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4 Publications visible to you, out of a total of 4
Abundant clock proteins point to missing molecular regulation in the plant circadian clock

Abstract (Expand)

Understanding the biochemistry behind whole-organism traits such as flowering time is a longstanding challenge, where mathematical models are critical. Very few models of plant gene circuits use the … absolute units required for comparison to biochemical data. We refactor two detailed models of the plant circadian clock from relative to absolute units. Using absolute RNA quantification, a simple model predicted abundant clock protein levels in Arabidopsis thaliana , up to 100,000 proteins per cell. NanoLUC reporter protein fusions validated the predicted levels of clock proteins in vivo . Recalibrating the detailed models to these protein levels estimated their DNA-binding dissociation constants (Kd). We estimate the same Kd from multiple results in vitro , extending the method to any promoter sequence. The detailed models simulated the Kd range estimated from LUX DNA-binding in vitro but departed from the data for CCA1 binding, pointing to further circadian mechanisms. Our analytical and experimental methods should transfer to understand other plant gene regulatory networks, potentially including the natural sequence variation that contributes to evolutionary adaptation.

Authors: Uriel Urquiza-García, Nacho Molina, Karen J. Halliday, Andrew J. Millar

Date Published: 3rd Sep 2024

Publication Type: Journal

Testing the inferred transcription rates of a dynamic, gene network model in absolute units (in silico Plants 2021)

Abstract (Expand)

The circadian clock coordinates plant physiology and development. Mathematical clock models have provided a rigorous framework to understand how the observed rhythms emerge from disparate, molecular … processes. However, models of the plant clock have largely been built and tested against RNA timeseries data in arbitrary, relative units. This limits model transferability, refinement from biochemical data and applications in synthetic biology. Here, we incorporate absolute mass units into a detailed model of the clock gene network in Arabidopsis thaliana. We re-interpret the established P2011 model, highlighting a transcriptional activator that overlaps the function of REVEILLE 8/LHY-CCA1-LIKE 5. The U2020 model incorporates the repressive regulation of PRR genes, a key feature of the most detailed clock model KF2014, without greatly increasing model complexity. We tested the experimental error distributions of qRT-PCR data calibrated for units of RNA transcripts/cell and of circadian period estimates, in order to link the models to data more appropriately. U2019 and U2020 models were constrained using these data types, recreating previously-described circadian behaviours with RNA metabolic processes in absolute units. To test their inferred rates, we estimated a distribution of observed, transcriptome-wide transcription rates (Plant Empirical Transcription Rates, PETR) in units of transcripts/cell/hour. The PETR distribution and the equivalent degradation rates indicated that the models’ predicted rates are biologically plausible, with individual exceptions. In addition to updated clock models, FAIR data resources and a software environment in Docker, this validation process represents an advance in biochemical realism for models of plant gene regulation.

Authors: Uriel Urquiza Garcia, Andrew J Millar

Date Published: 5th Aug 2021

Publication Type: Journal

Expanding the bioluminescent reporter toolkit for plant science with NanoLUC

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

Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure

Abstract (Expand)

Our understanding of the complex, transcriptional feedback loops in the circadian clock mechanism has depended upon quantitative, timeseries data from disparate sources. We measure clock gene RNA … profiles in Arabidopsis thaliana seedlings, grown with or without exogenous sucrose, or in soil-grown plants and in wild-type and mutant backgrounds. The RNA profiles were strikingly robust across the experimental conditions, so current mathematical models are likely to be broadly applicable in leaf tissue. In addition to providing reference data, unexpected behaviours included co-expression of PRR9 and ELF4, and regulation of PRR5 by GI. Absolute RNA quantification revealed low levels of PRR9 transcripts (peak approx. 50 copies cell(-1)) compared with other clock genes, and threefold higher levels of LHY RNA (more than 1500 copies cell(-1)) than of its close relative CCA1. The data are disseminated from BioDare, an online repository for focused timeseries data, which is expected to benefit mechanistic modelling. One data subset successfully constrained clock gene expression in a complex model, using publicly available software on parallel computers, without expert tuning or programming. We outline the empirical and mathematical justification for data aggregation in understanding highly interconnected, dynamic networks such as the clock, and the observed design constraints on the resources required to make this approach widely accessible.

Authors: A. Flis, A. P. Fernandez, T. Zielinski, V. Mengin, R. Sulpice, K. Stratford, A. Hume, A. Pokhilko, M. M. Southern, D. D. Seaton, H. G. McWatters, M. Stitt, K. J. Halliday, A. J. Millar

Date Published: 16th Oct 2015

Publication Type: Not specified

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