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      Multiple bHLH Proteins form Heterodimers to Mediate CRY2-Dependent Regulation of Flowering-Time in Arabidopsis

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          Abstract

          Arabidopsis thaliana cryptochrome 2 (CRY2) mediates light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with CRY2 in response to blue light to activate the transcription of FT ( Flowering Locus T). In vitro, CIB1 binds to the canonical E-box (CACGTG, also referred to as G-box) with much higher affinity than its interaction with non-canonical E-box (CANNTG) DNA sequences. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the non-canonical E-box sequences. Here, we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. Our genetic analysis demonstrates that CIB1, CIB2, CIB4, and CIB5 act redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated flowering. More importantly, CIB1 and other CIBs proteins form heterodimers, and some of the heterodimers have a higher binding affinity than the CIB homodimers to the non-canonical E-box in the in vitro DNA-binding assays. This result explains why in vitro CIB1 and other CIBs bind to the canonical E-box (G-box) with a higher affinity, whereas they are all associated with the non-canonical E-boxes at the FT promoter in vivo. Consistent with the hypothesis that different CIB proteins play similar roles in the CRY2-midiated blue light signaling, the expression of CIB proteins is regulated specifically by blue light. Our study demonstrates that CIBs function redundantly in regulating CRY2-dependent flowering, and that different CIBs form heterodimers to interact with the non-canonical E-box DNA in vivo.

          Author Summary

          Arabidopsis thaliana blue light receptor cryptochromes (CRYs) mediate light control of flowering time by interacting with CIB1 (CRY2-interacting bHLH1) in response to blue light. However, it remains unclear how the blue light-dependent CRY2-CIB1 interaction affects the FT transcription. We report here that in addition to CIB1, CRY2 also interact with CIB1 related bHLH proteins, CIBs. These CIBs act redundantly with CIB1 to activate the transcription of FT and flowering. More importantly, CIB1 and the CIBs can form heterodimers and some of those heterodimers have a higher binding affinity to the non-canonical E-box, although their homodimers all prefer canonical E-box (G-box), so they can bind to the non-canonical E-Box sequences of the FT promoter. This is the first example in plants that heterodimerization of bHLH can change the DNA binding affinity or specificity. CIB proteins are involved in blue light signaling and they are specifically stabilized by blue light.

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          Most cited references42

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          Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

          Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.
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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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              Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.

              We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                October 2013
                October 2013
                10 October 2013
                : 9
                : 10
                : e1003861
                Affiliations
                [1 ]National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
                [2 ]Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California, United States of America
                University of California Berkeley and USDA/ARS, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: HL CL. Performed the experiments: YL XL KL HL. Analyzed the data: YL XL HL. Contributed reagents/materials/analysis tools: KL. Wrote the paper: HL.

                Article
                PGENETICS-D-13-01073
                10.1371/journal.pgen.1003861
                3794922
                24130508
                06b92b13-29e5-44bb-883f-e6a1786114cb
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 23 April 2013
                : 20 August 2013
                Page count
                Pages: 12
                Funding
                This work is supported by National Natural Science Foundation of China (31270285 and 31322006), the Hundred Talents Program of Chinese Academy of Sciences, Shanghai Pujiang Program (12PJ1409800), and also US National Institutes of Health (GM56265 to CL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article

                Genetics
                Genetics

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