Metabolic Modelling
We adapted a metabolic model by Arnold and Nikoloski (2014) to allow for the diurnal accumulation of carbon compounds. Specifically, we ensured that cytosolic fumarate could be produced from cytosolic malate and added “export reactions” to the model (describing diurnal storage pools) for malate, fumarate and starch in addition to the already existing sucrose export. We generated four models: Wild type Col-0 in 20oC and 4oC conditions andfum2 in 20oC and 4oC conditions. We constrained the models using metabolite assays such that the beginning of day concentrations of fumarate, malate, and starch subtracted from their respective end of day concentrations equated to the diurnal flux over the eight-hour photoperiod. Furthermore, we assumed a constant rate of photosynthesis (Dyson et al. , 2016) throughout the day and converted the measured rates of photosynthesis to diurnal fluxes of carbon intake. We then used proteomics data to further constrain the upper bounds of the flux reactions (Ramon, Gollub, & Stelling, 2018). For each metabolic reaction we checked whether all of the corresponding proteins were available in the data set; if so, then those reactions were given an upper bounds of additive value of all of the identified proteins in case multiple isoforms exist. Given that the proteomics data is relative and not quantitative we scaled all of the proteomics constraints such that we were able to obtain model solutions across all four models. We used single flux solution, from a flux balance analysis maximizing carbon assimilation via Rubisco within feasible model constraints, in order to eliminate non-essential reactions which generate loops within the model, using the loopless function in the cobra (version 0.10.1) package. We then conducted flux sampling using the CHRR algorithm in the MATLAB toolbox as outlined in Herrmann, Dyson, Vass, Johnson, and Schwartz (2019).
All models, code and data used to conduct the computational analyses are available at https://github.com/HAHerrmann/FluxSamplingCol0Fum2 and have been archived in Zenodo (DOI: 10.5281/zenodo.3366934).