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Qin Wang - One of the best experts on this subject based on the ideXlab platform.

  • the universally conserved residues are not universally required for stable protein expression or functions of Cryptochromes
    Molecular Biology and Evolution, 2020
    Co-Authors: Wenjin He, Tiantian Su, Qin Wang
    Abstract:

    Universally conserved residues (UCRs) are invariable amino acids evolutionarily conserved among members of a protein family across diverse kingdoms of life. UCRs are considered important for stability and/or function of protein families, but it has not been experimentally examined systematically. Cryptochromes are photoreceptors in plants or light-independent components of the circadian clocks in mammals. We experimentally analyzed 51 UCRs of Arabidopsis Cryptochrome 2 (CRY2) that are universally conserved in eukaryotic Cryptochromes from Arabidopsis to human. Surprisingly, we found that UCRs required for stable protein expression of CRY2 in plants are not similarly required for stable protein expression of human hCRY1 in human cells. Moreover, 74% of the stably expressed CRY2 proteins mutated in UCRs retained wild-type-like activities for at least one photoresponses analyzed. Our finding suggests that the evolutionary mechanisms underlying conservation of UCRs or that distinguish UCRs from non-UCRs determining the same functions of individual Cryptochromes remain to be investigated.

  • a cry bic negative feedback circuitry regulating blue light sensitivity of arabidopsis
    Plant Journal, 2017
    Co-Authors: Zhaohe Yang, Xu Wang, Qin Wang, Zecheng Zuo, Yunjeong Han, Qing Liu, Bobin Liu, Jing Wang
    Abstract:

    Cryptochromes are blue light receptors that regulate various light responses in plants. Arabidopsis Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) mediate blue light inhibition of hypocotyl elongation and long-day (LD) promotion of floral initiation. It has been reported recently that two negative regulators of Arabidopsis Cryptochromes, Blue light Inhibitors of Cryptochromes 1 and 2 (BIC1 and BIC2), inhibit Cryptochrome function by blocking blue light-dependent Cryptochrome dimerization. However, it remained unclear how Cryptochromes regulate the BIC gene activity. Here we show that Cryptochromes mediate light activation of transcription of the BIC genes, by suppressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), resulting in activation of the transcription activator ELONGATED HYPOCOTYL 5 (HY5) that is associated with chromatins of the BIC promoters. These results demonstrate a CRY-BIC negative-feedback circuitry that regulates the activity of each other. Surprisingly, phytochromes also mediate light activation of BIC transcription, suggesting a novel photoreceptor co-action mechanism to sustain blue light sensitivity of plants under the broad spectra of solar radiation in nature.

  • molecular basis for blue light dependent phosphorylation of arabidopsis Cryptochrome 2
    Nature Communications, 2017
    Co-Authors: Weixian Deng, Mingxin Piao, Yaxing Li, Xu Wang, Qin Wang, William D Barshop
    Abstract:

    Plant Cryptochromes undergo blue light-dependent phosphorylation to regulate their activity and abundance, but the protein kinases that phosphorylate plant Cryptochromes have remained unclear. Here we show that photoexcited Arabidopsis Cryptochrome 2 (CRY2) is phosphorylated in vivo on as many as 24 different residues, including 7 major phosphoserines. We demonstrate that four closely related Photoregulatory Protein Kinases (previously referred to as MUT9-like kinases) interact with and phosphorylate photoexcited CRY2. Analyses of the ppk123 and ppk124 triple mutants and amiR4k artificial microRNA-expressing lines demonstrate that PPKs catalyse blue light-dependent CRY2 phosphorylation to both activate and destabilize the photoreceptor. Phenotypic analyses of these mutant lines indicate that PPKs may have additional substrates, including those involved in the phytochrome signal transduction pathway. These results reveal a mechanism underlying the co-action of Cryptochromes and phytochromes to coordinate plant growth and development in response to different wavelengths of solar radiation in nature.

  • Photoactivation and inactivation of Arabidopsis Cryptochrome 2
    Science, 2016
    Co-Authors: Qin Wang, Lianfeng Gu, Zhaohe Yang, Takeshi Yoshizumi, Xu Wang, Liang Yang
    Abstract:

    Cryptochromes are blue-light receptors that regulate development and the circadian clock in plants and animals. We found that Arabidopsis Cryptochrome 2 (CRY2) undergoes blue light–dependent homodimerization to become physiologically active. We identified BIC1 (blue-light inhibitor of Cryptochromes 1) as an inhibitor of plant Cryptochromes that binds to CRY2 to suppress the blue light–dependent dimerization, photobody formation, phosphorylation, degradation, and physiological activities of CRY2. We hypothesize that regulated dimerization governs homeostasis of the active Cryptochromes in plants and other evolutionary lineages.

  • arabidopsis cry2 and ztl mediate blue light regulation of the transcription factor cib1 by distinct mechanisms
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Qin Wang, David E Somers, Xiaoying Zhao, Takato Imaizumi, Elaine M. Tobin
    Abstract:

    Plants possess multiple photoreceptors to mediate light regulation of growth and development, but it is not well understood how different photoreceptors coordinate their actions to jointly regulate developmental responses, such as flowering time. In Arabidopsis, the photoexcited Cryptochrome 2 interacts with the transcription factor Cryptochrome-INTERACTING basic helix–loop–helix 1 (CIB1) to activate transcription and floral initiation. We show that the CIB1 protein expression is regulated by blue light; CIB1 is highly expressed in plants exposed to blue light, but levels of the CIB1 protein decreases in the absence of blue light. We demonstrate that CIB1 is degraded by the 26S proteasome and that blue light suppresses CIB1 degradation. Surprisingly, although Cryptochrome 2 physically interacts with CIB1 in response to blue light, it is not the photoreceptor mediating blue-light suppression of CIB1 degradation. Instead, two of the three light–oxygen–voltage (LOV)-domain photoreceptors, ZEITLUPE and LOV KELCH PROTEIN 2, but not FLAVIN-BINDING KELCH REPEAT 1, are required for the function and blue-light suppression of degradation of CIB1. These results support the hypothesis that the evolutionarily unrelated blue-light receptors, Cryptochrome and LOV-domain F-box proteins, mediate blue-light regulation of the same transcription factor by distinct mechanisms.

Chandra L. Tucker - One of the best experts on this subject based on the ideXlab platform.

  • optogenetic control of gene expression using Cryptochrome 2 and a light activated degron
    Methods of Molecular Biology, 2020
    Co-Authors: Carmen N Hernandezcandia, Chandra L. Tucker
    Abstract:

    Optogenetic tools allow for use of light as an external input to control cellular processes. When applied to regulate the function of transcription factors, optogenetic approaches provide a tunable, reversible, and bidirectional method to control gene expression. Herein, we present a detailed method to induce gene expression in mammalian cells using the light dependent dimerization of Cryptochrome 2 (CRY2) and CIB1 to complement a split transcription factor. We also describe a protocol to disrupt gene expression with light by fusing a dimeric transcription factor to CRY2. When combined with a light-induced degron attached to the gene product, this method allows for rapid modulation of target protein abundance.

  • Advances in optogenetic regulation of gene expression in mammalian cells using Cryptochrome 2 (CRY2).
    Methods (San Diego Calif.), 2019
    Co-Authors: Carmen N. Hernández-candia, Christina L. Wysoczynski, Chandra L. Tucker
    Abstract:

    Abstract Synthetic regulation of gene expression provides a powerful approach to reprogram molecular and cellular processes and test the function of specific genes and gene products. In the last decade, optogenetic systems that allow light-dependent gene regulation have become valuable tools, providing tight spatiotemporal control of protein levels. Here we discuss and build on recent optogenetic approaches for regulating gene expression in mammalian cells using Cryptochrome 2 (CRY2), a photoreceptor protein from Arabidopsis. We provide detailed protocols for using light to manipulate activity of a CRY2-based engineered photoactivatable Cre DNA recombinase, and to induce or disrupt transcription factor function. In addition, we provide instructions and software for building an inexpensive Rasberry-Pi-based programable LED device for optogenetic experiments, delivering pulsed light with customized control of illumination duration, frequency, and intensity.

  • Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis Cryptochrome 2.
    Nucleic Acids Research, 2017
    Co-Authors: Gopal P. Pathak, Jessica I. Spiltoir, Camilla Höglund, Lauren R. Polstein, Sari Heine-koskinen, Jari Rossi, Charles A. Gersbach, Chandra L. Tucker
    Abstract:

    : Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. In previous work, we used Cryptochrome 2 (CRY2) and CIB1, Arabidopsis proteins that interact upon light illumination, to regulate transcription with light in yeast. While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus. The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators. We used this knowledge to develop two different approaches to regulate cellular protein levels with light: a system using CRY2 and CIB1 to induce protein expression with light through stimulation of transcription, and a system using CRY2 and a LOV-fused degron to simultaneously block transcription and deplete protein levels with light. These tools will allow precise, bi-directional control of gene expression in a variety of cells and model systems.

  • optimized second generation cry2 cib dimerizers and photoactivatable cre recombinase
    Nature Chemical Biology, 2016
    Co-Authors: Amir Taslimi, Gopal P. Pathak, Robert M Hughes, Brian D Zoltowski, Jose G Miranda, Chandra L. Tucker
    Abstract:

    Arabidopsis thaliana Cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use in controlling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2-CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2-CIB system by identifying versions of CRY2-CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates an α13-α14 turn motif within plant CRYs whose perturbation alters signaling-state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second-generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range, allowing robust recombination following exposure to a single, brief pulse of light.

  • An optimized optogenetic clustering tool for probing protein interaction and function
    Nature communications, 2014
    Co-Authors: Amir Taslimi, Justin D. Vrana, Daniel Chen, Sofya Borinskaya, Bruce J. Mayer, Matthew J. Kennedy, Chandra L. Tucker
    Abstract:

    The Arabidopsis photoreceptor Cryptochrome 2 (CRY2) was previously used as an optogenetic module, allowing spatiotemporal control of cellular processes with light. Here we report the development of a new CRY2-derived optogenetic module, 'CRY2olig', which induces rapid, robust, and reversible protein oligomerization in response to light. Using this module, we developed a novel protein interaction assay, Light-Induced Co-clustering, that can be used to interrogate protein interaction dynamics in live cells. In addition to use probing protein interactions, CRY2olig can also be used to induce and reversibly control diverse cellular processes with spatial and temporal resolution. Here we demonstrate disrupting clathrin-mediated endocytosis and promoting Arp2/3-mediated actin polymerization with light. These new CRY2-based approaches expand the growing arsenal of optogenetic strategies to probe cellular function.

Chentao Lin - One of the best experts on this subject based on the ideXlab platform.

  • blue light dependent interaction of cry2 with spa1 regulates cop1 activity and floral initiation in arabidopsis
    Current Biology, 2011
    Co-Authors: Zecheng Zuo, Hongtao Liu, Bin Liu, Xuanming Liu, Chentao Lin
    Abstract:

    Cryptochromes are blue light receptors that mediate light regulation of gene expression in all major evolution lineages, but the molecular mechanism underlying Cryptochrome signal transduction remains not fully understood. It has been reported that Cryptochromes suppress activity of the multifunctional E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) to regulate gene expression in response to blue light. But how plant Cryptochromes mediate light suppression of COP1 activity remains unclear. We report here that Arabidopsis CRY2 (Cryptochrome 2) undergoes blue light-dependent interaction with the COP1-interacting protein SUPPRESSOR OF PHYTOCHROME A 1 (SPA1). We demonstrate that SPA1 acts genetically downstream from CRY2 to mediate blue light suppression of the COP1-dependent proteolysis of the flowering-time regulator CONSTANS (CO). We further show that blue light-dependent CRY2-SPA1 interaction stimulates CRY2-COP1 interaction. These results reveal for the first time a wavelength-specific mechanism by which a Cryptochrome photoreceptor mediates light regulation of protein degradation to modulate developmental timing in Arabidopsis.

  • derepression of the nc80 motif is critical for the photoactivation of arabidopsis cry2
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Dror Shalitin, Maskit Maymon, John Klejnot, Hongyun Yang, Xiaoying Zhao, Xuanming Liu, Javier Lopez, Krishnaprasad T Bendehakkalu, Chentao Lin
    Abstract:

    Cryptochromes are blue light receptors that regulate photomorphogenesis in plants and the circadian clock in animals and plants. Arabidopsis Cryptochrome 2 (CRY2) mediates blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation. CRY2 undergoes blue light-induced phosphorylation, which was hypothesized to be associated with CRY2 photoactivation. To further investigate how light activates CRY2, we analyzed the physiological activities and phosphorylation of various CRY2 fusion proteins in transgenic plants. Our results showed that an 80-residue motif, referred to as NC80, was sufficient to confer the physiological function of CRY2. The GUS-NC80 fusion protein expressed in transgenic plants is constitutively active but unphosphorylated, suggesting that the blue light-induced CRY2 phosphorylation causes a conformational change to derepress the NC80 motif. Consistent with this hypothesis, the CRY2 C-terminal tail was found to be required for the blue light-induced CRY2 phosphorylation but not for the CRY2 activity. We propose that the PHR domain and the C-terminal tail of the unphosphorylated CRY2 form a “closed” conformation to suppress the NC80 motif in the absence of light. In response to blue light, the C-terminal tail of CRY2 is phosphorylated and electrostatically repelled from the surface of the PHR domain to form an “open” conformation, resulting in derepression of the NC80 motif and signal transduction to trigger photomorphogenic responses.

  • regulation of photoperiodic flowering by arabidopsis photoreceptors
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Todd C. Mockler, Hongyun Yang, Dhavan Parikh, Yingchia Cheng, Sarah Dolan, Chentao Lin
    Abstract:

    Photoperiodism is a day-length-dependent seasonal change of physiological or developmental activities that is widely found in plants and animals. Photoperiodic flowering in plants is regulated by photosensory receptors including the red/far-red light-receptor phytochromes and the blue/UV-A light-receptor Cryptochromes. However, the molecular mechanisms underlying the specific roles of individual photoreceptors have remained poorly understood. Here, we report a study of the day-length-dependent response of Cryptochrome 2 (cry2) and phytochrome A (phyA) and their role as day-length sensors in Arabidopsis. The protein abundance of cry2 and phyA showed a diurnal rhythm in plants grown in short-day but not in plants grown in long-day. The short-day-specific diurnal rhythm of cry2 is determined primarily by blue light-dependent cry2 turnover. Consistent with a proposition that cry2 and phyA are the major day-length sensors in Arabidopsis, we show that phyA mediates far-red light promotion of flowering with modes of action similar to that of cry2. Based on these results and a finding that the photoperiodic responsiveness of plants depends on light quality, a model is proposed to explain how individual phytochromes and Cryptochromes work together to confer photoperiodic responsiveness in Arabidopsis.

  • the arabidopsis blue light receptor Cryptochrome 2 is a nuclear protein regulated by a blue light dependent post transcriptional mechanism
    Plant Journal, 1999
    Co-Authors: Hongwei Guo, Hien Duong, Chentao Lin
    Abstract:

    Cryptochrome 2 is a flavin-type blue light receptor mediating floral induction in response to photoperiod and a blue light-induced hypocotyl growth inhibition. cry2 is required for the elevated expression of the flowering-time gene CO in response to long-day photoperiods, but the molecular mechanism underlying the function of cry2 is not clear. The carboxyl domain of cry2 bears a basic bipartite nuclear localization signal, and the cry2 protein was co-fractionated with the nucleus. Analysis of transgenic plants expressing a fusion protein of CRY2 and the reporter enzyme GUS (GUS-CRY2) indicated that the GUS-CRY2 fusion protein accumulated in the nucleus of transgenic plants grown in dark or light. The C-terminal domain of cry2 that contains the basic bipartite nuclear localization signal was sufficient to confer nuclear localization of the fusion protein. Phenotypic analysis of transgenic plants expressing the fusion protein GUS-CRY2 demonstrated that GUS-CRY2 acts as a functional photoreceptor in vivo, mediating the blue light-induced inhibition of hypocotyl elongation. These results strongly suggest that cry2 is a nuclear protein. Although no obvious light regulation was found for the nuclear compartmentation of GUS-CRY2 fusion protein, the abundance of GUS-CRY2 was regulated by blue light in a way similar to that of cry2.

  • regulation of flowering time by arabidopsis photoreceptors
    Science, 1998
    Co-Authors: Hongwei Guo, Hongyun Yang, Todd C. Mockler, Chentao Lin
    Abstract:

    The shift in plants from vegetative growth to floral development is regulated by red–far-red light receptors (phytochromes) and blue–ultraviolet A light receptors (Cryptochromes). A mutation in the Arabidopsis thaliana CRY2 gene encoding a blue-light receptor apoprotein (CRY2) is allelic to the late-flowering mutant, fha . Flowering in cry2/fha mutant plants is only incompletely responsive to photoperiod. Cryptochrome 2 (cry2) is a positive regulator of the flowering-time gene CO , the expression of which is regulated by photoperiod. Analysis of flowering in cry2 and phyB mutants in response to different wavelengths of light indicated that flowering is regulated by the antagonistic actions of phyB and cry2.

Ute Hoecker - One of the best experts on this subject based on the ideXlab platform.

  • Cryptochrome 2 competes with cop1 substrates to repress cop1 ubiquitin ligase activity during arabidopsis photomorphogenesis
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Jathish Ponnu, Tabea Riedel, Eva Penner, Andrea Schrader, Ute Hoecker
    Abstract:

    In plants, the Cryptochrome photoreceptors suppress the activity of the COP1/SPA ubiquitin ligase to initiate photomorphogenesis in blue light. Both CRY1 and CRY2 interact with the COP1/SPA complex in a blue light-dependent manner. The mechanisms underlying the inhibition of COP1 activity through direct interactions with photoactivated CRYs are not fully understood. Here we tested the hypothesis that CRY2 inhibits COP1 by displacing the degradation substrates from COP1. To this end, we analyzed the role of a conserved valine-proline (VP) motif in the C-terminal domain of CRY2 (CCT2), which resembles the core COP1-WD40-binding sequences present in the substrates of COP1. We show that the VP motif in CRY2 is essential for the interaction of CRY2 with COP1 in yeast two-hybrid assays and in planta Mutations in the VP motif of CRY2 abolished the CRY2 activity in photomorphogenesis, indicating the importance of VP. The interaction between COP1 and its VP-containing substrate PAP2 was prevented in the presence of coexpressed CRY2, but not in the presence of CRY2 carrying a VP mutation. Thus, since both PAP2 and CRY2 engage VP motifs to bind to COP1, these results demonstrate that CRY2 outcompetes PAP2 for binding to COP1. We further found that the previously unknown interaction between SPA1-WD and CCT2 occurs via the VP motif in CRY2, suggesting structural similarities in the VP-binding pockets of COP1-WD40 and SPA1-WD40 domains. A VP motif present in CRY1 is also essential for binding to COP1. Thus, CRY1 and CRY2 might share this mechanism of COP1 inactivation.

  • photoreceptor specificity in the light induced and cop1 mediated rapid degradation of the repressor of photomorphogenesis spa2 in arabidopsis
    PLOS Genetics, 2015
    Co-Authors: Song Chen, Niels Lory, Johannes Stauber, Ute Hoecker
    Abstract:

    The Arabidopsis COP1/SPA E3 ubiquitin ligase is a key negative regulator that represses light signaling in darkness by targeting transcription factors involved in the light response for degradation. The COP1/SPA complex consists of COP1 and members of the four-member SPA protein family (SPA1-SPA4). Genetic analysis indicated that COP1/SPA2 function is particularly strongly repressed by light when compared to complexes carrying the other three SPAs, thereby promoting a light response after exposure of plants to extremely low light. Here, we show that the SPA2 protein is degraded within 5–15 min after exposure of dark-grown seedlings to a pulse of light. Phytochrome photoreceptors are required for the rapid degradation of SPA2 in red, far-red and also in blue light, whereas Cryptochromes are not involved in the rapid, blue light-induced reduction in SPA2 protein levels. These results uncover a photoreceptor-specific mechanism of light-induced inhibition of COP1/SPA2 function. Phytochrome A (phyA) is required for the severe blue light responsiveness of spa triple mutants expressing only SPA2, thus confirming the important role of phyA in downregulating SPA2 function in blue light. In blue light, SPA2 forms a complex with Cryptochrome 1 (cry1), but not with Cryptochrome 2 (cry2) in vivo, indicating that the lack of a rapid blue light response of the SPA2 protein is only in part caused by a failure to interact with Cryptochromes. Since SPA1 interacts with both cry1 and cry2, these results provide first molecular evidence that the light-regulation of different SPA proteins diverged during evolution. SPA2 degradation in the light requires COP1 and the COP1-interacting coiled-coil domain of SPA2, supporting that SPA2 is ubiquitinated by COP1. We propose that light perceived by phytochromes causes a switch in the ubiquitination activity of COP1/SPA2 from ubiquitinating downstream substrates to ubiquitinating SPA2, which subsequently causes a repression of COP1/SPA2 function.

Hongyun Yang - One of the best experts on this subject based on the ideXlab platform.

  • photoexcited cry2 interacts with cib1 to regulate transcription and floral initiation in arabidopsis
    Science, 2008
    Co-Authors: Xuhong Yu, Kunwu Li, John Klejnot, Hongyun Yang, Dominique N Lisiero
    Abstract:

    Cryptochromes (CRY) are photolyase-like blue-light receptors that mediate light responses in plants and animals. How plant Cryptochromes act in response to blue light is not well understood. We report here the identification and characterization of the Arabidopsis CIB1 (Cryptochrome-interacting basic-helix-loop-helix) protein. CIB1 interacts with CRY2 (Cryptochrome 2) in a blue light–specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 binds to G box (CACGTG) in vitro with a higher affinity than its interaction with other E-box elements (CANNTG). However, CIB1 stimulates FT messenger RNA expression, and it interacts with chromatin DNA of the FT gene that possesses various E-box elements except G box. We propose that the blue light–dependent interaction of Cryptochrome(s) with CIB1 and CIB1-related proteins represents an early photoreceptor signaling mechanism in plants.

  • arabidopsis Cryptochrome 2 completes its posttranslational life cycle in the nucleus
    The Plant Cell, 2007
    Co-Authors: Xuhong Yu, Maskit Maymon, Dror Shalitin, John Klejnot, Hongyun Yang, Xiaoying Zhao, Javier Lopez
    Abstract:

    CRY2 is a blue light receptor regulating light inhibition of hypocotyl elongation and photoperiodic flowering in Arabidopsis thaliana. The CRY2 protein is found primarily in the nucleus, and it is known to undergo blue light–dependent phosphorylation and degradation. However, the subcellular location where CRY2 exerts its function or undergoes blue light–dependent phosphorylation and degradation remains unclear. In this study, we analyzed the function and regulation of conditionally nuclear-localized CRY2. Our results show that CRY2 mediates blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation in the nucleus. Consistent with this result and a hypothesis that blue light–dependent phosphorylation is associated with CRY2 function, we demonstrate that CRY2 undergoes blue light–dependent phosphorylation in the nucleus. CRY2 phosphorylation is required for blue light–dependent CRY2 degradation, but only a limited quantity of CRY2 is phosphorylated at any given moment in seedlings exposed to blue light, which explains why continuous blue light illumination is required for CRY2 degradation. Finally, we showed that CRY2 is ubiquitinated in response to blue light and that ubiquitinated CRY2 is degraded by the 26S proteasome in the nucleus. These findings demonstrate that a photoreceptor can complete its posttranslational life cycle (from protein modification, to function, to degradation) inside the nucleus.

  • derepression of the nc80 motif is critical for the photoactivation of arabidopsis cry2
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Dror Shalitin, Maskit Maymon, John Klejnot, Hongyun Yang, Xiaoying Zhao, Xuanming Liu, Javier Lopez, Krishnaprasad T Bendehakkalu, Chentao Lin
    Abstract:

    Cryptochromes are blue light receptors that regulate photomorphogenesis in plants and the circadian clock in animals and plants. Arabidopsis Cryptochrome 2 (CRY2) mediates blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation. CRY2 undergoes blue light-induced phosphorylation, which was hypothesized to be associated with CRY2 photoactivation. To further investigate how light activates CRY2, we analyzed the physiological activities and phosphorylation of various CRY2 fusion proteins in transgenic plants. Our results showed that an 80-residue motif, referred to as NC80, was sufficient to confer the physiological function of CRY2. The GUS-NC80 fusion protein expressed in transgenic plants is constitutively active but unphosphorylated, suggesting that the blue light-induced CRY2 phosphorylation causes a conformational change to derepress the NC80 motif. Consistent with this hypothesis, the CRY2 C-terminal tail was found to be required for the blue light-induced CRY2 phosphorylation but not for the CRY2 activity. We propose that the PHR domain and the C-terminal tail of the unphosphorylated CRY2 form a “closed” conformation to suppress the NC80 motif in the absence of light. In response to blue light, the C-terminal tail of CRY2 is phosphorylated and electrostatically repelled from the surface of the PHR domain to form an “open” conformation, resulting in derepression of the NC80 motif and signal transduction to trigger photomorphogenic responses.

  • regulation of photoperiodic flowering by arabidopsis photoreceptors
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Todd C. Mockler, Hongyun Yang, Dhavan Parikh, Yingchia Cheng, Sarah Dolan, Chentao Lin
    Abstract:

    Photoperiodism is a day-length-dependent seasonal change of physiological or developmental activities that is widely found in plants and animals. Photoperiodic flowering in plants is regulated by photosensory receptors including the red/far-red light-receptor phytochromes and the blue/UV-A light-receptor Cryptochromes. However, the molecular mechanisms underlying the specific roles of individual photoreceptors have remained poorly understood. Here, we report a study of the day-length-dependent response of Cryptochrome 2 (cry2) and phytochrome A (phyA) and their role as day-length sensors in Arabidopsis. The protein abundance of cry2 and phyA showed a diurnal rhythm in plants grown in short-day but not in plants grown in long-day. The short-day-specific diurnal rhythm of cry2 is determined primarily by blue light-dependent cry2 turnover. Consistent with a proposition that cry2 and phyA are the major day-length sensors in Arabidopsis, we show that phyA mediates far-red light promotion of flowering with modes of action similar to that of cry2. Based on these results and a finding that the photoperiodic responsiveness of plants depends on light quality, a model is proposed to explain how individual phytochromes and Cryptochromes work together to confer photoperiodic responsiveness in Arabidopsis.

  • Regulation of Arabidopsis Cryptochrome 2 by blue-light-dependent phosphorylation.
    Nature, 2002
    Co-Authors: Dror Shalitin, Maskit Maymon, Hongyun Yang, Todd C. Mockler, Garry C. Whitelam
    Abstract:

    Cryptochromes are blue/ultraviolet-A light receptors that mediate various light responses in plants and animals1,2. But the initial photochemical reaction of Cryptochrome is still unclear. For example, although most photoreceptors are known to undergo light-dependent protein modification such as phosphorylation3,4, no blue-light dependent phosphorylation has been reported for a Cryptochrome. Arabidopsis Cryptochrome 2 (cry2) mediates light regulation of seedling development and photoperiodic flowering5,6. The physiological activity and cellular level of cry2 protein are light-dependent5,6,7,8, and protein–protein interactions are important for cry2 function9,10. Here we report that cry2 undergoes a blue-light-dependent phosphorylation, and that cry2 phosphorylation is associated with its function and regulation. Our results suggest that, in the absence of light, cry2 remains unphosphorylated, inactive and stable; absorption of blue light induces the phosphorylation of cry2, triggering photomorphogenic responses and eventually degradation of the photoreceptor.