BioModelsAndrew J MillarNon-curatedotherL3V1https://www.ebi.ac.uk/biomodels/MODEL2512170001falseBioModelsSBMLModelsU2019 5 Urquiza 2025 Arabidopsis circadian clock2025MODEL2512170001Uriel Urquiza-García, Nacho Molina, Karen J Halliday, Andrew J MillarUriel Urquiza-García10.1038/s44320-025-00086-5, 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 (K_d). We estimate the same K_d from multiple results in vitro, extending the method to any promoter sequence. The detailed models simulated the K_d 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.. 4, 21. Centre for Engineering Biology and School of Biological Sciences, C. H. Waddington Building, University of Edinburgh, King's Buildings, Edinburgh, EH9 3BF, UK.andrew.millar@ed.ac.uk10.1038/s44320-025-00086-5Institute of Molecular Plant Sciences, University of EdinburghfalseU2019_5 - Urquiza 2025 Arabidopsis circadian clockSBML L3V1 format model U2019.5 of the clock gene circuit in the plant Arabidopsis thaliana, inspired by the P2011 model, published in Pokhilko et al Mol Syst Biol 2012 (Biomodels BD000..0412). The U2019 version uses very similar interactions to P2011, where the combined LHY/CCA1 component indirectly activates the wave of PRR gene expression, whereas the U2020 version uses both indirect activation and inter-PRR repression. This .5 version has parameters scaled to yield absolute units for both RNAs and proteins (copy number per cell), whereas the .3 versions of each model are scaled to match only the absolute RNA level data (BioModels MODEL2212200001 and MODEL2212200002). SBML files were verified using COPASI software. The full process is described in Urquiza Garcia et al. Molecular Systems Biology, 2025.2025-12-172026-09-182025-12-17MODEL2512170001MODEL251217000110.1038/s44320-025-00086-5