29 results on '"Batschauer, Alfred"'
Search Results
2. DASH-type cryptochromes - solved and open questions.
- Author
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Kiontke S, Göbel T, Brych A, and Batschauer A
- Subjects
- Animals, Humans, Arabidopsis metabolism, Cryptochromes metabolism, Deoxyribodipyrimidine Photo-Lyase metabolism, Drosophila metabolism, Synechocystis metabolism
- Abstract
Drosophila, Arabidopsis, Synechocystis, human (DASH)-type cryptochromes (cry-DASHs) form one subclade of the cryptochrome/photolyase family (CPF). CPF members are flavoproteins that act as DNA-repair enzymes (DNA-photolyases), or as ultraviolet(UV)-A/blue light photoreceptors (cryptochromes). In mammals, cryptochromes are essential components of the circadian clock feed-back loop. Cry-DASHs are present in almost all major taxa and were initially considered as photoreceptors. Later studies demonstrated DNA-repair activity that was, however, restricted to UV-lesions in single-stranded DNA. Very recent studies, particularly on microbial organisms, substantiated photoreceptor functions of cry-DASHs suggesting that they could be transitions between photolyases and cryptochromes.
- Published
- 2020
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3. The Arabidopsis cryptochrome 2 I404F mutant is hypersensitive and shows flavin reduction even in the absence of light.
- Author
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Araguirang GE, Niemann N, Kiontke S, Eckel M, Dionisio-Sese ML, and Batschauer A
- Subjects
- Arabidopsis Proteins chemistry, Arabidopsis Proteins radiation effects, Cryptochromes chemistry, Cryptochromes radiation effects, Light, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Phenotype, Protein Conformation, Signal Transduction, Transcription Factors genetics, Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Cryptochromes genetics, Cryptochromes metabolism, Flavins metabolism
- Abstract
Main Conclusion: The cryptochrome photoreceptor mutant cry2I404F exhibits hyperactivity in the dark, hypersensitivity in different light conditions, and in contrast to the wild-type protein, its flavin chromophore is reducible even in the absence of light. Plant cryptochromes (cry) are blue-light photoreceptors involved in multiple signaling pathways and various photomorphogenic responses. One biologically hyperactive mutant of a plant cryptochrome that was previously characterized is Arabidopsis cry1L407F (Exner et al. in Plant Physiol 154:1633-1645, 2010). Protein sequence alignments of different cryptochromes revealed that L407 in cry1 corresponds to I404 in cry2. Point mutation of Ile to Phe in cry2 in this position created a novel mutant. The present study provided a baseline data on the elucidation of the properties of cry2I404F. This mutant was still able to bind ATP-triggering conformational changes, as confirmed by partial tryptic digestion and thermo-FAD assays. Surprisingly, the FAD cofactor of cry2I404F was reduced by the addition of reductant even in the absence of light. In vivo, cry2I404F exhibited a cop phenotype in the dark and hypersensitivity to various light conditions compared to cry2 wild type. Overall, these data suggest that the hypersensitivity to red and blue light and hyperactivity of this novel mutant in the dark can be mostly accounted to structural alterations brought forth by the Ile to Phe mutation at position 404 that allows reduction of the flavin chromophore even in the absence of light.
- Published
- 2019
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4. ATP boosts lit state formation and activity of Arabidopsis cryptochrome 2.
- Author
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Eckel M, Steinchen W, and Batschauer A
- Subjects
- Arabidopsis physiology, Arabidopsis radiation effects, Arabidopsis Proteins genetics, Cryptochromes genetics, Light, Oxidation-Reduction, Recombinant Proteins, Adenosine Triphosphate metabolism, Arabidopsis genetics, Arabidopsis Proteins metabolism, Cryptochromes metabolism, Signal Transduction
- Abstract
Cryptochrome (cry) blue light photoreceptors have important roles in the regulation of plant development. Their photocycle includes redox changes of their flavin adenine dinucleotide (FAD) chromophore, which is fully oxidised in the dark state and semi-reduced in the signalling-active lit state. The two Arabidopsis thaliana cryptochromes, cry1 and cry2, and the plant-type cryptochrome CPH1 from Chlamydomonas rheinhardtii bind ATP and other nucleotides. Binding of ATP affects the photocycle of these photoreceptors and causes structural alterations. However, the exact regions that undergo structural changes have not been defined, and most importantly it is not known whether ATP binding affects the biological activity of these photoreceptors in planta. Here we present studies on the effect of ATP on Arabidopsis cry2. Recombinant cry2 protein showed a high affinity for ATP (K
D of 1.09 ± 0.48 μm). Binding of ATP and other adenines promoted photoreduction of the FAD chromophore in vitro and caused structural changes, particularly in α-helix 21 which links the photosensory domain with the C-terminal extension. The constructed cry2Y399A mutant was unable to bind ATP and did not show enhancement of photoreduction by ATP. When this mutant gene was expressed in Arabidopsis null cry2 mutant plants it retained some biological activity, which was, however, lower than that of the wild type. Our results indicate that binding of ATP to cry2, and most likely to other plant-type cryptochromes, is not essential but boosts the formation of the signalling state and biological activity., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)- Published
- 2018
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5. Hyperactivity of the Arabidopsis cryptochrome (cry1) L407F mutant is caused by a structural alteration close to the cry1 ATP-binding site.
- Author
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Orth C, Niemann N, Hennig L, Essen LO, and Batschauer A
- Subjects
- Adenosine Triphosphate chemistry, Amino Acid Substitution, Arabidopsis Proteins antagonists & inhibitors, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Binding Sites, Binding, Competitive, Biocatalysis, Cryptochromes antagonists & inhibitors, Cryptochromes chemistry, Cryptochromes genetics, Enzyme Inhibitors chemistry, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacology, Hot Temperature adverse effects, Indazoles chemistry, Indazoles metabolism, Indazoles pharmacology, Ligands, Molecular Docking Simulation, Mutagenesis, Site-Directed, Peptide Fragments antagonists & inhibitors, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments metabolism, Protein Conformation, Protein Denaturation, Protein Interaction Domains and Motifs, Protein Stability, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Structural Homology, Protein, Adenosine Triphosphate metabolism, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Cryptochromes metabolism, Models, Molecular, Mutation
- Abstract
Plant cryptochromes (cry) act as UV-A/blue light receptors. The prototype, Arabidopsis thaliana cry1, regulates several light responses during the life cycle, including de-etiolation, and is also involved in regulating flowering time. The cry1 photocycle is initiated by light absorption by its FAD chromophore, which is most likely fully oxidized (FAD
ox ) in the dark state and photoreduced to the neutral flavin semiquinone (FADH°) in its lit state. Cryptochromes lack the DNA-repair activity of the closely related DNA photolyases, but they retain the ability to bind nucleotides such as ATP. The previously characterized L407F mutant allele of Arabidopsis cry1 is biologically hyperactive and seems to mimic the ATP-bound state of cry1, but the reason for this phenotypic change is unclear. Here, we show that cry1 L407F can still bind ATP, has less pronounced photoreduction and formation of FADH° than wild-type cry1, and has a dark reversion rate 1.7 times lower than that of the wild type. The hyperactivity of cry1 L407F is not related to a higher FADH° occupancy of the photoreceptor but is caused by a structural alteration close to the ATP-binding site. Moreover, we show that ATP binds to cry1 in both the dark and the lit states. This binding was not affected by cry1's C-terminal extension, which is important for signal transduction. Finally, we show that a recently discovered chemical inhibitor of cry1, 3-bromo-7-nitroindazole, competes for ATP binding and thereby diminishes FADH° formation, which demonstrates that both processes are important for cry1 function., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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6. Flavin Adenine Dinucleotide and N 5 ,N 10 -Methenyltetrahydrofolate are the in planta Cofactors of Arabidopsis thaliana Cryptochrome 3.
- Author
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Göbel T, Reisbacher S, Batschauer A, and Pokorny R
- Subjects
- Chromatography, Reverse-Phase, Cryptochromes genetics, Escherichia coli genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Immunoprecipitation, Plants, Genetically Modified, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Spectrometry, Fluorescence, Arabidopsis enzymology, Cryptochromes metabolism, Flavin-Adenine Dinucleotide metabolism, Tetrahydrofolates metabolism
- Abstract
Members of the cryptochrome/photolyase family (CPF) of proteins utilize noncovalently bound light-absorbing cofactors for their biological function. Usually, the identity of these cofactors is determined after expression in heterologous systems leaving the question unanswered whether these cofactors are identical to the indigenous ones. Here, cryptochrome 3 from Arabidopsis thaliana was expressed as a fusion with the green fluorescent protein in Arabidopsis plants. Besides the confirmation of the earlier report of its localization in chloroplasts, our data indicate that fractions of the fusion protein are present in the stroma and associated with thylakoids, respectively. Furthermore, it is shown that the fusion protein expressed in planta contains the same cofactors as the His
6 -tagged protein expressed in Escherichia coli, that is, flavin adenine dinucleotide and N5 ,N10 -methenyltetrahydrofolate. This demonstrates that the heterologously expressed cryptochrome 3, characterized in a number of previous studies, is a valid surrogate of the corresponding protein expressed in plants. To our knowledge, this is also a first conclusive analysis of cofactors bound to an Arabidopsis protein belonging to the CPF and purified from plant tissue., (© 2016 The Authors. Photochemistry and Photobiology published by Wiley Periodicals, Inc. on behalf of American Society for Photobiology.)- Published
- 2017
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7. Shadow on the Plant: A Strategy to Exit.
- Author
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Fankhauser C and Batschauer A
- Subjects
- Arabidopsis metabolism, Arabidopsis Proteins metabolism, Basic Helix-Loop-Helix Transcription Factors metabolism, Cryptochromes metabolism
- Abstract
The light spectrum perceived by plants is affected by crowding, which results in the shade avoidance syndrome (SAS). Findings presented by Pedmale et al. bring cryptochromes to the forefront of SAS and elucidate a fascinating molecular crosstalk between photoreceptor systems operating in different wavebands., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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8. Cellular metabolites enhance the light sensitivity of Arabidopsis cryptochrome through alternate electron transfer pathways.
- Author
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Engelhard C, Wang X, Robles D, Moldt J, Essen LO, Batschauer A, Bittl R, and Ahmad M
- Subjects
- Amino Acid Substitution, Animals, Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Cryptochromes genetics, Electron Transport, Insecta, Light, Models, Molecular, Mutation, Missense, Oxidation-Reduction, Tryptophan chemistry, Arabidopsis radiation effects, Arabidopsis Proteins metabolism, Cryptochromes metabolism, Flavins metabolism, Signal Transduction
- Abstract
Cryptochromes are blue light receptors with multiple signaling roles in plants and animals. Plant cryptochrome (cry1 and cry2) biological activity has been linked to flavin photoreduction via an electron transport chain comprising three evolutionarily conserved tryptophan residues known as the Trp triad. Recently, it has been reported that cry2 Trp triad mutants, which fail to undergo photoreduction in vitro, nonetheless show biological activity in vivo, raising the possibility of alternate signaling pathways. Here, we show that Arabidopsis thaliana cry2 proteins containing Trp triad mutations indeed undergo robust photoreduction in living cultured insect cells. UV/Vis and electron paramagnetic resonance spectroscopy resolves the discrepancy between in vivo and in vitro photochemical activity, as small metabolites, including NADPH, NADH, and ATP, were found to promote cry photoreduction even in mutants lacking the classic Trp triad electron transfer chain. These metabolites facilitate alternate electron transfer pathways and increase light-induced radical pair formation. We conclude that cryptochrome activation is consistent with a mechanism of light-induced electron transfer followed by flavin photoreduction in vivo. We further conclude that in vivo modulation by cellular compounds represents a feature of the cryptochrome signaling mechanism that has important consequences for light responsivity and activation., (© 2014 American Society of Plant Biologists. All rights reserved.)
- Published
- 2014
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9. Lifetimes of Arabidopsis cryptochrome signaling states in vivo.
- Author
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Herbel V, Orth C, Wenzel R, Ahmad M, Bittl R, and Batschauer A
- Subjects
- Animals, Arabidopsis genetics, Arabidopsis radiation effects, Arabidopsis Proteins genetics, Cell Line, Cryptochromes genetics, Flavin-Adenine Dinucleotide metabolism, Gene Expression Regulation, Plant, Genes, Reporter, Insecta, Mutation, Photoreceptors, Plant genetics, Photoreceptors, Plant metabolism, Plants, Genetically Modified, Seedlings genetics, Seedlings physiology, Seedlings radiation effects, Time Factors, Arabidopsis physiology, Arabidopsis Proteins metabolism, Cryptochromes metabolism, Flavin-Adenine Dinucleotide analogs & derivatives, Light, Light Signal Transduction
- Abstract
One crucial component in light signaling is the quantity of photoreceptor present in the active signaling state. The lifetime of the signaling state of a photoreceptor is limited because of thermal or otherwise back reversion of the chromophore to the ground state, and/or degradation of the photoreceptor in the light-activated state. It was previously shown that the lit state of plant cryptochromes contains flavin-neutral semiquinone, and that the half-lives of the lit state were in the range of 3-4 min in vitro. However, it was unknown how long-lived the signaling states of plant cryptochromes are in situ. Based on the loss of degradation of cry2 after prolonged dark incubation and loss of reversibility of photoactivated cry1 by a pulse of green light, we estimate the in vivo half-lives of the signaling states of cry1 and cry2 to be in the range of 5 and 16 min, respectively. Based on electron paramagnetic resonance measurements, the lifetime of the Arabidopsis cry1 lit state in insect cells was found to be ~6 min, and thus very similar to the lifetime of the signaling state in planta. Thus, the signaling state lifetimes of plant cryptochromes are not, or are only moderately, stabilized in planta., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)
- Published
- 2013
- Full Text
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10. Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A.
- Author
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Weidler G, Zur Oven-Krockhaus S, Heunemann M, Orth C, Schleifenbaum F, Harter K, Hoecker U, and Batschauer A
- Subjects
- Arabidopsis genetics, Cell Cycle Proteins genetics, Cell Nucleus metabolism, Fluorescence Resonance Energy Transfer, Microscopy, Fluorescence methods, Mutation, Phytochrome A genetics, Protein Serine-Threonine Kinases metabolism, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Cell Cycle Proteins metabolism, Cryptochromes metabolism, Phytochrome A metabolism, Protein Serine-Threonine Kinases genetics
- Abstract
The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.
- Published
- 2012
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11. A gain-of-function mutation of Arabidopsis cryptochrome1 promotes flowering.
- Author
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Exner V, Alexandre C, Rosenfeldt G, Alfarano P, Nater M, Caflisch A, Gruissem W, Batschauer A, and Hennig L
- Subjects
- Alleles, Amino Acid Sequence, Arabidopsis genetics, Arabidopsis Proteins chemistry, Cryptochromes chemistry, Light, Models, Molecular, Molecular Dynamics Simulation, Molecular Sequence Data, Sequence Homology, Amino Acid, Arabidopsis physiology, Arabidopsis Proteins genetics, Cryptochromes genetics, Flowers, Point Mutation
- Abstract
Plants use different classes of photoreceptors to collect information about their light environment. Cryptochromes are blue light photoreceptors that control deetiolation, entrain the circadian clock, and are involved in flowering time control. Here, we describe the cry1-L407F allele of Arabidopsis (Arabidopsis thaliana), which encodes a hypersensitive cryptochrome1 (cry1) protein. Plants carrying the cry1-L407F point mutation have elevated expression of CONSTANS and FLOWERING LOCUS T under short-day conditions, leading to very early flowering. These results demonstrate that not only the well-studied cry2, with an unequivocal role in flowering promotion, but also cry1 can function as an activator of the floral transition. The cry1-L407F mutants are also hypersensitive toward blue, red, and far-red light in hypocotyl growth inhibition. In addition, cry1-L407F seeds are hypersensitive to germination-inducing red light pulses, but the far-red reversibility of this response is not compromised. This demonstrates that the cry1-L407F photoreceptor can increase the sensitivity of phytochrome signaling cascades. Molecular dynamics simulation of wild-type and mutant cry1 proteins indicated that the L407F mutation considerably reduces the structural flexibility of two solvent-exposed regions of the protein, suggesting that the hypersensitivity might result from a reduced entropic penalty of binding events during downstream signal transduction. Other nonmutually exclusive potential reasons for the cry1-L407F gain of function are the location of phenylalanine-407 close to three conserved tryptophans, which could change cry1's photochemical properties, and stabilization of ATP binding, which could extend the lifetime of the signaling state of cry1.
- Published
- 2010
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12. Light-induced activation of class II cyclobutane pyrimidine dimer photolyases.
- Author
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Okafuji A, Biskup T, Hitomi K, Getzoff ED, Kaiser G, Batschauer A, Bacher A, Hidema J, Teranishi M, Yamamoto K, Schleicher E, and Weber S
- Subjects
- Amino Acid Sequence, Animals, Arabidopsis Proteins chemistry, Coenzymes metabolism, Deoxyribodipyrimidine Photo-Lyase chemistry, Electron Spin Resonance Spectroscopy, Electron Transport, Enzyme Activation radiation effects, Flavin-Adenine Dinucleotide metabolism, Free Radicals metabolism, Models, Molecular, Molecular Sequence Data, Photochemical Processes radiation effects, Protein Conformation, Spectrophotometry, Ultraviolet, Xenopus laevis, Arabidopsis enzymology, Arabidopsis Proteins metabolism, Deoxyribodipyrimidine Photo-Lyase metabolism, Light, Oryza enzymology
- Abstract
Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD photolyase and (6-4) photolyase. By ENDOR spectroscopy, the local environment of the flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FAD(ox), have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD photolyase, light-induced generation of FADH from FAD(ox), and subsequently FADH(-) from FADH, proceeds in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I photolyases, but completely lacking from the equivalent positions of class II photolyase sequences. Nevertheless, class II photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution., ((c) 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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13. Photoreduction of the folate cofactor in members of the photolyase family.
- Author
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Moldt J, Pokorny R, Orth C, Linne U, Geisselbrecht Y, Marahiel MA, Essen LO, and Batschauer A
- Subjects
- Arabidopsis Proteins, Cryptochromes, Electrons, Flavoproteins metabolism, Light, Mitochondria metabolism, Models, Biological, Models, Molecular, Molecular Conformation, Mutagenesis, Site-Directed, Pyrimidine Dimers chemistry, Spectrometry, Mass, Electrospray Ionization, Spectrophotometry methods, Tetrahydrofolates chemistry, Arabidopsis metabolism, Deoxyribodipyrimidine Photo-Lyase metabolism, Escherichia coli metabolism
- Abstract
Cryptochromes and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH(-)* to the DNA lesion and electron back-transfer to semireduced FADH(o) after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.
- Published
- 2009
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14. Increased DNA repair in Arabidopsis plants overexpressing CPD photolyase.
- Author
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Kaiser G, Kleiner O, Beisswenger C, and Batschauer A
- Subjects
- Arabidopsis enzymology, Base Sequence, DNA Primers, Plants, Genetically Modified, Ultraviolet Rays, Arabidopsis genetics, Arabidopsis Proteins genetics, DNA Repair, Deoxyribodipyrimidine Photo-Lyase genetics
- Abstract
Ultraviolet-B (UV-B, 280-320 nm) radiation may have severe negative effects on plants including damage to their genetic information. UV protection and DNA-repair mechanisms have evolved to either avoid or repair such damage. Since autotrophic plants are dependent on sunlight for their energy supply, an increase in the amount of UV-B reaching the earth's surface may affect the integrity of their genetic information if DNA damage is not repaired efficiently and rapidly. Here we show that overexpression of cyclobutane pyrimidine dimer (CPD) photolyase (EC 4.1.99.3) in Arabidopsis thaliana (L.), which catalyses the reversion of the major UV-B photoproduct in DNA (CPDs), strongly enhances the repair of CPDs and results in a moderate increase of biomass production under elevated UV-B.
- Published
- 2009
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15. Recognition and repair of UV lesions in loop structures of duplex DNA by DASH-type cryptochrome.
- Author
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Pokorny R, Klar T, Hennecke U, Carell T, Batschauer A, and Essen LO
- Subjects
- Arabidopsis genetics, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Cryptochromes, Crystallography, X-Ray, DNA Damage physiology, DNA Damage radiation effects, DNA Repair radiation effects, DNA, Plant genetics, DNA, Plant metabolism, DNA, Single-Stranded genetics, DNA, Single-Stranded metabolism, Deoxyribodipyrimidine Photo-Lyase genetics, Deoxyribodipyrimidine Photo-Lyase metabolism, Protein Structure, Secondary physiology, Protein Structure, Tertiary physiology, Pyrimidine Dimers metabolism, Ultraviolet Rays adverse effects, Arabidopsis enzymology, Arabidopsis Proteins chemistry, DNA Repair physiology, DNA, Plant chemistry, DNA, Single-Stranded chemistry, Deoxyribodipyrimidine Photo-Lyase chemistry, Pyrimidine Dimers chemistry
- Abstract
DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-situ-repaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.
- Published
- 2008
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16. The signaling state of Arabidopsis cryptochrome 2 contains flavin semiquinone.
- Author
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Banerjee R, Schleicher E, Meier S, Viana RM, Pokorny R, Ahmad M, Bittl R, and Batschauer A
- Subjects
- Arabidopsis physiology, Arabidopsis Proteins metabolism, Coenzymes metabolism, Cryptochromes, DNA Repair physiology, DNA Repair radiation effects, DNA, Single-Stranded chemistry, DNA, Single-Stranded metabolism, Electron Spin Resonance Spectroscopy, Flavin-Adenine Dinucleotide chemistry, Flavin-Adenine Dinucleotide metabolism, Flavoproteins metabolism, Light, Oxidation-Reduction radiation effects, Pyrimidine Dimers chemistry, Pyrimidine Dimers metabolism, Spectrophotometry, Ultraviolet, Arabidopsis chemistry, Arabidopsis Proteins chemistry, Coenzymes chemistry, Flavin-Adenine Dinucleotide analogs & derivatives, Flavoproteins chemistry, Signal Transduction physiology, Signal Transduction radiation effects
- Abstract
Cryptochrome (Cry) photoreceptors share high sequence and structural similarity with DNA repair enzyme DNA-photolyase and carry the same flavin cofactor. Accordingly, DNA-photolyase was considered a model system for the light activation process of cryptochromes. In line with this view were recent spectroscopic studies on cryptochromes of the CryDASH subfamily that showed photoreduction of the flavin adenine dinucleotide (FAD) cofactor to its fully reduced form. However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA, which is absent for other members of the cryptochrome/photolyase family. Thus, CryDASH may have functions different from cryptochromes. The photocycle of other members of the cryptochrome family, such as Arabidopsis Cry1 and Cry2, which lack DNA repair activity but control photomorphogenesis and flowering time, remained elusive. Here we have shown that Arabidopsis Cry2 undergoes a photocycle in which semireduced flavin (FADH(.)) accumulates upon blue light irradiation. Green light irradiation of Cry2 causes a change in the equilibrium of flavin oxidation states and attenuates Cry2-controlled responses such as flowering. These results demonstrate that the active form of Cry2 contains FADH(.) (whereas catalytically active photolyase requires fully reduced flavin (FADH(-))) and suggest that cryptochromes could represent photoreceptors using flavin redox states for signaling differently from DNA-photolyase for photorepair.
- Published
- 2007
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17. Cryptochrome 3 from Arabidopsis thaliana: structural and functional analysis of its complex with a folate light antenna.
- Author
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Klar T, Pokorny R, Moldt J, Batschauer A, and Essen LO
- Subjects
- Alanine, Amino Acid Sequence, Binding Sites, Cryptochromes, Electrons, Flavin-Adenine Dinucleotide metabolism, Folic Acid chemistry, Folic Acid metabolism, Glutamine, Models, Molecular, Molecular Sequence Data, Mutant Proteins chemistry, Mutant Proteins metabolism, Protein Structure, Secondary, Spectrophotometry, Static Electricity, Structure-Activity Relationship, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Arabidopsis Proteins metabolism, Deoxyribodipyrimidine Photo-Lyase chemistry, Deoxyribodipyrimidine Photo-Lyase metabolism, Folic Acid analogs & derivatives, Light-Harvesting Protein Complexes metabolism
- Abstract
Cryptochromes are almost ubiquitous blue-light receptors and act in several species as central components of the circadian clock. Despite being evolutionary and structurally related with DNA photolyases, a class of light-driven DNA-repair enzymes, and having similar cofactor compositions, cryptochromes lack DNA-repair activity. Cryptochrome 3 from the plant Arabidopsis thaliana belongs to the DASH-type subfamily. Its crystal structure determined at 1.9 Angstroms resolution shows cryptochrome 3 in a dimeric state with the antenna cofactor 5,10-methenyltetrahydrofolate (MTHF) bound in a distance of 15.2 Angstroms to the U-shaped FAD chromophore. Spectroscopic studies on a mutant where a residue crucial for MTHF-binding, E149, was replaced by site-directed mutagenesis demonstrate that MTHF acts in cryptochrome 3 as a functional antenna for the photoreduction of FAD.
- Published
- 2007
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18. Functional and expression analysis of Arabidopsis SPA genes during seedling photomorphogenesis and adult growth.
- Author
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Fittinghoff K, Laubinger S, Nixdorf M, Fackendahl P, Baumgardt RL, Batschauer A, and Hoecker U
- Subjects
- Arabidopsis anatomy & histology, Arabidopsis radiation effects, Arabidopsis Proteins genetics, Cell Cycle Proteins genetics, Cryptochromes, Flavoproteins metabolism, Gene Expression Profiling, Gene Expression Regulation, Developmental radiation effects, Phytochrome A metabolism, Protein Kinases genetics, Protein Kinases metabolism, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis Proteins metabolism, Cell Cycle Proteins metabolism, Gene Expression Regulation, Plant radiation effects, Light, Seedlings growth & development, Seedlings radiation effects
- Abstract
The four members of the Suppressor of phyA-105 (SPA) gene family function to inhibit photomorphogenesis in dark- and light-grown seedlings. Additionally, SPA1-SPA4 regulate elongation growth of adult plants. In these processes, SPA2, SPA3 and SPA4 have overlapping but distinct functions. Here, we have further investigated the role of SPA1 which is partially masked by functional redundancy. We show that SPA1 represses not only red, far-red and blue light responses in a PHYA-dependent fashion, but also acts to suppress light signaling in darkness. We demonstrate that deletion-derivatives of SPA1 lacking the complete N-terminus or part of the kinase-like domain retain SPA1 function in light- and dark-grown seedlings, while deletion of the constitutive photomorphogenesis 1 (COP1)-interacting coiled-coil domain eliminates SPA1 activity. This suggests that the coiled-coil domain and the WD-repeat domain of SPA1 are sufficient for SPA1 function. An analysis of spa2 spa3 spa4 triple mutants demonstrates that SPA1, like SPA2, is sufficient for normal etiolation of dark-grown seedlings. In light-grown seedlings and adult plants, in contrast, SPA1 function is divergent from SPA2 function, with SPA1 playing the predominant role. Levels of SPA1, SPA3 and SPA4 transcript are increased by red, far-red and blue light, consistent with a role of these three SPA genes in light-grown seedlings. The abundance of SPA2 mRNA, in contrast, is not altered by light. Taken together, the analysis of SPA transcript levels suggests that differences in SPA gene expression patterns contribute to divergence in SPA1-SPA4 function.
- Published
- 2006
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19. Crystallization and preliminary X-ray analysis of cryptochrome 3 from Arabidopsis thaliana.
- Author
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Pokorny R, Klar T, Essen LO, and Batschauer A
- Subjects
- Arabidopsis Proteins, Catalysis, Cloning, Molecular, Cryptochromes, Crystallization, Crystallography, X-Ray, Deoxyribodipyrimidine Photo-Lyase chemistry, Dimerization, Electrophoresis, Polyacrylamide Gel, Escherichia coli metabolism, Flavin-Adenine Dinucleotide chemistry, Light, Methylenetetrahydrofolate Dehydrogenase (NAD+) chemistry, Protein Conformation, Protein Structure, Tertiary, Recombinant Proteins, Spectrophotometry, Structure-Activity Relationship, Synchrotrons, Ultraviolet Rays, X-Ray Diffraction, Arabidopsis metabolism, Flavoproteins chemistry
- Abstract
Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome-antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 A. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.
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- 2005
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20. Novel ATP-binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome-1.
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Bouly JP, Giovani B, Djamei A, Mueller M, Zeugner A, Dudkin EA, Batschauer A, and Ahmad M
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- Animals, Arabidopsis Proteins, Baculoviridae physiology, Biological Clocks, Biotechnology methods, Cell Line, Cryptochromes, Flavins antagonists & inhibitors, Flavins metabolism, Flavoproteins genetics, Gene Expression, Humans, Light, Nucleotides metabolism, Oxidants chemistry, Oxidants pharmacology, Phosphorus Radioisotopes, Phosphorylation, Receptors, G-Protein-Coupled, Recombinant Proteins genetics, Recombinant Proteins metabolism, Serine metabolism, Spodoptera, Adenosine Triphosphate metabolism, Arabidopsis metabolism, Drosophila Proteins, Eye Proteins, Flavoproteins metabolism, Photoreceptor Cells, Invertebrate, Protein Binding physiology
- Abstract
Cryptochromes are blue-light photoreceptors sharing sequence similarity to photolyases, a class of flavoenzymes catalyzing repair of UV-damaged DNA via electron transfer mechanisms. Despite significant amino acid sequence similarity in both catalytic and cofactor-binding domains, cryptochromes lack DNA repair functions associated with photolyases, and the molecular mechanism involved in cryptochrome signaling remains obscure. Here, we report a novel ATP binding and autophosphorylation activity associated with Arabidopsis cry1 protein purified from a baculovirus expression system. Autophosphorylation occurs on serine residue(s) and is absent in preparations of cryptochrome depleted in flavin and/or misfolded. Autophosphorylation is stimulated by light in vitro and oxidizing agents that act as flavin antagonists prevent this stimulation. Human cry1 expressed in baculovirus likewise shows ATP binding and autophosphorylation activity, suggesting this novel enzymatic activity may be important to the mechanism of action of both plant and animal cryptochromes.
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- 2003
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21. An Arabidopsis protein closely related to Synechocystis cryptochrome is targeted to organelles.
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Kleine T, Lockhart P, and Batschauer A
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- Amino Acid Sequence, Arabidopsis cytology, Arabidopsis genetics, Cryptochromes, DNA metabolism, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, Flavin-Adenine Dinucleotide metabolism, Flavoproteins metabolism, Molecular Sequence Data, Phylogeny, Protein Transport, Receptors, G-Protein-Coupled, Sequence Alignment, Sequence Homology, Amino Acid, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Arabidopsis Proteins metabolism, Chloroplasts metabolism, Cyanobacteria chemistry, Deoxyribodipyrimidine Photo-Lyase chemistry, Deoxyribodipyrimidine Photo-Lyase metabolism, Drosophila Proteins, Eye Proteins, Flavoproteins chemistry, Mitochondria metabolism, Photoreceptor Cells, Invertebrate
- Abstract
Cryptochromes (CRYs) are blue/UV-A photoreceptors related to the DNA repair enzyme DNA photolyase. They have been found in plants, animals and most recently in the cyanobacterium Synechocystis. Closely related to the Synechocystis cryptochrome is the Arabidopsis gene At5g24850. Here, we show that the encoded protein of At5g24850 binds flavin adenine dinucleotide (FAD). It has no photolyase activity, and is likely to function as a photoreceptor. We have named it At-cry3 to distinguish it from the other Arbabidopsis cryptochrome homologues At-cry1 and At-cry2. At-cry3 carries an N-terminal sequence, which mediates import into chloroplasts and mitochondria. Furthermore, we show that At-cry3 binds DNA. DNA binding was also demonstrated for the Synechocystis cryptochrome, indicating that both photoreceptors could have similar modes of action. Based on the finding of a new cryptochrome class in bacteria and plants, it has been suggested that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. However, our phylogenetic analyses are also consistent with an alternative explanation that the presence of cryptochromes in the plant nuclear genome is the result of dual horizontal gene transfer. That is, CRY1 and CRY2 genes may originate from an endosymbiotic ancestor of modern-day alpha-proteobacteria, while the CRY3 gene may originate from an endosymbiotic ancestor of modern-day cyanobacteria.
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- 2003
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22. Einfluss von Nukleotiden auf die biologische Aktivität von Cryptochrom 1 aus Arabidopsis thaliana
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Orth, Christian and Batschauer, Alfred (Prof. Dr.)
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ADP ,ATP ,Biowissenschaften, Biologie ,ddc:570 ,Arabidopsis ,sense organs ,Nukleotide ,Cryptochrom 1 ,Life sciences - Abstract
Cryptochromes (cry) are blue light receptors that together with photolyases form the so- called cryptochrome/photolyase family (CPF) with members occuring in all three domains of life. All members of the CPF show high structural similarities within the N-terminal photosensory (PHR, photolyase homologous region) domain that non-covalently binds the FAD cofactor. But their biologigal functions are different. While photolyases are enzymes that repair UV-damaged DNA in a light dependent manner, Cryptochromes lost the ability to repair DNA but gained important functions as photoreceptors to regulate a wide variety of biological responses. The two classical cryptochromes, cry1 and cry2, that are encoded in the model plant Arabidopsis thaliana have their main functions as blue-light photoreceptors that regulate photomorphogenesis and photoperiodic flowering. For the formation of the cryptochrome´s active state the photoexcitation of the flavin cofactor and a subsequent electron transfer towards the excited flavin is required. This allows the formation of the photoreceptors active state that, in case of plant cryptochromes, contains the flavin cofactor in its semireduced radical form FADH°. The transition from the resting state of the flavin in darkness to the active state is called „photoreduction“. For cry1 it was demonstrated for the first time in 2003 that plant cryptochromes bind ATP (Bouly et al., 2003). One year later the crystal structure of the PHR domain of Arabidopsis thaliana was published. Crystals were soaked with the non-hydrolyzable ATP analogon AMP-PNP and the co-crystal structure showed that the nucleotide is associated within the FAD cavity by a central tyrosine (Brautigam et al., 2004). Since then, not much is known about the nucleotide binding properties of cryptochromes. In 2010 a mutant of cry1 with a hyperactivity regarding blu-light responses was described (Exner et al., 2010). The mutation was caused by a leucine to phenylalanin exchange at position 407 of the cry1 PHR and was therefore called cry1L407F mutant. This mutant showed a cop-phenotype in the dark and based on in-silico structure modeling it was hypothesized that this mutant might mimic the signal active state of cry1 in darkness and that ATP binding might also be involved in this hyperactivity. The central aspect of this work was to investigate the effects of the binding of ATP and other nucleotides to the PHR domain of cry1 wild type and cry1L407F mutant protein. To have a suitable negative control in the performed assays, an ATP non-binding cry1 mutant was generated by using targeted mutagenesis. In this mutant, called cry1Y402A, the tyrosin on position 402, which is essential for ATP-binding in cry1, was exchanged to an alanin. The obtained data demonstrated that ATP enhances the photoreduction of both cry1WT and cry1L407F by stabilizing the active state of the photoreceptors. In protein stability assays it was shown that ATP binding also increases the stability of cry1. The data further indicated that the hyperactivity of the cry1L407F mutant is most likely caused by a structural alteration that is diffrent from the structure of cry1 with ATP bound. Furthermore a luciferase-based ATP-binding assay was established to approach the KD of ATP and cry1. This provided a basis for later-stage ITC meassurements in which a KD of 3.3 µM was determined for ATP binding to cry1 . Although many of the aspects of ATP binding of cryptochromes could be addressed the biological function of ATP binding remains unclear., Während der Durchführung dieser Arbeit wurden verschiedene in-vitro Techniken und Assays benutzt, um die Nukleotidbindung von Cryptochrom 1 aus Arbidopsis thaliana näher zu charakterisieren. Die Fähigkeit von pflanzlichen Cryptochromen, ATP zu binden wurde bereits in 2003 beschrieben (Bouly et al., 2003) und auch die Bindestelle für ATP konnte in einer Kokristallstruktur von Cryptochrom 1 und AMP-PNP identifiziert werden (Brautigam et al., 2004). Jedoch wurde die ATP-Bindung von Cryptochrom 1 hinsichtlich ihrer Auswirkungen auf die Stabilität der PHR-Domäne nicht weiter untersucht, ebenso nicht die Spezifität der Nukleotide. Im Zuge dieser Arbeit konnte gezeigt werden, dass die PHR-Domäne durch die Bindung von ATP und ADP eine kompaktere Struktur einnimmt, welche sowohl den Prozess der Photoreduktion des FAD beschleunigt, als auch das Protein in seiner Gesamtheit stabilisiert. Der stabilisierende Effekt konnte sowohl auf thermischer Ebene über eine Erhöhung der Schmelztemperatur als auch auf molekularer Ebene durch eine erhöhte Resistenz gegenüber der Protease Trypsin nachgewiesen werden. Des Weiteren wurde über Schmelzpunktbestimmung und Photoreduktionsexperimente der Einfluss weiterer Nukleotide wie NAD(H) und NADP(H) auf diese Prozesse getestet. Die Ergebnisse zeigten, dass diese Nukleotide keine signifikanten Effekte auf die Photoreduktion und die Proteinstabilität von cry 1 haben. Daher ist auch eine Bindung dieser Nukleotide eher unwahrscheinlich. Es konnte in dieser Arbeit ferner gezeigt werden, dass die Bindung von Nukleotiden spezifisch für ATP oder dessen Derivate ist. Die Tatsache, dass selbst das strukturell sehr ähnliche GTP in Stabilitätsassays keinen Effekt auf die PHR Domäne von Cryptochrom 1 hat, spricht für die Selektivität der Bindung von Adenin-Derivaten. Die Verwendung der cry1Y402A PHR Mutante in den Assays zeigte, dass das Tyrosin an Position 402 essentiell für die ATP-Bindung von Cryptochrom 1 ist, da diese Mutante in keinem der Assays auf die Zugabe von ATP (oder anderen Nukleotiden) reagierte. Für die cry1L407F PHR Mutante konnte gezeigt werden, dass die Hypersensitivität unabhängig von der ATP-Bindung nicht durch eine verstärkte Photoreduktion zurückzuführen ist. Auch das Mimikrieren eines ATP-gebundenen Zustandes konnte anhand der vorliegenden Daten ausgeschlossen werden. Vielmehr ist eine strukturelle Veränderung in der Nähe der ATP-Bindestelle für die Hyperaktivität verantwortlich, wobei der genaue Mechanismus der der Hyperaktivität zu Grunde liegt, nicht abschließend geklärt werden konnte.
- Published
- 2021
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23. Promoter elements of the mustard CHS1 gene are sufficient for light regulation in transgenic plants
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Kaiser, Thomas, Emmler, Karlheinz, Kretsch, Thomas, Weisshaar, Bernd, Schäfer, Eberhard, and Batschauer, Alfred
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- 1995
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24. Blue-Light-Induced Changes in Arabidopsis Cryptochrome 1 Probed by FTIR Difference Spectroscopy.
- Author
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Kottke, Tilman, Batschauer, Alfred, Ahmad, Margaret, and Heberle, Joachim
- Subjects
- *
CRYPTOCHROMES , *ARABIDOPSIS , *SPECTRUM analysis , *PHOTORECEPTORS , *CIRCADIAN rhythms , *HOMOLOGY (Biology) , *DROSOPHILA - Abstract
Cryptochromes are blue-light photoreceptors that regulate a variety of responses in animals and plants, including circadian entrainment in Drosophila and photomorphogenesis in Arabidopsis. They comprise a photolyase homology region (PHR) of about 500 amino acids and a C-terminal extension of varying length. In the PHR domain, flavin adenine dinucleotide (FAD) is noncovalently bound. The presence of a second chromophore, such as methenyltetrahydrofolate, in animal and plant cryptochromes is still under debate. Arabidopsis cryptochrome 1 (CRY1) has been intensively studied with regard to function and interaction of the protein in vivo and in vitro. However, little is known about the pathway from light absorption to signal transduction on the molecular level. We investigated the full-length CRY1 protein by Fourier transform infrared (FTIR) and UV/vis difference spectroscopy. Starting from the fully oxidized state of the chromophore FAD, a neutral flavoprotein radical is formed upon illumination in the absence of any exogenous electron donor. A preliminary assignment of the chromophore bands is presented. The FTIR difference spectrum reveals only moderate changes in secondary structure of the apoprotein in response to the photoreduction of the chromophore. Deprotonation of an aspartic or glutamic acid, probably D396, accompanies radical formation, as is deduced from the negative band at 1734 cm-1 in D2O. The main positive band at 1524 cm-1 in the FTIR spectrum shows a strong shift to lower frequencies as compared to other flavoproteins. Together with the unusual blue-shift of the absorption in the visible range to 595 nm, this clearly distinguishes the radical form of CRY1 from those of structurally highly homologous DNA photolyases. As a consequence, the direct comparison of cryptochrome to photolyase in terms of photoreactivity and mechanism has to be made with caution. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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25. Nuclear localization of the Arabidopsis blue light receptor cryptochrome 2.
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Kleiner, Oliver, Kircher, Stefan, Harter, Klaus, and Batschauer, Alfred
- Subjects
ARABIDOPSIS ,CRYPTOCHROMES ,PHOTORECEPTORS ,GREEN fluorescent protein - Abstract
Summary: The cryptochrome blue light photoreceptor family of Arabidopsis thaliana consists of two members, CRY1 and CRY2 (PHH1). CRY2 contains a putative nuclear localization signal (NLS) within its C-terminal region. We examined whether CRY2 is localized in the nucleus and whether the C-terminal region of CRY2 is involved in nuclear targeting. Total cellular and nuclear protein extracts from Arabidopsis were subjected to immunoblot analysis with CRY2-specific antibodies. Strong CRY2 signals were obtained in the nuclear fraction. Fusion proteins consisting of the green fluorescent protein (GFP) and different fragments of CRY2 were expressed in parsley protoplasts and the localization of the fusion proteins was determined by fluorescence and confocal laser scanning microscopy. GFP-fusions containing the entire CRY2 protein or its C-terminal region were found exclusively in the nucleus. We conclude from these results that CRY2 is localized in the nucleus and that nuclear localization is mediated by the C-terminal region of CRY2. [ABSTRACT FROM AUTHOR]
- Published
- 1999
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26. Class II DNA photolyase from Arabidopsis thaliana contains FAD as a cofactor.
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Kleiner, Oliver, Butenandt, Jens, Carell, Thomas, and Batschauer, Alfred
- Subjects
PYRIMIDINES ,ARABIDOPSIS ,ESCHERICHIA coli - Abstract
Studies the overexpression of Arabidopsis cyclobutane pyrimidine dimer (CPD)-photolyase in Escherichia coli. Purification and analysis of photolyases for chromophore composition and enzymatic activity; Characterization of the chromophore; Reaction mechanism of photolyases.
- Published
- 1999
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27. Funktionelle Analyse von Cryptochrom 3 aus Arabidopsis thaliana
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Reisbacher, Stefan and Batschauer, Alfred (Prof. Dr.)
- Subjects
Photoreceptor ,Cryptochrom ,Deoxyribodipyrimidin-Photolyase ,fungi ,Arabidopsis ,food and beverages ,DNA repair ,Germination ,Life sciences -- Biowissenschaften, Biologie ,Life sciences ,Cryptochrome ,Ackerschmalwand ,Biowissenschaften, Biologie ,ddc:570 ,Photorezeptoren ,Samenkeimung ,Photolyase ,2009 - Abstract
Cryptochromes are blue/UV-A light photoreceptors, which are closely related to photolyases but do not show DNA repair activity. Apart from the classical plant cryptochromes cry1 and cry2, a third Cryptochrom was identified in Arabidopsis thaliana. Cryptochrome 3 (cry3) belongs to the subfamily of the DASH cryptochromes and was shown to be localized in the chloroplasts and mitochondria. Although cry3 has been characterized quite well considering protein structure and biochemical properties, the biological function of this protein in the plant remains unknown until today. In this thesis the biological function of cry3 in the plant has been investigated. The localization of cry3 in chloroplasts and mitochondria could be confirmed by immunological studies on organellar fractions of Arabidopsis cell culture and immunolocalization studies using gold labelled secondary antibodies. Therefore any artefacts of the previous localization studies by overexpression and GFP fusion of cry3 can be excluded. At transcript level CRY3 expression is regulated by light. During the early phase of deetiolation cry3 is transiently induced mainly by far-red light. Phytochrome A was identified as the responsible photoreceptor for this regulation of cry3. PIF1 and PIF3 are also involved in this phytochrome dependent process. In adult plants grown in light / dark cycles, CRY3 expression shows a circadian regulation, which is also affected by the photoperiod. For functional analysis of cry3, several seed collections were screened for cry3 mutants and transgenic cry3 lines were produced. For further functional analysis cry3 overexpressor lines, RNAi knock-down lines, a cry3 knock-out transposon line and several T-DNA insertion lines with altered CRY3 expression and truncated cry3 c-term are now available. Two of these mutant lines are affected in seed germination under limiting light conditions. However this phenotype could not be proven beyond any doubt. Apart from this reduced germination rate the analyzed cry3 lines did not show any clear phenotypic differences compared to wild type plants. Cry3 does not seem to affect blue, green or UV light dependent regulation of plastid genes in Arabidopsis. Growth of Arabidopsis plants treated with elevated levels of UV-B light is not affected by cry3, although cry3 has been described to repair UV lesions in vitro in single stranded DNA and in double stranded DNA with loop structures. The results of this thesis clearly disagree with a function for cry3 as a DNA repair enzyme, because the PCR based repair assay described here could not detect any effect of cry3 on the repair activity in chloroplasts, mitochondria or in the nucleus., Cryptochrome sind Blau-/UVA-Photorezeptoren, die eng mit den Photolyasen verwandt sind, aber keine DNA-Reparaturaktivität besitzen. Neben den „klassischen“ pflanzlichen Cryptochromen cry1 und cry2 wurde in Arabidopsis thaliana mit Cryptochrom 3 (cry3) ein drittes Cryptochrom identifiziert. Dieses Cryptochrom aus der Familie der DASH-Cryptochrome wurde als plastiden- und mitochondrienlokalisiertes Protein beschrieben. Obwohl Cryptochrom 3 schon sehr gut strukturell und biochemisch charakterisiert ist, konnte die biologische Funktion dieses Proteins in der Pflanze bisher noch nicht aufgeklärt werden. In dieser Arbeit wurde daher die biologische Funktion von cry3 in der Pflanze untersucht. Die cry3-Lokalisation in Chloroplasten und Mitochondrien konnte durch Zellfraktionierung und Immunolokalisationsstudien eindeutig bestätigt werden. Damit konnten Artefakte bei den bisherigen Untersuchungen durch die Überexpression und GFP-Fusion von cry3 ausgeschlossen werden. Die CRY3-Expression wird auf der Transkript-Ebene durch Licht reguliert. CRY3 wird während der Deetiolierungsphase hauptsächlich durch dunkelrotes Licht transient induziert. Phytochrom A konnte als der hauptverantwortliche Photorezeptor für diese Reaktion identifiziert werden. An dieser phytochromabhängigen Regulation sind auch PIF1 und PIF3 beteiligt. In ergrünten Pflanzen unterliegt die CRY3-Expression einer circadianen Regulation, welche anscheinend auch durch die Photoperiode beeinflusst wird. Zur funktionellen Analyse von Cryptochrom 3 wurden cry3-Mutanten in verschiedenen Mutanten-Kollektionen identifiziert und außerdem verschiedene cry3-Linien hergestellt. Zur weiteren Untersuchung der cry3-Funktion steht eine cry3-Überexpressionslinie, eine RNAi-knock-down-Linie, eine cry3-knock-out-Linie mit einer Transposon-Insertion, verschiedene T-DNA-Insertionslinien mit veränderter CRY3-Expression bzw. verkürztem cry3 C-Terminus, sowie eine putative knock-out-Linie mit einer Punktmutation in der splicing Erkennungssequenz zur Verfügung. Zwei dieser Mutantenlinien waren in ihrer Keimungsfähigkeit unter spezifischen Bedingungen beeinträchtigt. Der Keimungsphänotyp konnte in dieser Arbeit allerdings nicht eindeutig belegt werden. Abgesehen von dieser reduzierten Keimrate waren bei den untersuchten transgenen cry3-Linien keine offensichtlichen phänotypischen Unterschiede festzustellen. Auch ein Einfluss von cry3 auf die Blau-, Grün- und UV-Licht-abhängige Regulation von plastidenkodierten Genen konnte nicht nachgewiesen werden. Das Wachstum von Arabidopsis unter erhöhter UV-B-Bestrahlung wird durch cry3 nicht beeinflusst. Für cry3 ist zwar in vitro eine Photolyasefunktion bei einzelsträngiger DNA und doppelsträngiger DNA mit loop-Strukturen beschrieben worden, dennoch sprechen die Ergebnisse dieser Arbeit gegen eine in vivo Funktion von cry3 als DNA-Reparaturenzym, weil durch einen PCR-basierten Reparatur-assay kein Einfluss von cry3 auf die DNA-Reparatur in Chloroplasten und Mitochondrien nachgewiesen werden konnte.
- Published
- 2009
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28. Modes of action of cryptochrome 2 from Arabidopsis thaliana
- Author
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Muñoz Viana, Rafael and Batschauer, Alfred (Dr.)
- Subjects
animal structures ,Cryptochrom ,fungi ,Arabidopsis ,Development ,Tabak ,Life sciences ,Life sciences -- Biowissenschaften, Biologie ,Cryptochrome ,%22">Schmalwand ,Lichtabsorption ,Biowissenschaften, Biologie ,Chromophore ,2007 ,ddc:570 ,Schmalwand ,sense organs - Abstract
Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light developmental responses in plants. Arabidopsis thaliana cryptochrome 2 (Atcry2) is the major photoreceptor mediating blue light regulation of flowering induction. Although the biological role of crys in plants is well-known, the initial photochemistry underlying cryptochrome activation and regulation remain poorly understood. In the present work we addressed several aspects in the early activation events of cry 2. Many members of the photoreceptor families and components of the light signalling transduction pathway dimerize. Therefore, we studied wheter Atcry2 dimerizes, too. Immunoprecipitation studies of transgenic Arabidopsis extracts expressing both, cry2-GFP and cry2 revealed that full-lenght Atcry2 is a homodimer in vivo in a light-independent fashion. The identity of the domains involved in cry2 homodimerization were investigated, and both CNT2 and CCT2 were found as monomers. Because of the sequence similarity of cry2 with cry1, heterodimerization between cry1 and cry2 was also studied, but no cry1-cry2 heterodimers were found in our experiments. The in vivo effect of dimerization was investigated using Arabidopsis transgenic lines expression either CCT2-GFP or cry2-GFP in addition of the endogenous cry2. Cry2-GFP dimers showed phosphorylation and degradation under blue light in the same way as the endogenous cry2, whereas under the same conditions the CCT2-GFP monomers remained stable and unphosphorylated. Moreover, cry2-GFP was able to promote early flowering in plants kept under short day conditions. Whereas under the same conditions CCT2-GFP expressing transgenic Arabidopsis flowered as late as wild-type. Crys purified from Escherichia coli contain two chromophores, which were identified as flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF), whereas the presence of MTHF was not found for Atcrys purified from an eukaryotic source as insect cells. The detailed knowledge of which chromophore(s) are attached under natural conditions is important for the interpretation of spectroscopic data of these receptors. Therefore, we overexpressed epitope-tagged cry2 in planta. Specific immuno-precipitation of the tagged cry2 protein allowed purification of sufficient amounts of the photoreceptor to identify its chromophores. Based on fluorescence emission data we found that cry2 binds indeed FAD and MTHF in planta. In addition, energy transfer from MTHF to FAD was observed. Because of their similarity in aminoacid sequence and structure, photolyases have been taken as a model for crys photocycle. However, crys were shown to undergo a photocycle in which semireduced flavin (FADHo) accumulates upon blue light irradiation in contrast to photolyase that accumulates fully reduced FADH-. Green light irradiation of cry2 causes a change in the equilibrium of flavin oxidation states, and attenuates cry2-controlled responses such as flowering. Here, we provided in vivo evidence for semireduced flavin (FADHo) being the active FAD redox state in Atcry2 by analysis of the expression of flowering genes, linking in vitro with physiological studies. In order to address further insight into the role of phosphorylation on Atcry2 activity, cry2 protein purification from the plant source following mass spectroscopy was performed. Pitifully, the obtained amounts were too small to allow clear results. Genes that are specifically blue-light induced in cell cultures were identified by PCR and qRT-PCR that can be used in future studies as reporters for transient studies monitoring cry activity., Cryptochrome sind mit DNA-Photolyasen eng verwandte UV-A/Blaulicht-Photorezeptoren, die zahlreiche Entwicklungsprozesse in Pflanzen steuern. Arabidopsis thaliana Cryptochrom 2 (cry2) spielt hierbei eine zentrale Rolle bei der photoperiodischen Regulation der Blühinduktion. Obwohl die biologischen Funktionen von Cryptochromen in Pflanzen gut untersucht sind, sind deren photochemischen Prozesse, die zur Aktivierung führen, wenig verstanden. In der vorliegenden Arbeit wurden zahlreiche Aspekte der Aktivierung von cry2 untersucht. Zahlreiche pflanzlichen Photorezeptoren und Komponenten der Lichtsignaltransduktion dimerisieren. Entsprechend wurde hier untersucht, ob dies auch für cry2 der Fall ist. Immunpräzipitationsstudien an Proteinextrakten transgener Arabidopsis Pflanzen, die cry2-GFP exprimieren, zeigten, dass cry2 in vivo als Dimer vorliegt. Licht scheint keinen Einfluss auf die Dimerisierung zu haben. Bei der Analyse, welche Domänen von cry2 für Dimerisierung notwendig sind, konnten keine eindeutigen Befunde erzielt werden, da sowohl die N-terminale Domäne (CNT2) als auch die C-terminale Domäne (CCT2) als Monomer gefunden wurden. Aufgrund der hohen Sequenzähnlichkeit von cry2 mit cry1 wurde untersucht, ob diese Heterodimere bilden. Die vorliegenden Befunde gaben darauf keinen Hinweis. Um die Rolle der Dimerisierung von cry2 auf dessen biologische Funktion zu untersuchen, wurden transgene Linien untersucht, die neben dem endogenen cry2 zusätzlich CCT2-GFP oder cry2-GFP exprimieren. Cry2-GFP Dimere wurden lichtabhängig phosphoryliert und abgebaut, ähnlich dem endogenen cry2. Im Gegensatz hierzu, blieben die CCT2-GFP Monomere unphosphoryliert und stabil. Weiterhin führte die Expression von cry2-GFP zu einer Beschleunigung des Blühens unter Kurztag-Bedingungen im Vergleich zum Wildtyp, nicht aber die Expression von CCT2-GFP. Pflanzliche Cryptochrome, die in E. coli exprimiert wurden, tragen zwei Chromophore (FAD und MTHF). Nach Expression in eukaryontischen Systemen wie Insektenzellen konnte bislang allerdings keine Bindung von MTHF an cry nachgewiesen werden. Kenntnisse darüber, welche Chromophore an cry in planta gebunden sind, sind für die Interpretation der spektroskopischen Daten dieser Photorezeptoren bedeutsam. Deshalb wurde in der vorliegenden Arbeit Epitop-markiertes cry2 in Pflanzen exprimiert und spezifisch über Immunpräzipitation in Mengen aufgereinigt, die eine Identifizierung der gebundenen Chromophore ermöglichte. Die Ergebnisse von Fluoreszenz-Emissionsspektroskopie zeigten, dass cry2 in planta sehr wahrscheinlich beide Cofaktoren, FAD und MTHF, bindet. Zusätzlich ergaben diese Untersuchungen Hinweis auf Energietransfer von MTHF auf FAD. Augrund der Ähnlichkeit von Photolyasen und Cryptochromem in ihrer Aminosäuresequenz and Struktur wurde die Photochemie von Photolyasen als Modell für den Photozyklus von Cryptochromen benutzt. Allerdings zeigten in vitro Untersuchungen, dass Cryptochrome nach Anregung mit Blaulicht semireduziertes Flavin (FADHo) akkumulieren, im Gegensatz zu Photolyasen, die vollständig reduziertes Flavin (FADH-) bilden. Zu Blaulicht zusätzlich gegebenes Grünlicht verschiebt das Gleichgewicht der Oxidationszustände von Flavin hin zu oxidiertem FAD und reprimiert cry2-kontrollierte Prozesse wie die Induktion der Blütenbildung. Hier durchgeführte Untersuchungen über die Wirkung von Grünlicht auf die Expression von Blühgenen lieferten zusätzliche Hinweise darauf, dass cry2 mit semireduziertem Flavin den aktiven Zustand dieses Photorezeptors repräsentiert. Zum weiteren Verständnis der Phosphorylierung von cry2 wurde versucht, das Protein aus Pflanzen in Mengen zu isolieren, die für nachfolgende massenspektrometrische Analysen ausreichend sind. Leider wurde dieses Ziel nicht erreicht. Weiterhin wurden die Expression zahlreicher Gene in Arabidopsis Zellkulturen durch PCR- und quantitative real-time PCR-Analysen daraufhin untersucht, ob sie spezifisch durch Blaulicht reguliert werden. Einige Gene konnten hierbei identifiziert werden, die in zukünftigen Untersuchungen als Reporter genutzt werden können, um die Aktivität von Wildtyp und Mutanten Cryptochromen zu untersuchen.
- Published
- 2007
29. Der Blaulicht-Photorezeptor Cryptochrom 2 aus Arabidopsis thaliana: Lichtabhängige Phosphorylierung und Interaktionspartner in der Signaltransduktion
- Author
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Müller, Markus Bernd and Batschauer, Alfred Prof. Dr.
- Subjects
signal-transduction ,animal structures ,Cryptochrom ,Flavine ,phosphorylation ,Photorezeptor ,flavin ,Life sciences ,%22">Schmalwand ,arabidopsis ,Biowissenschaften, Biologie ,Lichtabsorption ,ddc:570 ,sense organs ,cryptochrome - Abstract
Althougth the physiological functions of plant cryptochromes have been thoroughly investigated and their near relatives, the photolyases have been studied on the molecular level in very detail, the knowledge about the signal transduction of the cryptochromes however is relatively poor. Following the aim to find out more about these molecular processes, this work presents insights into some aspects, using Arabidopsis cry2 as a model. In this thesis, a light dependent posphorylation of cry2 has been discovered and further investigated. The recorded action-spectrum of this phosphorylation shows clear characteristics of a flavin-spectrum. Attempts to identifiy the responsible kinase have not been finished so far. A potential autophosphorylation activity connected with cry2 has been discussed, but could not be proven, finally. Further experiments with heterologouesly expressed cry2 in yeast resulted in data that suggest a role of cry2 as light dependent transcription activator. Also a DNA-binding activity of in vitro-transcribed and -translated cry2 has been demonstrated. Furthermore, the subcellular localization of two putative interaction partners of cry2 ? At5g26280 and At2g02230 - has been described. At2g02230 has been identified as functional F-box protein. Studies to elucidate the degradation pathway of cry2 could not strengthen the model of a proteasomal degradation.Althougth the physiological functions of plant cryptochromes have been thoroughly investigated and their near relatives, the photolyases have been studied on the molecular level in very detail, the knowledge about the signal transduction of the cryptochromes however is relatively poor. Following the aim to find out more about these molecular processes, this work presents insights into some aspects, using Arabidopsis cry2 as a model. In this thesis, a light dependent posphorylation of cry2 has been discovered and further investigated. The recorded action-spectrum of this phosphorylation shows clear characteristics of a flavin-spectrum. Attempts to identifiy the responsible kinase have not been finished so far. A potential autophosphorylation activity connected with cry2 has been discussed, but could not be proven, finally. Further experiments with heterologouesly expressed cry2 in yeast resulted in data that suggest a role of cry2 as light dependent transcription activator. Also a DNA-binding activity of in vitro-transcribed and -translated cry2 has been demonstrated. Furthermore, the subcellular localization of two putative interaction partners of cry2 ? At5g26280 and At2g02230 - has been described. At2g02230 has been identified as functional F-box protein. Studies to elucidate the degradation pathway of cry2 could not strengthen the model of a proteasomal degradation., Obwohl über die physiologische Bedeutung der pflanzlichen Cryptochrome bereits viel bekannt ist, und deren nahe Verwandte, die Photolyasen auch molekular sehr detailliert, einschließlich der Struktur auf atomarer Ebene, charakterisiert sind, sind die Mechanismen der Signalweiterleitung bei den Cryptochromen bis heute nur wenig erforscht. Dem Ziel folgend, diese molekularen Prozesse aufzuklären, wurde in dieser Arbeit Arabidopsis- Cryptochrom 2 untersucht. Es wurde dabei die lichtabhängige Phosphorylierung von cry2 entdeckt und näher untersucht. Das Aktionspektrum dieser Phosphorylierung ähnelt dabei stark einem Flavinspektrum. Versuche, die für diesen Prozess verantwortliche Kinase zu identifizieren, konnten nicht abgeschlossen werden. Eine Autophosphorylierungsaktivität wurde in Kooperation mit Margaret Ahmad (Paris) für cry1 gefunden, ist für cry2 bislang aber nicht gezeigt. In Hefe heterolog exprimiertes cry2 zeigt lichtabhängige Effekte, die auf mögliche Eigenschaften von cry2 als Transkriptionsaktivator schließen lassen. Zudem konnte eine DNA- Bindung für in vitro- transkribiertes und -translatiertes cry2 gezeigt werden. Untersuchungen zum Abbauweg von cry2 konnten unsere These eines proteasomalen Abbau von cry2 nicht bestätigen. Die subzelluläre Lokalisation zweier putativer Interaktionspartner von cry2 - At5g26280 und At2g02230 - wurde über konfokale Laserscan- Mikroskopie untersucht. At2g2230 konnte zudem als funktionelles F-Box Protein identifiziert werden.
- Published
- 2003
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