Biology subjects

Millar, A. J.

Publications and source records attributed to Millar, A. J..

5 recordsLinked to original sources

bioRxiv2018

Expanding the bioluminescence reporter toolkit for plant chronobiology with NanoLUC

Bioluminescence has been an important tool for gathering circadian data with the main reporter gene exploited firefly luciferase (LUC). In some circumstances the rapid inactivation of LUC could be disadvantageous, e.g. reporting total protein levels through reporter translational fusions. In the latter scenario the commercially available Nano luciferase (NanoLUC) might offer and advantage, however no data in plant has been provided so far. We tested NanoLUC under different research scenarios were LUC has been used, for example enzyme purification, expression in transient plant systems and in stable transgenic lines. We show that NanoLUC is active in these experimental scenarios. We also created a set of NanoLUC variants for example MBP-NanoLUC-3xFlag-10xHis version of NanoLUC can be easily purified and stable for several days (half-life 37.2 at 4{degrees}C) and can be used for generating calibration curves for quantifying protein as the signal is linear over a large dynamic range. In addition, we show that NanoLUC can report in-planta protein levels on circadian time scale thanks to the stability of furimazine. Therefore, opening the possibility of using NanoLUC for reporting protein dynamics in seedlings. With this new technology, we explored the dynamics of protein BROTHER OF LUX ARRHYTHMO (BOA), which has been suggested in mathematical models to be a rhythmic protein from RNA data. Using an automatic plate-reader, we were able to track BOAp:BOA-NL for an extended period of time by just adding a mix of furimazine with Triton X-100, as it is done with LUC-reporter lines. In our experimental context BOA protein does not present strong oscillatory dynamics similar to what has been reported for Phytocrhome B for which rhythmic accumulation of transcript can be observed while total protein levels remain constant under diurnal conditions. Our results support the use NanoLUC for studying the dynamics of plant proteins for extended period of time under different circumstances.

plant biology↗

bioRxiv2018

A multi-model Framework for the Arabidopsis life cycle

Linking our understanding of biological processes at different scales is a major conceptual challenge in biology, which is aggravated by differences in research methods. Modelling can be a useful approach to consolidating our understanding across traditional research domains. The laboratory model species Arabidopsis thaliana is very widely used to study plant growth processes and has also been tested more recently in eco-physiology and population genetics. However, approaches from crop modelling that might link these domains are rarely applied to Arabidopsis. Here, we combine plant growth models with phenology models from eco-physiology, using the agent-based modelling language Chromar. We introduce a simpler Framework Model of vegetative growth for Arabidopsis, FM-lite. By extending this model to include inflorescence and fruit growth and seed dormancy, we present a whole-life-cycle, multi-model FM-life, which allows us to simulate at the population level in various genotype x environment scenarios. Environmental effects on plant growth distinguish between the simulated life history strategies that were compatible with previously-described Arabidopsis phenology. Our results simulate reproductive success that is founded on the broad range of physiological processes familiar from crop models and suggest an approach to simulate evolution directly in future.\n\nHighlightA whole-life-cycle multi-model for Arabidopsis thaliana combines phenology and physical growth models to explain reproductive success in different genotype x environment scenarios.

plant biology↗

bioRxiv2018

Circadian protein regulation in the green lineage I. A phospho-dawn anticipates light onset before proteins peak in daytime.

Diel regulation of protein levels and protein modification had been less studied than transcript rhythms. Here, we compare transcriptome data under light-dark cycles to partial proteome and phosphoproteome data, assayed using shotgun mass-spectrometry, from the alga Ostreococcus tauri, the smallest free-living eukaryote. 10% of quantified proteins but two-thirds of phosphoproteins were rhythmic. Mathematical modelling showed that light-stimulated protein synthesis can account for the observed clustering of protein peaks in the daytime. Prompted by night-peaking and apparently dark-stable proteins, we also tested cultures under prolonged darkness, where the proteome changed less than under the diel cycle. The dark-stable, prasinophyte-specific proteins were also reported to accumulate when O. tauri formed lipid droplets. In the phosphoproteome, 39% of rhythmic phospho-sites reached peak levels just before dawn. This anticipatory phosphorylation suggests that a clock-regulated phospho-dawn prepares green cells for daytime functions. Acid-directed and proline-directed protein phosphorylation sites were regulated in antiphase, implicating the clock-related, casein kinases 1 and 2 in phase-specific regulation, alternating with the CMGC protein kinase family. Understanding the dynamic phosphoprotein network should be facilitated by the minimal kinome and proteome of O. tauri. The data are available from ProteomeXchange, with identifiers PXD001734, PXD001735 and PXD002909. This submission updates a previous version, posted on bioRxiv on 4th April 2018, as https://www.biorxiv.org/content/10.1101/287862v1 HighlightThe phosphorylation of most protein sites was rhythmic under light-dark cycles, and suggested circadian control by particular kinases. Day-peaking, rhythmic proteins likely reflect light-stimulated protein synthesis in this microalga.

plant biology↗

bioRxiv2018

Mechanistic model of temperature influence on flowering through whole-plant accumulation of FT

We assessed temperature influence on flowering by incorporating temperature-responsive flowering mechanisms across developmental age into an existing model. Temperature influences both the leaf production rate and expression of FLOWERING LOCUS T (FT), a photoperiodic flowering regulator, in leaves. The Arabidopsis Framework Model incorporated temperature influence on leaf growth but ignored the consequences of leaf growth on and direct temperature influence of FT expression. We measured FT production in differently aged leaves and modified the model, adding the mechanistic temperature influence on FT transcription, and linking FT to leaf growth. Our simulations suggest that in long days, the developmental timing (leaf number) at which the reproductive transition occurs is influenced by day length and temperature through FT, while temperature influences the rate of leaf production and the time (in days) the transition occurs. Further, we demonstrated that FT is mainly produced in the first 10 leaves in the Columbia ecotype, and that FT accumulation alone cannot explain flowering in conditions in which flowering is delayed. Our simulations supported our hypotheses that: 1) temperature regulation of FT, accumulated with leaf growth, is a component of thermal time, and 2) incorporating mechanistic temperature regulation of FT can improve model predictions in fluctuating temperatures.

plant biology↗

bioRxiv2017

Linking circadian time to growth rate quantitatively via carbon metabolism

Predicting a multicellular organisms phenotype quantitatively from its genotype is challenging, as genetic effects must propagate across scales. Circadian clocks are intracellular regulators that control temporal gene expression patterns and hence metabolism, physiology and behaviour. Here we explain and predict canonical phenotypes of circadian timing in a multicellular, model organism. We used diverse metabolic and physiological data to combine and extend mathematical models of rhythmic gene expression, photoperiod-dependent flowering, elongation growth and starch metabolism within a Framework Model for the vegetative growth of Arabidopsis thaliana, sharing the model and data files in a structured, public resource. The calibrated model predicted the effect of altered circadian timing upon each particular phenotype in clock-mutant plants under standard laboratory conditions. Altered night-time metabolism of stored starch accounted for most of the decrease in whole-plant biomass, as previously proposed. Mobilisation of a secondary store of malate and fumarate was also mis-regulated, accounting for any remaining biomass defect. We test three candidate mechanisms for the accumulation of these organic acids. Our results link genotype through specific processes to higher-level phenotypes, formalising our understanding of a subtle, pleiotropic syndrome at the whole-organism level, and validating the systems approach to understand complex traits starting from intracellular circuits. This work updates the first biorXiv version, February 2017, https://doi.org/10.1101/105437, with an expanded description and additional analysis of the same core data sets and the same FMv2 model, summary tables and supporting, follow-on data from three further studies with further collaborators. This biorXiv revision constitutes the second version of this report.

systems biology↗