The Arabidopsis Framework Model version 2 predicts the organism-level effects of circadian clock gene mis-regulation

Predicting a multicellular organism’s 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. The three candidate mechanisms tested did not explain this organic acid accumulation. 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

Three Isoforms of Isoamylase Contribute Different Catalytic Properties for the Debranching of Potato Glucans

Isoamylases are debranching enzymes that hydrolyze α-1,6 linkages in α-1,4/α-1,6–linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism

Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch

Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)–mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering—an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications

Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans

Isoamylases are debranching enzymes that hydrolyze � -1,6 linkages in � -1,4/ � -1,6–linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism

Prediction and analysis of phenotypes in the Arabidopsis clock mutant prr7prr9 using the Framework Model v2 (FMv2)

This upload contains or links to the biological data, FMv2 model and simulations for the Chew et al. 2017 paper (bioRxiv https://doi.org/10.1101/105437 ), updated 2022 as bioRxiv https://doi.org/10.1101/105437v2, mostly testing and simulating the effect of a slow circadian clock in the prr7prr9 double mutant compared to the Col wild type plants, with controls in lsf1 and prr7 single mutants. Effects tested comprise gene expression, hypocotyl elongation, flowering time, major carbon metabolite levels, gas exchange and growth in terms of leaf number, area and biomass. This is one of the outputs from the EU TiMet project, https://fairdomhub.org/projects/92. Several data files contain results generated in the same studies, but not covered by the publication. For example, additional time points (18 or 21 days of growth), many additional metabolites, and additional genotypes including pgm, lhy cca1, and in one case, toc1 and gi. This data archive was updated during submisson to the journal _in Silico _Plants in 2022, and is formatted as a Research Object, generated by the Snapshot function of FairdomHub, based on Investigation https://fairdomhub.org/investigations/123. The same Snapshot is shared on FairdomHub and Zenodo. We request that users gives appropriate credit to the authors of any data released here, as a norm of academic practice, including data released under CC-0 licence on the FairdomHub. CC-0 was not available on Zenodo or DataShare.Chew, Yin Hoon; Seaton, Daniel D.; Mengin, Virginie; Flis, Anna; Mugford, Sam T.; George, Gavin M.; Moulin, Michael; Hume, Alastair; Zeeman, Samuel C.; Fitzpatrick, Teresa B.; Smith, Alison M.; Stitt, Mark; Millar, Andrew J.. (2022). Prediction and analysis of phenotypes in the Arabidopsis clock mutant prr7prr9 using the Framework Model v2 (FMv2), [dataset]. School of Biological Sciences. https://doi.org/10.7488/ds/3453

Uncovering the mode of action of engineered T cells in patient cancer organoids

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies