown cells had lower chlorophyll a/b ratios, lower respiration rates and lower light compensation points than high light grown cells. Maximum photosynthetic rate per cell dry weight was highest in low light conditions, indicating that the cells did not enlarge their photosynthetic machinery upon exposure to high light. Transfer of cultures to 800 μmol m⁻² s⁻¹ caused photoinhibition as indicated by a decrease in photosynthetic efficiency and by the occurrence of a slowly reversible quenching of variable chlorophyll fluorescence. Extension of the photoinhibitory treatment over six light dark cycles did not result in further dramatic changes of these parameters, whereas the chlorophyll content per dry weight and the chlorophyll a/b ratio decreased. Measurements of photochemical quenching showed that the capability of the cells to dissipate excessive energy had increased during the acclimation process. The presence of the xanthophyll cycle pigments and the operation of the cycle could be demonstrated. In agreement with the putative photoprotective function of antheraxanthin and zeaxanthin these pigments could only be detected under photoinhibitory conditions. Prolonged photoinhibitory treatment resulted in increases in the xanthophyll pigment concentration but not of the potential to deepoxidate violaxanthin. The limited potential of the cells to accumulate zeaxanthin and antheraxanthin might indicate that the xanthophyll cycle is not the main factor determining their resistance to high light stress.     

First record of colour polymorphism in the Neotropical apple snail Asolene platae: inheritance mechanism and evidence for multiple paternity

Asolene platae (Ampullariidae) is a dioecious freshwater snail with subaquatic gelatinous egg masses that dwells in the Río de la Plata basin (Argentina). The aim of this study was to describe the inheritance mechanism of the colour variations of the shell and soft parts of this snail, and to study their potential use as a genetic marker. The wild-type phenotype presents dark pigments in the soft parts and in shell bands, whereas the yellow phenotype lacks dark pigments in the soft parts and also most dark bands in the shell, except for a subsutural and a periumbilical band. The data showed that the lack of pigments in A. platae is a recessive homozygotic condition with a simple Mendelian inheritance mechanism. Females of the wild-type phenotype had a higher number of bands than the males. The pigment of the bands of both phenotypes is located in the calcareous matrix of the shell. Using the lack of pigments as a genetic marker we demonstrated the existence of biparental egg masses in A. platae, hitherto known in only one species within the Ampullariidae.     

Inhibition of Accumulation of Bacteriochlorophyll and Carotenoids by Blue Light in an Aerobic Photosynthetic Bacterium, Roseobacter denitrificans, during Anaerobic Respiration

The effects of light on the accumulation of bacteriochlorophylland carotenoids were investigated in an aerobic photosyntheticbacterium, Roseobacter denitrificans during anaerobic respiration.Accumulation of pigments occurred in darkness but not in whitelight, with the growth rate being similar under both dark andlight conditions. Once pigments had accumulated during growthin darkness, subsequent irradiation with white light did notresult in degradation of the accumulated pigments, an indicationthat the pigments were stabilized in the membranes. The presentresults, therefore, exclude the possibility of inhibition ofthe accumulation of the photosynthetic pigments by the photochemicaldegradation of the pigments in the presence of molecular oxygenand light (blue light). The action spectrum for the inhibitionof the accumulation of the pigments showed that light at 470nm was the most effective and light at wavelengths longer than500 nm had little inhibitory effect. Together with previousresults [Shimada et al. (1992) Plant Cell Physiol. 33: 471],the present data suggest that a signal-transduction system associatedwith an unidentified blue pigment(s) is involved in the inhibitionof the accumulation of the photosynthetic pigments in R. denitrificans. (Received May 6, 1992; Accepted September 21, 1992)     

Light-Induced Chloroplast Differentiation in Soybean Cells in Suspension Culture : Ultrastructural Changes during the Bleaching and Greening Cycles

Suspension cultures of SB-P cells of soybean (Glycine max) provide a novel, reproducible, and readily manipulable greening system useful for inducing chloroplast differentiation. The cells are subcultured and grown heterotrophically (3% sucrose) in the dark for at least three successive 14-day periods, subcultured and grown in the dark for 7 days more, and finally placed under white light and grown photoautotrophically. Chlorophyll begins to accumulate by 1 hour of light and continues up to 12 days. The chlorophyll a:chlorophyll b ratio is 3:1. Dark-grown cells contain a small amount of total carotenoids which increase 10-fold during greening. Chloroplast differentiation is strictly light dependent, with photosynthetic pigments accumulating in the light and being lost from cells returned to the dark. In the dark, the chloroplasts dedifferentiate to amyloplasts as the organized thylakoid network is lost and starch accumulates. Under continuous light, the amyloplasts differentiate into mature chloroplasts as the organelle elongates, becomes spanned by several bands of thylakoids, and undergoes grana formation. Chloroplast differentiation in SB-P cells is similar to that in intact angiosperms developing under normal light-dark cycles.