Photochemistry and Photobiology

Cover image for Vol. 90 Issue 5

Edited By: Jean Cadet

Impact Factor: 2.684

ISI Journal Citation Reports © Ranking: 2013: 36/74 (Biophysics); 161/291 (Biochemistry & Molecular Biology)

Online ISSN: 1751-1097

Virtual Issue on Photosensory Biology


This virtual issue of P&P collects some of the many relevant papers published in the Journal on diverse areas of photosensory biology during the last five years.
Edited by Francesco Lenci and Jean Cadet

Besides being a fundamental source of energy for all photosynthetic organisms and microorganisms, light is an environmental stimulus of primary importance for all living beings, terrestrial and aquatic, diurnal and nocturnal, prey and predator, alike. The importance of light as a stimulus applies for creatures provided with "eyes" and neural networks as well as for aneural life forms like plants, fungi and even unicellular microorganisms, such as bacteria, algae and protozoa.






Phototaxis of Haloarcula marismortui Revealed Through a Novel Microbial Motion Analysis Algorithm


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Lin, Y.-C., Fu, H.-Y., Yang, C.-S.
Photochemistry and Photobiology 86:5 (1084-1090), 2010

Haloarcula marismortui has been described to be nonmotile prior to the recent identification of flagellar filaments, suggesting the motile nature of H. marismortui. Here we observed the locomotion of freshly cultured H. marismortui cells and tracked the swimming trajectories via ImageJ. Trajectories of H. marismortui are intrinsically noisy, posing difficulties in motion analysis with previously established algorithms. By introducing the concept of “window vector,” a Microsoft Excel-VBA-implemented microbial motion analysis algorithm reported here was able to (1) discriminate nonswimming objects from swimming cells without empirical customization by applying a power-law relationship and (2) reduce the noise caused by Brownian motion, thus enhancing the accuracy of swim reversal identification. Based on this motion analysis algorithm, two recently identified sensory rhodopsins, HmSRI and HmSRII, were shown to mediate photoattractant and photorepellent responses, respectively, revealing the phototactic activity of H. marismortui, the only archaeon showing such phenomenon other than Halobacterium salinarum.

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In Vivo Effects on Photosynthesis Gene Expression of Base Pair Exchanges in the Gene Encoding the Light-responsive BLUF Domain of AppA in Rhodobacter Sphaeroides



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Metz, S., Hendriks, J., Jäger, A., Hellingwerf, K., Klug, G.
Photochemistry and Photobiology 86:4(882-889), 2010

The Rhodobacter sphaeroides protein AppA has the unique quality of sensing and transmitting light and redox signals. By acting as antirepressor to the PpsR protein, it acts as a major regulator in photosynthesis gene expression. In this study, we show that by introducing amino acid exchanges into the AppA protein, the in vivo activity as an antirepressor can be greatly altered. The tryptophan 104 to phenylalanine (W104F) base exchange greatly diminished blue-light sensitivity of the BLUF domain. From the obtained in vivo data, the difference in thermal recovery rate of the signaling state of the BLUF domain between the wild type and mutated protein was calculated, predicting an about 10-fold faster recovery in the mutant, which is consistent with in vitro data. Introduction of a tyrosine 21 to phenylalanine (Y21F) or to cysteine (Y21C) mutation led to a complete loss of AppA antirepressor activity, while additionally leading to an increase of photosynthesis gene expression after illumination with high blue-light quantities. Interestingly, this effect is not visible in a W104F/Y21F double mutant that again shows a wild-type–like behavior of the BLUF domain after blue-light illumination, thus restoring the activity of AppA.

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Flagellar Motions in Phototactic Steering in a Brown Algal Swarmer


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Matsunaga, S., Uchida, H., Iseki, M., Watanabe, M., Murakami, A.
Photochemistry and Photobiology 86:2 (374-381), 2010

Using infrared high-speed video microscopy, we observed light-triggered transitory flagellar motions in flagellate reproductive cells (swarmers) of a brown alga, Scytosiphon lomentaria, under primary helical swimming conditions before and during negative phototactic orientation to unilateral actinic light. The posterior flagellum, which is autofluorescent and thought to be light-sensing, was passively dragged in the dark and exhibited one to several rapid lateral beats during orientation changes for phototactic steering. Notably, a brief cessation of anterior flagellar beating was occasionally observed concomitantly with rapid beats of the posterior flagellum. This behavior caused a pause in helical body rotation, which may contribute to the accuracy of phototactic steering. Thus, coordinated regulation of the movement of the two flagella plays a crucial role in phototactic steering.

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Heteroblastic Development and Shade-avoidance in Response to Blue and Red Light Signals in Acacia implexa


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Forster, M. A., Bonser, S. P.
Photochemistry and Photobiology 85:6 (1375-1383), 2009

Information from blue (400–500 nm) and red (660–730 nm) wavelengths is used by plants to determine proximity of neighbors or actual shading. Plants undergo trait changes in order to out-compete neighbors or accommodate shading. Heteroblasty, the dramatic shift from one leaf type to another during juvenility, can be influenced by the light environment although it is unknown whether cues from blue or red (or both) are driving the developmental process. Seedlings of three populations of Acacia implexa (Mimosaceae) collected from low, medium and high rainfall habitats were grown in a factorial design of high/low blue and red light to determine how light signals affect heteroblasty and patterns of biomass allocation. Low blue light significantly delayed heteroblasty in the low rainfall population and low red light significantly delayed in the low and high rainfall populations. Low blue light increased stem elongation and decreased root biomass whereas low red light induced a strong shade-avoidance response. These results were consistent across populations although the low rainfall population showed greater trait variability in response to red light signals. We conclude that red light conveys a greater information signal than blue light that affects heteroblasty and seedling development in A. implexa.

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Blue Light-induced Chloroplast Avoidance and Phototropic Responses Exhibit Distinct Dose Dependency of PHOTOTROPIN2 in Arabidopsis thaliana


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Kimura, M., Kagawa, T.
Photochemistry and Photobiology 85:5 (1260-1264), 2009

PHOTOTROPIN2 (PHOT2) is a unique photoreceptor involved in chloroplast avoidance movement and also regulates blue light (BL) responses, such as phototropism and leaf flattening, together with PHOTOTROPIN1 (PHOT1) in Arabidopsis thaliana. Previous work showed that the defect of the phot2-1 mutant in chloroplast avoidance movement was a semidominant trait. In the present study, we examined PHOT2 dose dependency of BL responses using the phot1-5 phot2-1 double mutant expressing an AtPHOT2-GFP (P2G) fusion protein. Chloroplast avoidance and phototropic responses of P2G transgenic lines were enhanced in a manner dependent on the P2G levels, whereas the leaf flattening phenotype was simply complemented by P2G equivalent to the wild type (WT) PHOT2 level. The chloroplast avoidance velocity of P2G transgenic lines exhibited enhanced sensitivity to BL in comparison with WT. In contrast, the defect of the phototropic response was rescued by P2G expression equivalent only to the response of the phot1 mutant. These results collectively indicate that each BL response has distinct threshold levels of PHOT2 requirement.

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Characterization of an Unusual LOV Domain Protein in the α-Proteobacterium Rhodobacter sphaeroides


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Hendrischk, A.-K., Moldt, J., Frühwirth, S. W., Klug, G.
Photochemistry and Photobiology 85:5 (1254-1259), 2009

The facultatively phototrophic purple bacterium Rhodobacter sphaeroides 2.4.1 harbors a LOV (light, oxygen and voltage) domain protein, which shows a particular structure. LOV domains perceive blue light by a noncovalently bound flavin and transmit the signal to various coupled output domains. Proteins, that harbor a LOV core, function e.g. as phototropins or circadian clock regulators. Jα helices, which act as linker between the LOV core and the output domain, were shown to be involved in the light-dependent activation of the output domain. Like PpSB2 from Pseudomonas putida, the LOV domain protein of R. sphaeroides is not coupled to an effector domain and harbors an extended C-terminal α helix. We expressed the R. sphaeroides LOV domain recombinantly in Escherichia coli. The protein binds an FMN as a cofactor and shows a photocycle typical for LOV domain containing proteins. In R. sphaeroides, we detected the protein as well in the cytoplasm as in the membrane fraction, which was not reported for other bacterial LOV domain proteins.

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UV-B Action Spectrum for UVR8-Mediated HY5 Transcript Accumulation in Arabidopsis



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Brown, B. A., Headland, L. R., Jenkins, G. I.
Photochemistry and Photobiology 85:5 (1147-1155), 2009

Arabidopsis thalianaUV RESISTANCE LOCUS8 (UVR8) is a UV-B-specific signaling component that mediates low fluence, photomorphogenic responses to UV-B. It is required for UV-B-induced expression of the gene encoding the ELONGATED HYPOCOTYL5 (HY5) transcription factor. HY5 is a key effector of responses mediated by UVR8. In mature leaf tissue, HY5 transcript accumulation occurred rapidly in response to a brief UV-B treatment and no induction was observed in a uvr8 mutant over a broad range of UV wavelengths. In response to monochromatic light, maximal transcript accumulation occurred in wild-type plants at wavelengths 280–300 nm. HY5 transcript accumulation showed reciprocity between the fluence rate and duration of UV-B exposure, and on this basis conditions were chosen to generate an action spectrum for the UVR8 signaling pathway. Dose–response curves were produced for a range of UV wavelengths using 20 min exposure to UV and harvesting tissue 2 h after the start of illumination. Experiments using mutants defective in sinapate ester and flavonoid biosynthesis indicated that the presence of UV-absorbing compounds did not affect the construction of an action spectrum under the conditions employed. The action spectrum for the induction of HY5 by the UVR8 pathway showed a main peak at 280 nm with a smaller peak at 300 nm. The data are discussed in relation to the proposed mechanisms of UV-B photoreception.

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Photosensory Functions of Channelrhodopsins in Native Algal Cells


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Sineshchekov, O. A., Govorunova, E. G., Spudich, J. L.
Photochemistry and Photobiology 85:2 (556-563), 2009

Photomotility responses in flagellate alga are mediated by two types of sensory rhodopsins (A and B). Upon photoexcitation they trigger a cascade of transmembrane currents which provide sensory transduction of light stimuli. Both types of algal sensory rhodopsins demonstrate light-gated ion channel activities when heterologously expressed in animal cells, and therefore they have been given the alternative names channelrhodopsin 1 and 2. In recent publications their channel activity has been assumed to initiate the transduction chain in the native algal cells. Here we present data showing that: (1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; (2) the primary function of Type B sensory rhodopsin (channelrhodopsin-2) is biochemical activation of secondary Ca2+-channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; (3) Type A sensory rhodopsin (channelrhodopsin-1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low-efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells.

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The Photoreactions of Recombinant Phytochrome CphA from the Cyanobacterium Calothrix PCC7601: A Low-Temperature UV–Vis and FTIR Study


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Schwinté, P., Gärtner, W., Sharda, S., Mroginski, M.-A., Hildebrandt, P., Siebert, F.
Photochemistry and Photobiology 85:1 (239-249), 2009

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV–Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV–Vis and FTIR experiments. Three intermediates have been trapped at −25, −45 and −120°C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at −70 and −140°C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore–protein interactions.

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Interactions Between Chromophore and Protein in Phytochrome Identified by Novel Oxa-, Thia- and Carba-Chromophores



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Bongards, C., Gärtner, W.
Photochemistry and Photobiology 84:5 (1109-1117), 2008

Six new bilin chromophores of the plant photoreceptor phytochrome have been synthesized, carrying at the photoisomerizing ring D an oxygen or a sulfur atom or a methylene group instead of the pyrrole nitrogen atom. These furanone-, thiophenone- or cyclopentenone-containing compounds bound covalently to the recombinant apophytochrome phyA of Avena sativa. The novel chromoproteins showed hypsochromically shifted absorption spectra with respect to native phytochrome and a strongly diminished photochemical activity, but a three- to four-fold higher fluorescence quantum yield. These results demonstrate that, on the one hand, also ring D-modified chromophores can be forced into a partially extended structure, required for incorporation into the apoprotein binding pocket and covalent binding. On the other hand, the modifications introduced into ring D of the chromophores strongly impede the formation of stable far red-absorbing forms of plant photoreceptor phytochrome (Pfr-form) of the chromoproteins, highlighting especially the role of the pyrrole nitrogen atom and hydrogen bonding for the precise interactions between that par

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Involvement of Electron Transfer in the Photoreaction of Zebrafish Cryptochrome-DASH



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Zikihara, K., Ishikawa, T., Todo, T., Tokutomi, S.
Photochemistry and Photobiology 84: 4 (1016-1023), 2008 

Photoreaction of a blue-light photoreceptor Cryptochrome-DASH (Cry-DASH), a new member of the Cryptochrome family, from zebrafish was studied by UV–visible absorption spectroscopy in aqueous solutions at 293 K. Zebrafish Cry-DASH binds two chromophores, a flavin adenine dinucleotide (FAD) and a N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF) noncovalently. The bound FAD exists in the oxidized form (FADox) in the dark. Blue light converts FADox to the neutral radical form (FADH?). Formed FADH? is transformed to the fully reduced form FADH2 (or FADH−) by successive light irradiation, or reverts to FADox. FADH2 (or FADH−) reverts to FADH? or possibly to FADox directly. The effect of dithiothreitol suggests a possible electron transfer between FAD in zebrafish Cry-DASH and reductants in the external medium. This is the first report on the photoreaction pathway and kinetics of a vertebrate Cry-DASH family protein.

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Low-temperature Spectroscopy of Met100Ala Mutant of Photoactive Yellow Protein


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Imamoto, Y., Harigai, M., Morimoto, T., Kataoka, M.
Photochemistry and Photobiology 84:4 (970-976), 2008

The trans-to-cis photoisomerization of the p-coumaroyl chromophore of photoactive yellow protein (PYP) triggers the photocycle. Met100, which is located in the vicinity of the chromophore, is a key residue for the cis-to-trans back-isomerization of the chromophore, which is a rate-determining reaction of the PYP photocycle. Here we characterized the photocycle of the Met100Ala mutant of PYP (M100A) by low temperature UV–visible spectroscopy. Irradiation of M100A at 80 K yielded a 380 nm species (M100ABL), while the corresponding intermediate of wild type (WT; PYPBL) is formed above 90 K. The amounts of redshifted intermediates produced from M100A (M100AB′ and M100AL) were substantially less than those from WT. While the near-UV intermediate (PYPM) is not formed from WT in glycerol samples at low temperature, M100AM was clearly observed above 190 K. These alterations of the photocycle of M100A were explained by the shift in the equilibrium between the intermediates. The carbonyl oxygen of the thioester linkage of the cis-chromophore in the photocycle intermediates is close to the phenyl ring of Phe96 (3.5 Å), which would be displaced by the mutation of Met100. These findings imply that the interaction between chromophore and amino acid residues near Met100 is altered during the early stage of the PYP photocycle.

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Identification of Six New Photoactive Yellow Proteins—Diversity and Structure–Function Relationships in a Bacterial Blue Light Photoreceptor



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Kumauchi, M., Hara, M. T., Stalcup, P., Xie, A., Hoff, W. D.
Photochemistry and Photobiology 84:4 (956-969), 2008

Photoactive yellow proteins (PYP) are bacterial photoreceptors with a Per-Arnt-Sim (PAS) domain fold. We report the identification of six new PYPs, thus nearly doubling the size of this protein family. This extends the taxonomic diversity of PYP-containing bacteria from photosynthetic to nonphotosynthetic bacteria, from aquatic to soil-dwelling organisms, and from Proteobacteria to Salinibacter ruber from the phylum Bacteriodetes. The new PYPs greatly increase the sequence diversity of the PYP family, reducing the most prevalent pair-wise identity from 45% to 25%. Sequence alignments and analysis indicate that all 14 PYPs share a common structure with 13 highly conserved residues that form the chromophore binding pocket. Nevertheless, the functional properties of the PYPs vary greatly—the absorbance maximum extends from 432 to 465 nm, the pKa of the chromophore varies from pH 2.8 to 10.2, and the lifetime of the presumed PYP signaling state ranges from 1 ms to 1 h. Thus, the PYP family offers an excellent opportunity to investigate how functional properties are tuned over a wide range, while maintaining the same overall protein structural fold. We discuss the implications of these results for structure–function relationships in the PYP family.

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Dynamics Change of Phoborhodopsin and Transducer by Activation: Study Using D75N Mutant of the Receptor by Site-directed Solid-state 13C NMR


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Kawamura, I., Yoshida, H., Ikeda, Y., Yamaguchi, S., Tuzi, S., Saitô, H., Kamo, N., Naito, A.
Photochemistry and Photobiology 84:4 (921-930), 2008

Photoactive yellow proteins (PYP) are bacterial photoreceptors with a Per-Arnt-Sim (PAS) domain fold. We report the identification of six new PYPs, thus nearly doubling the size of this protein family. This extends the taxonomic diversity of PYP-containing bacteria from photosynthetic to nonphotosynthetic bacteria, from aquatic to soil-dwelling organisms, and from Proteobacteria to Salinibacter ruber from the phylum Bacteriodetes. The new PYPs greatly increase the sequence diversity of the PYP family, reducing the most prevalent pair-wise identity from 45% to 25%. Sequence alignments and analysis indicate that all 14 PYPs share a common structure with 13 highly conserved residues that form the chromophore binding pocket. Nevertheless, the functional properties of the PYPs vary greatly—the absorbance maximum extends from 432 to 465 nm, the pKa of the chromophore varies from pH 2.8 to 10.2, and the lifetime of the presumed PYP signaling state ranges from 1 ms to 1 h. Thus, the PYP family offers an excellent opportunity to investigate how functional properties are tuned over a wide range, while maintaining the same overall protein structural fold. We discuss the implications of these results for structure–function relationships in the PYP family.

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Signal Transfer in Haloarchaeal Sensory Rhodopsin– Transducer Complexes


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Sasaki, J., Spudich, J. L.
Photochemistry and Photobiology 84:4 (863-868), 2008

Membrane-inserted complexes consisting of two photochemically reactive sensory rhodopsin (SR) subunits flanking a homodimer of a transducing protein subunit (Htr) are used by halophilic archaea for sensing light gradients to modulate their swimming behavior (phototaxis). The SR–Htr complexes extend into the cytoplasm where the Htr subunits bind a his-kinase that controls a phosphorylation system that regulates the flagellar motors. This review focuses on current progress primarily on the mechanism of signal relay within the SRII–HtrII complexes from Natronomonas pharaonis and Halobacterium salinarum. The recent elucidation of a photoactive site steric trigger crucial for signal relay, advances in understanding the role of proton transfer from the chromophore to the protein in SRII activation, and the localization of signal relay to the membrane-embedded portion of the SRII–HtrII interface, are beginning to produce a clear picture of the signal transfer process. The SR–Htr complexes offer unprecedented opportunities to resolve first examples of the chemistry of signal relay between membrane proteins at the atomic level, which would provide a major contribution to the general understanding of dynamic interactions between integral membrane proteins.

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Flavin-based Blue-light Photosensors: A Photobiophysics Update


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Losi, A.
Photochemistry and Photobiology 83:6 (1283-1300), 2007

This review deals with the biophysical aspects of flavin-based photosensors, comprising cryptochromes, LOV (Light, Oxygen and Voltage) and BLUF (Blue Light sensing Using FAD) proteins. Special emphasis is given to structural issues, photocycle quantum yields and energetics, mechanism of the light-triggered reactions, early stages in signal transduction and oligomeric states of the light sensing protein modules. For BLUF and LOV domains important parallels are emerging, despite their different α/β fold arrangement, whereas there is increasing evidence for a mechanicistic and functional splitting of the cryptochrome family.

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Photophysics and Multifunctionality of Hypericin-Like Pigments in Heterotrich Ciliates: A Phylogenetic Perspective


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Lobban, C. S., Hallam, S. J., Mukherjee, P., Petrich, J. W.
Photochemistry and Photobiology 83:5 (1074-1094), 2007

In this paper, we review the literature and present some new data to examine the occurrence and photophysics of the diverse hypericin-like chromophores in heterotrichs, the photoresponses of the cells, the various roles of the pigments and the taxa that might be studied to advance our understanding of these pigments. Hypericin-like chromophores are known chemically and spectrally so far only from the stentorids and Fabrea, the latter now seen to be sister to stentorids in the phylogenetic tree. For three hypericin-like pigments, the structures are known but these probably do not account for all the colors seen in stentorids. At least eight physiological groups of Stentor exist depending on pigment color and presence/absence of zoochlorellae, and some species can be bleached, leading to many opportunities for comparison of pigment chemistry and cell behavior. Several different responses to light are exhibited among heterotrichs, sometimes by the same cell; in particular, cells with algal symbionts are photophilic in contrast to the well-studied sciaphilous (shade-loving) species. Hypericin-like pigments are involved in some well-known photophobic reactions but other pigments (rhodopsin and flavins) are also involved in photoresponses in heterotrichs and other protists. The best characterized role of hypericin-like pigments in heterotrichs is in photoresponses and they have at least twice evolved a role as photoreceptors. However, hypericin and hypericin-like pigments in diverse organisms more commonly serve as predator defense and the pigments are multifunctional in heterotrichs. A direct role for the pigments in UV protection is possible but evidence is equivocal. New observations are presented on a folliculinid from deep water, including physical characterization of its hypericin-like pigment and its phylogenetic position based on SSU rRNA sequences. The photophysics of hypericin and hypericin-like pigments is reviewed. Particular attention is given to how their excited-state properties are modified by the environment. Dramatic changes in excited-state behavior are observed as hypericin is moved from the homogeneous environment of organic solvents to the much more structured surroundings provided by the complexes it forms with proteins. Among these complexes, it is useful to consider the differences between environments where hypericin is not found naturally and those where it is, notably, for example, in heterotrichs. It is clear that interaction with a protein modifies the photophysics of hypericin and understanding the molecular basis of this interaction is one of the outstanding problems in elucidating the function of hypericin and hypericin-like chromophores.

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PAR and UV Effects on Vertical Migration and Photosynthesis in Euglena gracilis


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Richter, P., Helbling, W., Streb, C., Häder, D.-P.
Photochemistry and Photobiology 83:4 (818-823), 2007

Recently it was shown that the unicellular flagellate Euglena gracilis changes the sign of gravitaxis from negative to positive upon excessive radiation. This sign change persists in a cell culture for hours even if subsequently transferred to dim light. To test the ecological relevance of this behavior, a vertical column experiment was performed (max. depth 65 cm) to test distribution, photosynthetic efficiency and motility in different horizons of the column (surface, 20, 40 and 65 cm). One column was covered with a UV cut-off filter, which transmits photosynthetically active radiation (PAR) only, the other with a filter which transmits PAR and UV. The columns were irradiated with a solar simulator (PAR 162 W m−2, UV-A 32.6 W m−2, UV-B 1.9 W m−2). The experiment was conducted for 10 days, normally with a light/dim light cycle of 12 h:12 h, but in some cases the light regime was changed (dim light instead of full radiation). Under irradiation the largest fraction of cells was found at the bottom of the column. The cell density decreased toward the surface. Photosynthetic efficiency, determined with a pulse amplitude modulated fluorometer, was negligible at the surface and increased toward the bottom. While the cell suspension showed a positive gravitaxis at the bottom, the cells in the 40 cm horizon were bimodally oriented (about the same percentage of cells swimming upward and downward, respectively). At 20 cm and at the surface the cells showed negative gravitaxis. Positive gravitaxis was more pronounced in the UV + PAR samples. At the surface and in the 20 and 40 cm horizons photosynthetic efficiency was better in the PAR-only samples than in the PAR + UV samples. At the bottom photosynthetic efficiency was similar in both light treatments. The data suggest that high light reverses gravitaxis of the cells, so that they move downward in the water column. At the bottom the light intensity is lower (attenuation of the water column and self shading of the cells) and the cells recover. After recovery the cells swim upward again until the negative gravitaxis is reversed again.

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Array of Aromatic Amino Acid Side Chains Located Near the Chromophore of Photoactive Yellow Protein


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Morishita, T., Harigai, M., Yamazaki, Y., Kamikubo, H., Kataoka, M., Imamoto, Y.
Photochemistry and Photobiology 83:2 (280-286), 2007 

The role of the array of aromatic amino acid side chains located close to the chromophore binding loop of photoactive yellow protein (PYP) was studied using the alanine-substitution mutagenesis. Phe92, Tyr94, Phe96 and Tyr98 were replaced with alanine (F92A, Y94A, F96A and Y98A, respectively), then these mutants were characterized by UV-visible absorption spectra, circular dichroism (CD) spectra, thermal stability and photocycle kinetics. Absorption maxima of F92A, Y94A, F96A and Y98A were 444, 442, 439 and 447 nm, respectively, different to wild type (WT) at 446 nm. Far-UV CD spectra of mutants other than F92A were different from WT, indicating that Tyr94, Phe96 and Tyr98 maintain the native secondary structure of PYP. Mid-point temperatures of thermal denaturation of F92A, Y94A and F96A, estimated by the CD signal at 222 nm, were 5–10°C lower than WT. Time constants of the photocycle estimated by flash-induced absorbance change were 0.36 s for WT and 1.4 s for Y98A, however, 100, 30 and 3000 times slower than WT for F92A, Y94A and F96A, respectively. Tyr98 is located in the loop region, whereas Phe92, Tyr94 and Phe96 are incorporated in the β4 strand, showing that aromatic amino acid residues in the β-sheet regulate the absorption spectrum, thermal stability and photocycle of PYP. Aromatic rings of Phe92, Tyr94 and Phe96 lie nearly perpendicular to the aromatic ring of Phe75 or chromophore. Possible weak hydrogen bonds between the aromatic ring hydrogen and π-electrons of these residues are discussed.


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Phytochrome-Mediated Inhibition of Coleoptile Growth in Rice: Age-dependency and Action Spectra


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Xie, X., Shinomura, T., Inagaki, N., Kiyota, S., Takano, M.
Photochemistry and Photobiology 83: 1 (131-138), 2007

Phytochrome has been shown to be the major photoreceptor involved in the photo-inhibition of coleoptile growth in Japonica-type rice (Oryza sativa L.). We have characterized this typical photomorphogenetic response of rice using mutants deficient in phytochrome A (phyA) and phytochrome B (phyB) and with respect to age-dependency and action spectra. Seedlings were irradiated with a pulse of light 40 h or 80 h after germination (i.e. at an early or late developmental stage) and the final coleoptile length of these seedlings was determined. A saturating pulse of red light (R) had a stronger effect when it was given in the late stage than in the early stage. It was found that the photo-inhibition is mediated by both the phyA and the phyB in the late stage but predominantly by phyB in the early stage. Consistent with many other reported responses, the photo-inhibition in the phyA mutant, which was observed in the early and late developmental stages and is thought to be mediated mainly by phyB, occurred in the low-fluence range (101–103 μmol m−2) of R and was far-red-light (FR)-reversible; the photo-inhibition in the phyB mutant, which was observed in the late developmental stage and is thought to be mediated mainly by phyA, occurred in the very-low-fluence range (10−2–100 μmol m−2) and was FR-irreversible. The action spectra (350–800 nm at 50 nm intervals) obtained at the two developmental stages using phyA and phyB mutants indicated that both the phyB-mediated low-fluence response and the phyA-mediated very-low-fluence response have a major peak at 650 nm and a minor peak at 400 nm.

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Phototropins and Blue Light-dependent Calcium Signaling in Higher Plants


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Harada, A., Shimazaki, K.-i.
Photochemistry and Photobiology 83:1 (102-111), 2007

Plants have several kinds of photoreceptors, which regulate growth and development. Recent investigations using Arabidopsis thaliana revealed that the newly found blue light receptor phototropins mediate phototropism, chloroplast relocation, stomatal opening, rapid inhibition of hypocotyl elongation and leaf expansion (1,2). Several physiological studies suggest that one of the intermediates in phototropin signaling is cytosolic Ca2+. Studies using phototropin mutants have demonstrated that phototropins induce an increase in cytosolic Ca2+ concentration. However, the function of Ca2+ in the phototropin-mediated signaling process remains largely unknown. This review presents findings about phototropin-mediated calcium mobilization and the involvement of calcium in blue light-dependent plant responses.

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Cryptochrome Signaling in Plants



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Li, Q.-H., Yang, H.-Q.
Photochemistry and Photobiology 83:1 (94-101), 2007  

Cryptochromes are blue light receptors that mediate various light-induced responses in plants and animals. They share sequence similarity to photolyases, flavoproteins that catalyze the repair of UV light-damaged DNA, but do not have photolyase activity. Arabidopsis cryptochromes work together with the red/far-red light receptor phytochromes to regulate various light responses, including the regulation of cell elongation and photoperiodic flowering, and are also found to act together with the blue light receptor phototropins to mediate blue light regulation of stomatal opening. The signaling mechanism of Arabidopsis cryptochromes is mediated through negative regulation of COP1 by direct CRY–COP1 interaction through CRY C-terminal domain. Arabidopsis CRY dimerized through its N-terminal domain and dimerization of CRY is required for light activation of the photoreceptor activity. Recently, significant progresses have been made in our understanding of cryptochrome functions in other dicots such as pea and tomato and lower plants including moss and fern. This review will focus on recent advances in functional and mechanism characterization of cryptochromes in plants. It is not intended to cover every aspect of the field; readers are referred to other review articles for historical perspectives and a more comprehensive understanding of this photoreceptor (1–8)


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Phytochrome-dependent Photomovement Responses Mediated by Phototropin Family Proteins in Cryptogam Plants



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Suetsugu, N., Wada, M.
Photochemistry and Photobiology 83:1 (87-93), 2007

In this review, we describe the regulation of photomovement responses by phototropin and phytochrome photoreceptors. The blue light receptor phototropin mediates various photomovement responses such as phototropism, chloroplast movement and stomatal opening. In cryptogamic plants including ferns, mosses and green alga, red as well as blue light mediates phototropism and chloroplast movement. The red/far-red light reversibility suggests the involvement of phytochrome in these responses. Thereby, plant growth is presumably promoted by coordinating these photomovements to capture efficiently light for photosynthesis.

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Structure and Photoreaction of Photoactive Yellow Protein, a Structural Prototype of the PAS Domain Superfamily


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Imamoto, Y., Kataoka, M.
Photochemistry and Photobiology 83:1 (40-49), 2007

Photoactive yellow protein (PYP) is a water-soluble photosensor protein found in purple photosynthetic bacteria. Unlike bacterial rhodopsins, photosensor proteins composed of seven transmembrane helices and a retinal chromophore in halophilic archaebacteria, PYP is a highly soluble globular protein. The α/β fold structure of PYP is a structural prototype of the PAS domain superfamily, many members of which function as sensors for various kinds of stimuli. To absorb a photon in the visible region, PYP has a p-coumaric acid chromophore binding to the cysteine residue via a thioester bond. It exists in a deprotonated trans form in the dark. The primary photochemical event is photo-isomerization of the chromophore from trans to cis form. The twisted cis chromophore in early intermediates is relaxed and finally protonated. Consequently, the chromophore becomes electrostatically neutral and rearrangement of the hydrogen-bonding network triggers overall structural change of the protein moiety, in which local conformational change around the chromophore is propagated to the N-terminal region. Thus, it is an ideal model for protein conformational changes that result in functional change, responding to stimuli and expressing physiological activity. In this paper, recent progress in investigation of the photoresponse of PYP is reviewed.

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Three Putative Photosensory Light, Oxygen or Voltage (LOV) Domains with Distinct Biochemical Properties from the Filamentous Cyanobacterium Anabaena sp. PCC 7120



Narikawa, R., Zikihara, K., Okajima, K., Ochiai, Y., Katayama, M., Shichida, Y., Tokutomi, S., Ikeuchi, M.
Photochemistry and Photobiology 82: 6 (1627-1633), 2006

Light, oxygen or voltage (LOV) domains function as blue-light sensors in the phototropin family of photoreceptors found in plants, algae and bacteria. We detected putative LOV domains (Alr3170-LOV, A112875-LOV and A1r1229-LOV) in the genome of a filamentous cyanobacterium, Anabaena sp. PCC 7120. These cyanobacterial LOV domains are closely clustered with the known LOV domains. Alr3170-LOV and A112875-LOV carry the conserved cysteine residue unique to the photoactive LOV, whereas A1r1229-LOV does not. We expressed these three LOV domains in Escherichia coli and purified them. In fact, Alr3170-LOV and A112875-LOV that are conserved in Nostoc punctiforme, a related species, bound flavin mononuclcotide and showed spectral changes unique to known LOV domains on illumination with blue light. A1r3170-LOV was completely photoreduced and dark reversion was slow, whereas A112875-LOV was slowly photoreduced and dark reversion was rapid. For comparison, AvA112875-LOV in a closely related A. variabilis was also studied as a homolog of A112875-LOV. Finally, we observed that A1r1229-LOV that is not conserved in N. punctiforme showed no flavin binding.

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Increasing UV-B Induces Biphasic Leaf Cell Expansion in Phaseolus vulgaris, Suggesting Multiple Mechanisms for Controlling Plant Growth



Barkant, L., Evans, M. A., Edwards, G. E.
Photochemistry and Photobiology 82:6 (1612-1620), 2006

Leaf expansion, comprising cell division and cell enlargement, is controlled by light quality and quantity. The role of UV-B irradiance on leaf cell enlargement has not been determined. We studied the effect of a wide range of UV-B irradiances on the cell-enlargement-driven expansion of Phaseolus vulgaris L., cv. Contender (bush bean) leaf dises. Our growth method allowed separation of the cell enlargement phase of leaf expansion from the cell division phase. In two series of experiments with different types of UV-B screening filters, the effect of increasing levels of UV-B on the area of excised P. vulgaris leaf discs was investigated. One set of experiments utilized polyester (UV-B-absorbing) and cellulose acetate (UV-B-transmitting) filters. The other set utilized UV-B-absorbing and UV-B-transmitting acrylic filters. Regardless of which type of filter was used for screening, high (above summer solstice) levels of supplemental UV-B inhibited cell enlargement in a linear, dose-dependent manner, resulting in smaller leaf discs than treatment with UV-B-absorbing filters. Conversely, low levels of supplemental UV-B enhanced cell enlargement in a linear, dose-dependent manner, resulting in larger leaf discs than did treatment with UV-B-absorbing filters. The results suggest a biphasic response to UV-B, and that there is an optimum UV-B level that results in maximum leaf expansion by cell enlargement.

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Heterologous Expression of Photoactivated Adenylyl Cyclase (PAC) Genes from the Flagellate Euglena gracilis in Insect Cells



Ntefidou, M., Lüdtke, T., Ahmad, M., Häder, D.-P.
Photochemistry and Photobiology 82:6 (1601-1605), 2006

 The unicellular, green flagellate wild-type Euglena gracilis (strain Z) possesses two genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACα (Mr 105 kDa) and two PACβ (90 kDa) subunits. In an effort to produce sufficient amounts of PAC proteins, several routes of over-expression have been tried including homologous expression in Euglena and heterologous expression in Escherichia coll. All these approaches were hampered by low yield or formation of inclusion bodies. Therefore we decided to attempt a heterologous expression in an insect cell line. PACα and PACβ were separately cloned in the transfer vector pBacPAK9 with a His tag attached. The transfer vector was subsequently cotransfected via baculovirus into the insect cells and amplified. For the expression both recombinant viruses (containing PACα and PACβ, respectively) were cotransfected simultaneously into insect cells. The expressed proteins were analyzed in Western blots using PACα and PACβ antibodies. Most of the proteins were found to be in soluble form in high yield. The recombinant PAC proteins were purified via their attached His tag on an anti-His resin. Adenylyl cyclase activity was quantified after blue-light excitation using a cAMP enzyme immunoassay kit.

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Fluorescence Resonance Energy Transfer Between Polyphenolic Compounds and Riboflavin Indicates a Possible Accessory Photoreceptor Function for Some Polyphenolic Compounds


Chandrakuntal, K., Thomas, N. M., Kumar, P. G., Laloraya, M., Laloraya, M. M.
Photochemistry and Photobiology 82:5 (1358-1364), 2006

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.

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Two Native Pools of Phytochrome A in Monocots: Evidence from Fluorescence Investigations of Phytochrome Mutants of Rice



Sineshchekov, V., Loskovich, A., Inagaki, N., Takano, M.
Photochemistry and Photobiology 82: 4 (1116-1122), 2006

Fluorescence investigations of phytochrome (phy) in rice (Oryza sativa L. cv. Nipponbare) mutants deficient in phyA, phyB and phyA plus phyB were performed. Total content of the pigment (Ptot) and its spectroscopic and photochemical characteristics were determined in different parts of the dark-grown and far-red light (FR)-grown coleoptiles. Spectroscopically, phyA in the phyB mutant was identical to phyA in the wild-type (WT) and the extent of the conversion from Pr to lumi-R at 85 K was the same for phyA in both lines and varied similarly, depending on the part of the coleoptile used. The latter finding proved that phyA in rice is heterogeneous and comprises two phyA populations, phyA′ and phyA″. Functional properties of phyA were also determined. In the dark the phyB mutant had a higher content of phyA, inactive protochlorophyllide (Pchlide633) and active protochlorophyllide (Pchlide655) than WT and its coleoptile was longer, indicating that phyB may affect the development of WT seedlings in the dark. Constant FR drastically reduced the content of phyA, Pchlide633 and Pchlide655 and brought about coleoptile shortening and appearance of the first leaf, whereas pulsed FR of equal fluence was less effective. This suggested that the reactions were primarily of the high irradiance responses type, which are likely to be mediated by phyA′. The effects on protochlorophyllide biosynthesis and growth responses type were more pronounced in the phyB mutant than in the WT seedlings, which can be connected with the higher phyA′ content in the phyB mutant and/or phyB interference with its action in WT seedlings. In the phyA mutant induction of Pchlide633 and Pchlide655 biosynthesis was observed under constant FR, indicating that phyC may be responsible for this effect.

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