Dark Regeneration Research Articles

In-situ characterization of electron-assisted regeneration of Cz-Si solar cells

We examine the regeneration kinetics of passivated emitter and rear solar cells (PERCs) fabricated on boron-doped p-type Czochralski-grown silicon wafers in darkness by electron injection via application of a forward bias voltage at elevated temperature (140 °C) in order to discriminate between electronic and photonic effects. Based on these dark regeneration experiments, we address the existing inconsistency regarding the measured linear dependence of the regeneration rate constant on the excess carrier density. Using the method of dark regeneration by current injection into the solar cell, we are able to measure the total recombination current of the solar cell at the actual regeneration temperature under applied voltage, i.e., at the physically relevant regeneration conditions. The direct comparison of the regeneration rate constant as a function of electronically injected carrier concentration in the dark and the regeneration rate constant during illumination clearly shows that the regeneration is a purely electronically stimulated effect and that photons are not directly involved.

Kinetics of methylene blue (MB) photocatalyzed reduction and dark regeneration in a colorimetric oxygen sensor

Performance data for a dye based, regenerable oxygen sensor (Mills and Lawrie [1], Mills et al. [2]) are analyzed to develop useful kinetic models for sensor photoactivation (dye reduction) and dark, oxygen detection (dye oxidation). The titania loaded, thin film sensor exhibits an apparent first order photoactivation of the dye, which we demonstrate (Section 3.2 and Fig. 4) is due to a kinetic disguise of a zero order photoreaction occurring through a non-uniformly illuminated sensor film. The observed zero order, slow recovery due to dye oxidation by dioxygen (O2 detection) appears best rationalized by a model assuming a near O2-impermeable skin developing on the sensor surface as solvent is evaporatively removed following sensor film casting and curing.

A critical element of the light-induced quaternary structural changes in YtvA-LOV.

YtvA, a photosensory LOV (light-oxygen-voltage) protein from Bacillus subtilis, exists as a dimer that previously appeared to undergo surprisingly small structural changes after light illumination compared with other light-sensing proteins. However, we now report that light induces significant structural perturbations in a series of YtvA-LOV domain derivatives in which the Jα helix has been truncated or replaced. Results from native gel analysis showed significant mobility changes in these derivatives after light illumination; YtvA-LOV without the Jα helix dimerized in the dark state but existed as a monomer in the light state. The absence of the Jα helix also affected the dark regeneration kinetics and the stability of the flavin mononucleotide (FMN) binding to its binding site. Our results demonstrate an alternative way of photo-induced signal propagation that leads to a bigger functional response through dimer/monomer conversions of the YtvA-LOV than the local disruption of Jα helix in the As-LOV domain.

Blue Light-induced Dimerization of Monomeric Aureochrome-1 Enhances Its Affinity for the Target Sequence

Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in stramenopile alga, Vaucheria frigida. AUREO1 contains a basic leucine zipper (bZIP) domain in the central region and a light-oxygen-voltage sensing (LOV) domain at the C terminus, and has been suggested to function as a light-regulated transcription factor. We have previously reported that preparations of recombinant AUREO1 contained the complete coding sequence (full-length, FL) and N-terminal truncated protein (ZL) containing bZIP and LOV domains, and suggested that wild-type ZL (ZLwt2) was in a dimer form with intermolecular disulfide linkages at Cys(162) and Cys(182) (Hisatomi, O., Takeuchi, K., Zikihara, K., Ookubo, Y., Nakatani, Y., Takahashi, F., Tokutomi, S., and Kataoka, H. (2013) Plant Cell Physiol. 54, 93-106). In the present study, we report the photoreactions, oligomeric structures, and DNA binding of monomeric cysteine to serine-mutated ZL (ZLC2S), DTT-treated ZL (DTT-ZL), and FL (DTT-FL). Recombinant AUREO1 showed similar spectral properties and dark regeneration kinetics to those of dimeric ZLwt2. Dynamic light scattering and size exclusion chromatography revealed that ZLC2S and DTT-ZL were monomeric in the dark state. Dissociation of intermolecular disulfide bonds of ZLwt2 was in equilibrium with a midpoint oxidation-redox potential of approximately -245 ± 15 mV. BL induced the dimerization of monomeric ZL, which subsequently increased its affinity for the target sequence. Also, DTT-FL was monomeric in the dark state and underwent BL-induced dimerization, which led to formation of the FL2·DNA complex. Taken together, our results suggest that monomeric AUREO1 is present in vivo, with dimerization playing a key role in its role as a BL-regulated transcription factor.

Stimulation of Shoot Regeneration on Linum Hypocotyl Segments by Thidiazuron and Its Response to Light and Calcium

The basal segment from hypocotyl of Linum usitatissimum L. seedling readily regenerates, to produce a large number of shoots, in a short period of 5 [ndash ] 7 d. This response was stimulated by a low concentration (0.1 [micro ]M) of thidiazuron (TDZ). TDZ was also effective in inducing regeneration in dark. A drastic reduction in regeneration response on hormone-free as well as TDZ-supplemented medium was found after inclusion of an inhibitor of calcium-uptake, lanthanum (La3+). An essentiality of calcium in the regeneration was also evidenced by an increased response with increasing concentration of calcium. At Ca2+ concentration insufficient for regeneration, inclusion of TDZ resulted in shoot formation.

Light-mediated retinoic acid production.

Retinoids serve two main functions in biology: retinaldehyde forms the chromophore bound to opsins, and retinoic acid (RA) is the activating ligand of transcription factors. These two functions are linked in the vertebrate eye: we describe here that illumination of the retina results in an increase in RA synthesis, as detected with a RA bioassay and by HPLC. The synthesis is mediated by retinaldehyde dehydrogenases which convert some of the chromophore all-trans retinaldehyde, released from bleached rhodopsin, into RA. As the eye contains high levels of retinaldehyde dehydrogenases, and as the oxidation of retinaldehyde is an irreversible reaction, RA production has to be considered an unavoidable by-product of light. Through RA synthesis, light can thus directly influence gene transcription in the eye, which provides a plausible mechanism for light effects that cannot be explained by electric activity. Whereas the function of retinaldehyde as chromophore is conserved from bacteria to mammals, RA-mediated transcription is fully evolved only in vertebrates. Invertebrates differ from vertebrates in the mechanism of chromophore regeneration: while in the invertebrate visual cycle the chromophore remains bound, it is released as free all-trans retinaldehyde from illuminated vertebrate rhodopsin. RA synthesis occurring as corollary of dark regeneration in the vertebrate visual cycle may have given rise to the expansion of RA-mediated transcriptional regulation.

The visual pigments of four deep-sea crustacean species.

The visual pigments of four mesopelagic crustacean species were studied at sea by means of microspectrophotometry. The absorbance maxima obtained for the visual pigments and their metarhodopsins, respectively, were: 493 nm and 481 nm (Systellaspis debilis), 485 nm and 480 nm (Acanthephyra curtirostris), 491 nm and 482 nm (A. smithi), and 495 nm and 487 nm (Sergestes tenuiremis). The spectral characteristics of the rhodopsins and metarhodopsins permit high photosensitivity and facilitate photoregeneration in a nearly monochromatic environment. Photic regeneration of rhodopsins from the deep-sea environment was demonstrated, and data were obtained which are consistent with the occurrence of dark regeneration. Specific optical density of the observed visual pigments was calculated for two species.

Dark regeneration of rhodopsin in crayfish photoreceptors.

The eyes of crayfish were exposed to lights of known spectral composition, and the course of regeneration was followed in the dark by measuring the content of rhodopsin and metarhodopsin in single rhabdoms isolated at various times after the adaptation, using an assay that is based on the fluorescence of metarhodopsin. Complete recovery requires several days in the dark after intense adaptation to orange light, but requires less than 2 d after blue light exposure. Following an orange light exposure with blue produces recovery kinetics characteristic of the blue light exposure alone. This quickening of recovery occurs whether the receptors are exposed to blue light either immediately or many hours after the original exposure to orange. Conversely, following blue light adaptation with orange leads to slow recovery, which is characteristic of orange alone. Recovery from long-wavelength adaptation is slower principally because many rhabdoms seem to delay the onset of regeneration. We suggest that the regeneration system is itself photosensitive, and after orange light adaptation the supply of active chromophore (presumably 11-cis retinal) limits the rate of recovery. Once started, recovery proceeds slowly and continuously, and the total pigment concentration (rhodopsin plus metarhodopsin) in the rhabdomeric membrane remains approximately constant. Within hours after intense adapting exposures, the rhabdoms become altered in appearance, the surfaces become coated with accessory pigment, and the bands of microvilli are less distinct. These changes persist until recovery of rhodopsin proceeds, which suggests that visual pigment regeneration results from addition of newly synthesized rhodopsin associated with membrane turn-over.

Dark regeneration of squid rhodopsin and isorhodopsin

Dark regeneration of squid rhodopsin and isorhodopsin

Microspectrophotometry ofDrosophila visual pigments: Determinations of conversion efficiency in R1?6 receptors

We used microspectrophotometry, together with some associated electrophysiological measurements, to characterize the photopigments of R1–6 receptors in living, white-eyedDrosophila. Measurements were from the deep pseudopupil, an optical visualization of photopigment-containing rhabdomeres. Transmission changes associated with photointerconversion of rhodopsin (R480) and its stable metarhodopsin (M570) are easily seen (Fig. 1). Such transmission changes were measured to produce a difference specrum (Fig. 2); the isosbestic wavelength is about 502 nm. A photoequilibrium spectrum (Fig. 3), analyzed together with sensitivity data (Fig. 4a), was used to determine the fraction of M570 in photoequilibria established with various monochromatic wavelengths. From this, the quantum efficiency for M570 to R480 conversion relative to R480 to M570 efficiency was determined to be about 0.71. No dark regeneration of M570 to R480 occurred within a period of 60min (Fig. 4a, b).

Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP, ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts. During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation. The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts. The fluorescence rise during a second illumination is still biphasic. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts. Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination. Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.

The visual pigment and visual cycle of the lobster,Homarus

The visual pigment of the American lobster,Homarus americanus, has been studied in individual isolated rhabdoms by microspectrophotometry. Lobster rhodopsin has λmax at 515 nm and is converted by light to a stable metarhodopsin with λmax at 490 nm. These figures are in good agreement with corresponding values obtained by Wald and Hubbard (1957) in digitonin extracts. Photoregeneration of rhodopsin to metarhodopsin is also observed. The absorbance spectrum of lobster metarhodopsin is invariant with pH in the range 5.4–9, indicating that even after isomerization of the chromophore fromcis totrans, the binding site of the chromophore remains sequestered from the solvent environment. Total axial density of the lobster rhabdom to unpolarized light is about 0.7. As described for several other Crustacea, aldehyde fixation renders the metarhodopsin susceptible to photobleaching, a process that is faster at alkaline than at neutral or acid pH. Small amounts of a photoproduct with λmax at 370 nm are occasionally seen. A slower dark bleaching of lobster rhabdoms (τ1/2−2 h) also occurs, frequently through intermediates with absorption similar to metarhodopsin. The molar extinction coefficient of metarhodopsin is about 1.2 times greater than that of rhodopsin, each measured at their respective λmax. Isomerization of the chromophore fromcis totrans is accompanied by a change in the orientation of the absorption vector of about 3°. The absorption vector of metarhodopsin is either tilted more steeply into the membrane or is less tightly oriented with respect to the microvillar axes. When living lobsters are kept at room temperature, light adaptation does not result in an accumulation of metarhodopsin. At 4 °C, however, the same adapting lights cause a reduction of rhodopsin and an increase in metarhodopsin. There is thus a temperature-sensitive regeneration mechanism that supplements photoregeneration. Following 1 ms, 0.1 joule xenon flashes that convert about 70% of the rhodopsin to metarhodopsin in vivo, dark regeneration occurs in the living eye with half-times of about 25 and 55 min at 22 °C and 15 °C respectively.

Microspectrophotometry of rhodopsin and metarhodopsin in the moth Galleria.

Fresh, frozen sections of the photoreceptor layer of the compound eye of the moth Galleria have been examined by microspectrophotometry, using 4 times 8 mum measuring beams that sampled from approximately two to four rhabdoms. The principal visual pigment absorbs maximally at 510 nm (P510), and on irradiation is converted to a thermally stable, pH-insensitive metarhodopsin with lambda max at 484 nm (M484) and a 43% increase in molar extinction coefficient. Subsequently, short wavelength irradiation of the metarhodopsin photoregenerates some P510, but the absence of an isosbestic point the cycle of spectral changes is consistent with the presence of smaller amounts of violet-or ultraviolet-sensitive visual pigment(s) that also are converted to a blue-absorbing metarhodopsin. Difference spectra for both P510 and M484 were measured, using hydroxylamine. The 484-nm metarhodopsin is reversibly converted to a form with lambda max at 363 nm by high concentrations of glycerol. Dark regeneration of rhodopsin in vivo after several minutes exposure of thoroughly dark-adapted animals to full sunlight requires several days.