Millar, A. J., Short, S. R., Chua, N. -H. & Kay, S. A. A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell 4, 1075-1087

Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York 10021-6399.
The Plant Cell (Impact Factor: 9.34). 10/1992; 4(9):1075-87. DOI: 10.1105/tpc.4.9.1075
Source: PubMed


A 320-bp fragment of the Arabidopsis cab2 promoter is sufficient to mediate transcriptional regulation by both phytochrome and the circadian clock. We fused this promoter fragment to the firefly luciferase (Luc) gene to create a real-time reporter for regulated gene expression in intact plants. Cab2::Luc transcript accumulated in the expected patterns and luciferase activity was closely correlated to cab2::Luc mRNA abundance in both etiolated and green seedlings. The concentration of the bulk of luciferase protein did not reflect these patterns but maintained a relatively constant level, implying that a post-translational mechanism(s) leads to the high-amplitude regulation of luciferase activity. We used a low-light video imaging system to establish that luciferase bioluminescence in vivo accurately reports the temporal and spatial regulation of cab2 transcription in single seedlings. The unique qualities of the firefly luciferase system allowed us to monitor regulated gene expression in real time in individual multicellular organisms. This noninvasive marker for temporal regulation at the molecular level constitutes a circadian phenotype, which may be used to isolate mutants in the circadian clock.

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Available from: Steve A Kay, Oct 03, 2015
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    • "In contrast to fluorescence, luciferase does not require light irradiation for excitation and is thus neither subject to photobleaching nor phototoxicity. Hence, luciferase is a suitable reporter for long term in vivo measurements that are extensively used to record and monitor circadian clock activity in bacteria, fungi, plants, flies and mammals [3], [6], [7], [8], [9]. "
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    ABSTRACT: We show that firefly luciferase is a stable protein when expressed at 25°C in Neurospora, which limits its use as transcription reporter. We created a short-lived luciferase by fusing a PEST signal to its C-terminus (LUC-PEST) and applied the LUC-PEST reporter system to record in vivo transcription dynamics associated with the Neurospora circadian clock and its blue-light photosensory system over the course of several days. We show that the tool is suitable to faithfully monitor rapid, but also subtle changes in transcription in a medium to high throughput format.
    PLoS ONE 12/2013; 8(12):e83660. DOI:10.1371/journal.pone.0083660 · 3.23 Impact Factor
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    • "We note that the former has been successfully used to measure period, phase and amplitude of 150 accessions in one clock study [85] [102]. However, these methods are not as accurate and precise nor as efficient, as high-throughput assay for measuring rhythms in plant that utilizes clock-controlled promoter luciferase (CCP::LUC) reporter fusions [103]. One caveat with luciferase based assays is that it is a transgenic approach that requires transformation of suitable CCP::LUC constructs. "
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    ABSTRACT: Circadian clocks are ubiquitous mechanisms that provide an adaptive advantage by predicting subsequent environmental changes. In the model plant Arabidopsis thaliana (Arabidopsis), our understanding of the complex genetic network among clock components has considerably increased during these past years. Modeling has predicted the possibility of additional component to systematically and functionally complete the clock system. Mutagenesis screens have in the past been successfully employed to detect such novel components. With the advancement in sequencing technologies and improvements in statistical approaches, the extensive natural variation present in Arabidopsis accessions has emerged as a powerful alternative in functional gene discovery. In this review article, we review the previous efforts in mapping natural alleles affecting various clock parameters and will discuss further potentials of such natural-variation studies in physiological and ecological contexts.
    Seminars in Cell and Developmental Biology 04/2013; 24(5). DOI:10.1016/j.semcdb.2013.03.006 · 6.27 Impact Factor
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    • "To identify factors that confer trans-activation of the AtHAK5 promoter, we constructed a promoter::reporter fusion with the AtHAK5 promoter and a luciferase reporter gene (LUC) that can be monitored using nondestructive imaging (Millar et al., 1992). A stable homozygous AtHAK5pro:LUC reporter line that was responsive to low potassium was transformed with the pDKS 2–7 vector (Turk et al., 2005) to create a population of randomly integrated activation-tagged lines. "
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    ABSTRACT: Plants respond to low-nutrient conditions through metabolic and morphology changes that increase their ability to survive and grow. The transcription factor RAP2.11 was identified as a component in the response to low potassium through regulation of the high-affinity K(+) uptake transporter AtHAK5 and other components of the low-potassium signal transduction pathway. RAP2.11 was identified through the activation tagging of Arabidopsis lines that contained a luciferase marker driven by the AtHAK5 promoter that is normally only induced by low potassium. This factor bound to a GCC-box of the AtHAK5 promoter in vitro and in vivo. Transcript profiling revealed that a large number of genes were up-regulated in roots by RAP2.11 overexpression. Many regulated genes were identified to be in functional categories that are important in low-K(+) signaling. These categories included ethylene signaling, reactive oxygen species production, and calcium signaling. Promoter regions of the up-regulated genes were enriched in the GCCGGC motif also contained in the AtHAK5 promoter. These results suggest that RAP2.11 regulates AtHAK5 expression under low-K(+) conditions and also contributes to a coordinated response to low-potassium conditions through the regulation of other genes in the low-K(+) signaling cascade.
    Molecular Plant 03/2012; 5(5):1042-57. DOI:10.1093/mp/sss003 · 6.34 Impact Factor
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