Galileo Estopare Araguirang1,2, Nils Niemann3, Stephan Kiontke3, Maike Eckel3, Maribel L Dionisio-Sese1,4, Alfred Batschauer5. 1. Graduate School, University of the Philippines Los Baños, College, 4031, Laguna, Philippines. 2. Department of Plant Adaptation, Leibniz-Institut für Gemüse- und Zierpflanzenbau (IGZ), Großbeeren, 14979, Germany. 3. Department of Plant Physiology and Photobiology, Faculty of Biology, Philipps-University Marburg, 35032, Marburg, Germany. 4. Plant Biology Division, Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños, College, 4031, Laguna, Philippines. 5. Department of Plant Physiology and Photobiology, Faculty of Biology, Philipps-University Marburg, 35032, Marburg, Germany. batschau@staff.uni-marburg.de.
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.
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.
Authors: Vivien Exner; Cristina Alexandre; Gesa Rosenfeldt; Pietro Alfarano; Mena Nater; Amedeo Caflisch; Wilhelm Gruissem; Alfred Batschauer; Lars Hennig Journal: Plant Physiol Date: 2010-10-06 Impact factor: 8.340
Authors: Sarah Burney; Nathalie Hoang; Michael Caruso; Elizabeth A Dudkin; Margaret Ahmad; Jean-Pierre Bouly Journal: FEBS Lett Date: 2009-03-25 Impact factor: 4.124