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      PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length

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          Abstract

          Seasonal reproduction in many organisms requires detection of day length. This is achieved by integrating information on the light environment with an internal photoperiodic time‐keeping mechanism. Arabidopsis thaliana promotes flowering in response to long days ( LDs), and CONSTANS ( CO) transcription factor represents a photoperiodic timer whose stability is higher when plants are exposed to light under LDs. Here, we show that PSEUDO RESPONSE REGULATOR ( PRR) proteins directly mediate this stabilization. PRRs interact with and stabilize CO at specific times during the day, thereby mediating its accumulation under LDs. PRR‐mediated stabilization increases binding of CO to the promoter of FLOWERING LOCUS T ( FT ), leading to enhanced FT transcription and early flowering under these conditions. PRRs were previously reported to contribute to timekeeping by regulating CO transcription through their roles in the circadian clock. We propose an additional role for PRRs in which they act upon CO protein to promote flowering, directly coupling information on light exposure to the timekeeper and allowing recognition of LDs.

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          Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

          Background Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments. Results In this report, we present a new procedure that we call the Tape-Arabidopsis Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis) are used to make a "Tape-Arabidopsis Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC) to investigate protein-protein interactions in vivo, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr. Conclusion The protoplasts generated by this new Tape-Arabidopsis Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time.
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            Targeted destabilization of HY5 during light-regulated development of Arabidopsis.

            Arabidopsis seedlings display contrasting developmental patterns depending on the ambient light. Seedlings grown in the light develop photomorphogenically, characterized by short hypocotyls and expanded green cotyledons. In contrast, seedlings grown in darkness become etiolated, with elongated hypocotyls and dosed cotyledons on an apical hook. Light signals, perceived by multiple photoreceptors and transduced to downstream regulators, dictate the extent of photomorphogenic development in a quantitative manner. Two key downstream components, COP1 and HY5, act antagonistically in regulating seedling development. HY5 is a bZIP transcription factor that binds directly to the promoters of light-inducible genes, promoting their expression and photomorphogenic development. COP1 is a RING-finger protein with WD-40 repeats whose nuclear abundance is negatively regulated by light. COP1 interacts directly with HY5 in the nucleus to regulate its activity negatively. Here we show that the abundance of HY5 is directly correlated with the extent of photomorphogenic development, and that the COP1-HY5 interaction may specifically target HY5 for proteasome-mediated degradation in the nucleus.
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              FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis.

              Precise timing of CONSTANS (CO) gene expression is necessary for day-length discrimination for photoperiodic flowering. The FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and GIGANTEA (GI) proteins regulate CO transcription in Arabidopsis. We demonstrate that FKF1 and GI proteins form a complex in a blue-light-dependent manner. The timing of this interaction regulates the timing of daytime CO expression. FKF1 function is dependent on GI, which interacts with a CO repressor, CYCLING DOF FACTOR 1 (CDF1), and controls CDF1 stability. GI, FKF1, and CDF1 proteins associate with CO chromatin. Thus, the FKF1-GI complex forms on the CO promoter in late afternoon to regulate CO expression, providing a mechanistic view of how the coincidence of light with circadian timing regulates photoperiodic flowering.
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                Author and article information

                Contributors
                coupland@mpipz.mpg.de
                Journal
                EMBO J
                EMBO J
                10.1002/(ISSN)1460-2075
                EMBJ
                embojnl
                The EMBO Journal
                John Wiley and Sons Inc. (Hoboken )
                0261-4189
                1460-2075
                07 March 2017
                03 April 2017
                07 March 2017
                : 36
                : 7 ( doiID: 10.1002/embj.v36.7 )
                : 904-918
                Affiliations
                [ 1 ] Department of Plant Developmental BiologyMax Planck Institute for Plant Breeding Research CologneGermany
                [ 2 ]Present address: International Christian University Mitaka, TokyoJapan
                [ 3 ]Present address: Biotechnology Center in Southern Taiwan (BCST) TainanTaiwan
                [ 4 ]Present address: Agricultural Biotechnology Research CenterAcademia Sinica Nankang, TaipeiTaiwan
                Author notes
                [*] [* ]Corresponding author. Tel: +49 221 5062 205; Fax: +49 221 5062 207; E‐mail: coupland@ 123456mpipz.mpg.de
                Author information
                http://orcid.org/0000-0001-6988-4172
                Article
                EMBJ201693907
                10.15252/embj.201693907
                5376961
                28270524
                5476d157-6be6-494b-9d09-c32146bffb12
                © 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license

                This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs 4.0 License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

                History
                : 20 January 2016
                : 31 January 2017
                : 01 February 2017
                Page count
                Figures: 13, Tables: 0, Pages: 15, Words: 12976
                Funding
                Funded by: Deutsche Forschungsgemeinschaft (DFG)
                Award ID: SFB635
                Funded by: Max Planck Society
                Funded by: European Research Council
                Award ID: Hylife
                Categories
                Article
                Articles
                Custom metadata
                2.0
                embj201693907
                03 April 2017
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.0.9 mode:remove_FC converted:03.04.2017

                Molecular biology
                arabidopsis,circadian clock,constitutive photomorphogenic 1,photoperiodic flowering,pseudo response regulator,plant biology,post-translational modifications, proteolysis & proteomics,transcription

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