2 items tagged with 'Luciferase'.
Abstract (Expand)
Protein data over circadian time scale is scarce for clock transcription factors. Further work in this direction is required for refining quantitative clock models. However, gathering highly resolved … dynamics of low-abundance transcription factors has been a major challenge in the field. In this work we provide a new tool that could help this major issue. Bioluminescence is an important tool for gathering data on circadian gene expression. It allows data collection over extended time periods for low signal levels, thanks to a large signal-to-noise ratio. However, the main reporter so far used, firefly luciferase (FLUC), presents some disadvantages for reporting total protein levels. For example, the rapid, post-translational inactivation of this luciferase will result in underestimation of protein numbers. A more stable reporter protein could in principle tackle this issue. We noticed that NanoLUC might fill this gap, given its reported brightness and the stability of both enzyme and substrate. However, no data in plant systems on the circadian time scale had been reported.
Authors: Uriel Urquiza-García, Andrew J. Millar
Date Published: 1st Dec 2019
Publication Type: Journal
DOI: 10.1186/s13007-019-0454-4
Citation: Plant Methods 15(1),68
Created: 28th Aug 2024 at 17:57, Last updated: 28th Aug 2024 at 17:59
Abstract (Expand)
The circadian clocks that drive daily rhythms in animals are tightly coupled among the cells of some tissues. The coupling profoundly affects cellular rhythmicity and is central to contemporary … understanding of circadian physiology and behavior. In contrast, studies of the clock in plant cells have largely ignored intercellular coupling, which is reported to be very weak or absent. We used luciferase reporter gene imaging to monitor circadian rhythms in leaves of Arabidopsis thaliana plants, achieving resolution close to the cellular level. Leaves grown without environmental cycles for up to 3 wk reproducibly showed spatiotemporal waves of gene expression consistent with intercellular coupling, using several reporter genes. Within individual leaves, different regions differed in phase by up to 17 h. A broad range of patterns was observed among leaves, rather than a common spatial distribution of circadian properties. Leaves exposed to light-dark cycles always had fully synchronized rhythms, which could desynchronize rapidly. After 4 d in constant light, some leaves were as desynchronized as leaves grown without any rhythmic input. Applying light-dark cycles to such a leaf resulted in full synchronization within 2-4 d. Thus, the rhythms of all cells were coupled to external light-dark cycles far more strongly than the cellular clocks were coupled to each other. Spontaneous desynchronization under constant conditions was limited, consistent with weak intercellular coupling among heterogeneous clocks. Both the weakness of coupling and the heterogeneity among cells are relevant to interpret molecular studies and to understand the physiological functions of the plant circadian clock.
Authors: B. Wenden, D. L. Toner, S. K. Hodge, R. Grima, A. J. Millar
Date Published: 13th Apr 2012
Publication Type: Not specified
PubMed ID: 22496591
Citation: Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6757-62. doi: 10.1073/pnas.1118814109. Epub 2012 Apr 10.
Created: 10th Mar 2017 at 16:19, Last updated: 8th Dec 2022 at 17:26