Figure Legends
Figure 1: Illustration of the reactions used to set up the kinetic model. Reactions include the metabolites triose phosphate (TP), phosphoenolpyruvate (PEP), pyruvate (Pyr), malate (Mal), fumarate (Fum) and starch. The rate of photosynthesis (CO2 uptake) and respiration were constrained using experimentally measured values as outline in the Material and Methods. Model parameters were fitted to match the observed malate, fumarate and starch concentrations over an 8 h photoperiod on the first day of temperature treatment. All other metabolites were not allowed to accumulate in the leaf and all remaining carbon was assumed to be exported from the leaf (C export).
Figure 2: Effect of temperature on carbon assimilation in Col-0, C24 and fum2.2 accessions of Arabidopsis. (a) Maximum capacity for CO2 assimilation (Pmax ) was measured at 20 °C and under light- and CO2-saturating conditions on adult plants grown and at 20 °C and then exposed to 5, 10, 15, 20 25, or 30 °C treatments for one week. (b-c) In-cabinet measurements of CO2 assimilation of plants grown in control conditions (white), and after one day (cyan or orange) or 7 days (blue or red) of cold (b) or warm (c) treatment. Mean standard errors of 4 biological replicates are shown. Different letters above the error bars indicate statistically different values (Analysis of variance, Turkey’s test with a confidence level of 0.95).
Figure 3: Diurnal accumulation of malate (a-b), fumarate (c-d) and starch shown in hexose equivalents (e-f) in Col-0, C24, andfum2.2 plants under control conditions (white) or after one day (cyan or orange) and seven days (blue or red) of cold (a,c,e) warm (b,d,f) treatments, respectively. Diurnal accumulation was calculated by subtracting the beginning of day concentrations from the end of day concentrations. The uncertainties of 3-4 end of day and beginning of day measurements are shown as mean error bars.
Figure 4: A simplified engineering system of plant primary metabolism connecting high-risk metabolites and their carbon sinks. All high-risk metabolites, as identified in Table 1 were included: R5P (ribose-5-phosphate), TP (glyceraldehyde 3-phosphate), PEP (phosphoenolpyruvate), Pyr (pyruvate), KG (α-ketoglutarate), F6P (fructose 6-phosphate), G6P (glucose 6-phosphate), OAA (Oxaloacetate) and Malate. Only branch and end point metabolites were included in the kinetic model and are shown in red.
Figure 5 : Diurnal export rates as predicted from kinetic modelling. Rates of photosynthesis and respiration were used to constrain the model and the remaining model parameters were estimated to fit the measured metabolite concentrations (Table S1, Fig’s. 1, 2, S1). Export rates estimated for control conditions (white), the first day of cold (cyan) and the first day of warm (orange) treatment, represent the remaining carbon not stored in the leaf.
Table 1: The ten most high-risk metabolites as identified by the Failure Mode and Effect Analysis (FMEA). An existing metabolic model (Arnold and Nikoloski, 2014; Herrmann et al ., 2019c) was converted to a graph of carbon metabolism as outlined in the methods. Metabolites with the highest risk factors (R ) and their cellular locations are shown. Possible cellular locations, as written in the genome-scale metabolic model, include the chloroplast, mitochondrion, cytosol and peroxisomes. \(\mathrm{R=S}_{M}\times P_{M}\), where\(S_{M}\mathrm{=C}_{M}\times N_{M}\), was calculated such that the severity is equal to the normalized number of neighbours (NM ) times the normalized betweenness centrality (CM ). PM is based on the length of the shortest path and the number of shortest paths that lead to a metaboliteM .