Photopigment Distribution Research Articles

Optimization and effects of different culture conditions on growth of Halomicronema hongdechloris – a filamentous cyanobacterium containing chlorophyll f

A chlorophyll f containing cyanobacterium, Halomicronema hongdechloris (H. hongdechloris) was isolated from a stromatolite cyanobacterial community. The extremely slow growth rate of H. hongdechloris has hindered research on this newly isolated cyanobacterium and the investigation of chlorophyll f-photosynthesis. Therefore, optimizing H. hongdechloris culture conditions has become an essential requirement for future research. This work investigated the effects of various culture conditions, essential nutrients and light environments to determine the optimal growth conditions for H. hongdechloris and the biosynthetic rate of chlorophyll f. Based on the total chlorophyll concentration, an optimal growth rate of 0.22 ± 0.02 day-1(doubling time: 3.1 ± 0.3 days) was observed when cells were grown under continuous illumination with far-red light with an intensity of 20 μE at 32°C in modified K + ES seawater (pH 8.0) with additional nitrogen and phosphor supplements. High performance liquid chromatography on H. hongdechloris pigments confirmed that chlorophyll a is the major chlorophyll and chlorophyll f constitutes ~10% of the total chlorophyll from cells grown under far-red light. Fluorescence confocal image analysis demonstrated changes of photosynthetic membranes and the distribution of photopigments in response to different light conditions. The total photosynthetic oxygen evolution yield per cell showed no changes under different light conditions, which confirms the involvement of chlorophyll f in oxygenic photosynthesis. The implications of the presence of chlorophyll f in H. hongdechloris and its relationship with the ambient light environment are discussed.

Changes in the colour of light cue circadian activity

The discovery of melanopsin, the non-visual opsin present in intrinsically photosensitive retinal ganglion cells (ipRGCs), has created great excitement in the field of circadian biology. Now, researchers have emphasized melanopsin as the main photopigment governing circadian activity in vertebrates. Circadian biologists have tested this idea under standard laboratory, 12h Light: 12h Dark, lighting conditions that lack the dramatic daily colour changes of natural skylight. Here we used a stimulus paradigm in which the colour of the illumination changed throughout the day, thus mimicking natural skylight, but luminance, sensed intrinsically by melanopsin containing ganglion cells, was kept constant. We show in two species of cichlid, Aequidens pulcher and Labeotropheus fuelleborni, that changes in light colour, not intensity, are the primary determinants of natural circadian activity. Moreover, opponent-cone photoreceptor inputs to ipRGCs mediate the sensation of wavelength change, and not the intrinsic photopigment, melanopsin. These results have implications for understanding the evolutionary biology of non-visual photosensory pathways and answer long-standing questions about the nature and distribution of photopigments in organisms, including providing a solution to the mystery of why nocturnal animals routinely have mutations that interrupt the function of their short wavelength sensitive photopigment gene.

Two-dimensional mapping of photopigment distribution and activity of Chloroflexus-like bacteria in a hypersaline microbial mat

Pigment analysis in an intact hypersaline microbial mat by hyperspectral imaging revealed very patchy and spatially uncorrelated distributions of photopigments Chl a and BChl a/c, which are characteristic photopigments for oxygenic (diatoms and cyanobacteria) and anoxygenic phototrophs (Chloroflexaceae). This finding is in contrast to the expectation that these biomarker pigments should be spatially correlated, as oxygenic phototrophs are thought to supply the Chloroflexaceae members with organic substrates for growth. We suggest that the heterogeneous occurrence is possibly due to sulfide, whose production by sulfate-reducing bacteria may be spatially heterogeneous in the partially oxic photic zone of the mat. We furthermore mapped the near-infra-red-light controlled respiration of Chloroflexaceae under light and dark conditions and found that Chloroflexaceae are responsible for a major part of oxygen consumption at the lower part of the oxic zone in the mat. The presence of Chloroflexaceae was further confirmed by FISH probe and 16S rRNA gene clone library analysis. We assume that species related to the genera Oscillochloris and 'Candidatus Chlorothrix', in contrast to those related to Chloroflexus and Roseiflexus, depend less on excreted photosynthates but more on the presence of free sulfide, which may explain their presence in deeper parts of the mat.

Structuring of epilithic biofilms by the caddisfly Tinodes rostocki: photosynthetic activity and photopigment distribution in and beside larval retreats

Tinodes rostocki larvae (Trichoptera: Psychomyiidae) cover large proportions of stream hard substrata with retreats constructed of mineral particles and larval silk. We consider these retreats as 3-dimensional extensions of the epilithic biofilm that may possess a distinct microenviron- ment, community metabolism and composition. Therefore, we compared the photosynthetic/respira- tory activities (O2 and pH microsensors) and the photopigment composition (HPLC) of larval retreats and the surrounding epilithic biofilms. In retreats, pigment contents and photosynthetic/respiratory activities were highest in sections with a visible microphytobenthic biofilm that were mostly the older parts of the retreats. In contrast, newly constructed sections of the retreats and the surrounding epilithic biofilm had approximately 5-fold lower values. The fucoxanthin-to-chlorophyll ratio of the retreat biofilm was high (fuco/chl a = 1.27) and indicated diatom dominance, which was not evident in the surrounding epilithic biofilm (fuco/chl a = 0.15). Experimental transplantation of larval retreats to microscope slides allowed microsensor measurements through the 500 to 700 µm-thick wall and inside the lumen. In the light, O2 concentration and pH values increased significantly across the wall and remained high in the lumen of the retreat, whereas in darkness O2 and pH depressions in both wall and lumen were moderate or even absent. Our data suggest that T. rostocki larvae construct and maintain retreats with a particular physico-chemical microenvironment that favours a distinct micro- bial community. Thereby, abundant T. rostocki larvae might significantly influence benthic primary production and heterotrophic metabolism in small streams.

Scanning laser densitometry and color perimetry demonstrate reduced photopigment density and sensitivity in two patients with retinal degeneration

Purpose. To test the feasibility of scanning laser densitometry with a modified Rodenstock scanning laser ophthalmoscope (SLO) to measure the rod and cone photopigment distribution in patients with retinal diseases. Methods. Scanning laser densitometry was performed using a modified Rodenstock scanning laser ophthalmoscope. The distribution of the photopigments was calculated from dark adapted and bleached images taken with the 514 nm laser of the SLO. This wavelength is absorbed by rod and cone photopigments. Discrimination is possible due to their different spatial distribution. Additionally, to measure retinal sensitivity profiles, dark adapted two color static perimetry with a Tübinger manual perimeter was performed along the horizontal meridian with 1° spacing. Results. A patient with retinitis pigmentosa had slightly reduced photopigment density within the central ±5° but no detectable photopigment for eccentricities beyond 5°. A patient with cone dystrophy had nearly normal pigment density beyond ±5°, but considerably reduced photopigment density within the central ±5°. Within the central ±5°, the patient with retinitis pigmentosa had normal sensitivity for the red stimulus and reduced sensitivity for the green stimulus. There was no measurable function beyond 7°. The patient with cone dystrophy had normal sensitivity for the green stimulus outside the foveal center and reduced sensitivity for the red stimulus at the foveal center. The results of color perimetry for this patient with a central scotoma were probably influenced by eccentric fixation. Conclusion. Scanning laser densitometry with a modified Rodenstock SLO is a useful method to assess the human photopigment distribution. Densitometry results were confirmed by dark adapted two color static perimetry. Photopigment distribution and retinal sensitivity profiles can be measured with high spatial resolution. This may help to measure exactly the temporal development of retinal diseases and to test the success of different therapeutic treatments. Both methods have limitations at the present state of development. However, some of these limitations can be overcome by further improving the instruments.

Variation in Sensitivity, Absorption and Density of the Central Rod Distribution with Eccentricity

Purpose: To assess the human rod photopigment distribution and sensitivity with high spatial resolution within the central ±15° and to compare the results of pigment absorption, sensitivity and rod density distribution (number of rods per square degree). Method: Rod photopigment density distribution was measured with imaging densitometry using a modified Rodenstock scanning laser ophthalmoscope. Dark-adapted sensitivity profiles were measured with green stimuli (17′ arc diameter, 1° spacing) using a Tübingen manual perimeter. Sensitivity profiles were plotted on a linear scale and rod photopigment optical density distribution profiles were converted to absorption profiles of the rod photopigment layer. Results: Both the absorption profile of the rod photopigment and the linear sensitivity profile for green stimuli show a minimum at the foveal center and increase steeply with eccentricity. The variation with eccentricity corresponds to the rod density distribution. Conclusion: Rod photopigment absorption profiles, retinal sensitivity profiles, and the rod density distribution are linearly related within the central ±15°. This is in agreement with theoretical considerations. Both methods, imaging retinal densitometry using a scanning laser ophthalmoscope and dark-adapted perimetry with small green stimuli, are useful for assessing the central rod distribution and sensitivity. However, at present, both methods have limitations. Suggestions for improving the reliability of both methods are given.

Modifying a Rodenstock scanning laser ophthalmoscope for imaging densitometry

The necessary modifications and technical requirements are described for using a commercially available scanning laser ophthalmoscope (Rodenstock Model 101 SLO) as an imaging densitometer to assess human photopigment distribution. The main requirements are a linear detector amplifier, fast shutters for the laser beams, and a trigger unit. Images must be compensated for varying laser intensity. Both rod and cone photopigments are measured with the 514-nm argon laser of the SLO. Discrimination is possible owing to the different spatial distribution. The cone pigment density peaks in the foveal center (D = 0.40) with a steep decrease with increasing eccentricity E (full width at half-maximum, 2.5 degrees ). Rod photopigment increases with increasing eccentricity (D = 0.23 for E = 11 degrees ). These values are in agreement with previous reported results obtained with scanning laser ophthalmoscopes specially designed for retinal densitometry and high stability.

Mapping cone photopigment optical density.

The distribution of cone photopigment across the retina affects the amount of light captured by cones at each retinal location. Cone photopigment optical density is measured in two ways, with reflectometry and/or with color matching. Color matching measures a higher optical density than does reflectometry. Control experiments confirm that large-field color matches measure photopigment optical density toward their outer edge. There is qualitative agreement as to photopigment distribution from both techniques near the fovea. Beyond 1 deg, color matching indicates little decrease in photopigment with increasing eccentricity, whereas retinal densitometry shows a steep decline in photopigment. The decrease in perifoveal optical density measured with reflectometry is attributed to the decrease in cone coverage from fovea to perifovea as rods and interphotoreceptor spaces increase. Differences among subjects in photopigment distribution near the fovea, measured with both techniques, reflect differences in the specialization of the foveal center for cone length and/or photopigment concentration per cone, which are factors influencing results from both techniques.

Experimental degradation of plant materials in Hudson river sediments

We examined photopigment degradation and transformation in sediment microcosms that received different detrital source materials (planktonic, littoral, terrestrial) in the presence or absence of amphidpods (Gammarus sp.). Additions of realistic quantities of particulate organic matter resulted in detectable changes in pigment concentration and composition despite insignificant changes in total organic matter. The transformation of chlorophyll a to total phaeophorbide was significantly higher in all high quality (high nitrogen) detritus treatments containing amphipods. The highest production of phaeophorbide was in the higher quality detritus (blue-green algae, Anabaena cylindrica, and macrophyte, Vallisneria americana) when compared to red maple (Acer rubrum). Phaeophytin formation was not related to amphipod grazing and thus may be determined more by microbial heterotrophic processes. The degradation product of the carotenoid lutein, lutein 5,6 expoxide, was formed in all treatments. Phaeopigment composition can be used to infer differences in heterotrophic activity and will help in the interpretation of photopigment distribution in field samples.

Lateral diffusion of photopigments in photoreceptor disk membrane vesicles by the dynamic Kerr effect.

The lateral diffusion of photopigment molecules in the photoreceptor disk membranes, osmotically swollen into spherical vesicles, has been investigated by dynamic Kerr effect measurements. Upon application of a rapidly reversing bipolar electric field to dilute aqueous suspensions of bovine disk membrane vesicles, the birefringence transient shows a characteristic rise and a deep dip corresponding to the first and second pulses, respectively. The birefringence transient is ascribed to the slowly induced dipole moment caused by electric field induced displacement of the photopigment distribution on the vesicular surface. The lateral translational diffusion coefficient is estimated from the time constant of the slowly induced dipole as D = (3.3 +/- 1.2) X 10(-9) cm2 s-1. When spermine, a cationic tetraamine, is bound to the disk membrane vesicles, the relaxation time of the slowly induced dipole is shown to become longer, indicating that the birefringence mechanism is indeed due to the field-induced photopigment displacement.

Membranes of Rhodopseudomonas spheroides: interactions of chromatophores with the cell envelope.

Under carefully controlled ionic conditions, large-scale preparations of highly purified chromatophores and cell envelopes were obtained from phototrophically grown Rhodopseudomonas spheroides by zonal ultracentrifugation. The majority of the bacteriochlorophyll a was located in a single, discrete chromatophore band, whereas the envelopes were nearly devoid of photopigment. The envelope fraction contained substantial quantities of succinic dehydrogenase and cytochromes, confirming that phototrophically grown cells contain a photopigment-deficient cytoplasmic membrane. Magnesium at concentrations of 1.0 mM or higher caused chromatophores to reversibly aggregate with the cell envelope. Significant aggregation was also promoted by other divalent metals (Co(2+) > Mn(2+) > Ca(2+) > Mg(2+)), but aggregation was less extensive with monovalent cations. These results account for the distribution of photopigments in two bands reported by others and further suggest that the photosynthetic apparatus of R. spheroides is located on membranes largely distinct from the cell wall-cytoplasmic membrane complex.