Figure legends
Figure 1: Maximum capacity of photosynthesis (a), measured at 20°C under CO2- and light-saturating conditions at the end of each day of the acclimation period, for the wild-type Col-0 (circles) and the fum2.2 mutant (triangles). Rates of net photosynthesis (b) and respiration (c) measured as CO2exchange in growth conditions on Col-0 and fum2.2 plants at 20°C (white) and at 4°C after the first day of transfer (dashed) and after seven days of transfer (grey). Error bars show standard mean error (n=3-5). Different labels on columns in (b) and (c) indicate significantly different values (ANOVA, P<0.05)
Figure 2: Concentrations of starch (a), fumarate (b) and malate (c) in leaves measured at the beginning (open symbols) and end (closed symbols) of the photoperiod in Col-0 (circles) and fum2.2(triangles) plants. Error bars show standard mean error (n=5-7).
Figure 3: Distribution of diurnally fixed carbon, calculated from Figures 1-3 and data in Dyson et al. (2016) for Col-0 (a,c) andfum2.2 (b,f) plants in control conditions, on the first day of cold treatment (D0) and after one week of cold treatment (D7). Beginning-of-day concentrations were subtracted from end-of-day concentrations to estimate total diurnal fluxes to different sinks. Measured carbon sinks are fumarate (orange), malate (green), diurnal respiration (blue) and starch (purple). Export (and other) values (c,d) were calculated by subtracting all other values from the total diurnal carbon capture via photosynthesis. Data for “sugar”, which include sucrose and glucose retained in the leaf, are included in the analysis but are not visible on the scale of this figure.
Figure 4: Total protein concentrations (a) as calculated from Bradford assays. Different labels on columns indicate significantly different values (ANOVA, P<0.05). Principal component analysis (b) of the log2 scaled protein intensities in leaves of Col-0 (circles) and fum2.2 (triangles) plants measured .in control conditions (open symbols) and after one week of cold treatment (closed symbols). Hierarchical clustering and heat-map (c) of the log2 scaled protein values. Fold changes for Cluster 1 (d), 2 (e), 3 (f) and 4 (g), relative to Col-0 controls, are shown for the two genotypes and conditions. Full proteomic dataset is available in Table S1.
Figure 5: Summary of the relative abundance of proteins for the Benson-Calvin cycle enzymes of Col-0 (white bars) and fum2.2(grey bars) plants in control conditions (solid colours) and on Day 7 of 4°C treatment (hatched bars), as shown in the legend on the bottom left. RuBP (ribulose bisphosphate), 3PG (3-phosphoglycerate), 1,3-BPG (1,3-bisphosphoglycerate), GA3P (glyceraldehyde 3-phosphate), DHAP (dihydroxy-acetone-phosphate), SDP (sedoheptulose-1,7-bisphosphate), FBP (fructose-1,6-bisphosphate), F6P (fructose-6-phosphate), SDP (sedoheptulose-1,7-bisphosphate), S7P (sedoheptulose-1-phosphate), Ru5P (ribulose-5-phopshate), X5P (xylulose-5-phosphate). Data represent the total summed signal for all unique detected peptides in each case. Error bars represent the standard mean error, with different letters indicating significantly different values
Figure 6: The two shortest feasible pathways for producing fumarate in the cytosol, identified using a network analysis and flux sampling (see Materials and Methods for more details). The two pathways differ in the form of carbon exported from the chloroplast to the cytosol compartments. RuBP (ribulose bisphosphate), PGA (3-phosphoglyceric acid), DPGA (2,3-diphosphoglyverate), TP (triose phosphate), 2PGA (2-phosphoglycerate), PEP (phosphoenolpyruvate carboxylase), Pyr (Pyruvate), OAA (Oxaloacetate), Mal (Malate), Fum (Fumarate).
Figure 7: Flux sampling results obtained from the Col-0 (black) and fum2.2 (red) models for the export of PGA (3-phosphoglyceric acid ; a-d) and the export of TP (triose phosphate; e-h) from the chloroplast. Models were constrained according to cold conditions (a,e), to control conditions but with the rate of photosynthesis on the first day of 4°C treatment (b,f) , to cold conditions on Day 7 of 4°C treatment (c,g) and to control conditions with the production of NADPH set to lowest feasible value (d,h). Each panel shows a frequency diagram, representing the frequency with which each solution value was achieved over repeated iterations of the modelling.
Figure 8: Seed yield in plants of wild type Col-0 andfum2.2 grown for 8 weeks at 20oC and then either maintained at 20oC for a further 7 days (white bars), or transferred to 4oC for the same time (grey bars), before being transferred to a 16h day, to induce flowering. For each plant, the number of seeds per silique was counted for 10 siliques per plant (a) and the number of siliques counted on 6 plants (b). Total seed yield per plant was estimated as the product of these numbers (c). Error bars shown represent the standard error of the mean. Different letters on the bars indicate significantly different values (ANOVA, P<0.05).