fum2.2
Results presented here show that acclimation to cold results in a substantial change in the metabolism of Col-0 plants, with a shift from diurnal to nocturnal carbon export from the leaf and an increase in leaf diurnal carbon storage. In fum2.2 a similar shift occurs, but with a different distribution of carbon between pools. Plants offum2.2 carry out significantly less photosynthesis in the cold but retain a greater proportion of fixed carbon in the leaf. Although the protein changes in fum2.2 are less marked than in Col-0, there is nevertheless evidence of metabolic changes over the week. To better understand the factors underlying these changes in the two genotypes, we adopted a modelling approach.
Modelling was performed using flux sampling (Herrmann et al. , 2019) based on a modified version of the model of Arnold and Nikoloski (2014; see Methods for details). We set up metabolic models for the Col-0 and fum2 genotypes and constrained them using experimental data to represent possible flux solutions under control conditions and on the 1st and 7th days of cold treatment. Constraining the model using the proteomic data allowed us to analyse the above observed difference in Col-0 and fum2 plants in response to cold, including changes in the electron transport proteins and Benson Calvin cycle enzymes, in a system context.
In order to determine feasible pathways by which assimilated carbon can be converted to cytosolic fumarate, we validated potential pathways against the flux sampling results as outlined in the Materials and Methods in order to see whether they were carrying a significant flux under the given model constraints. The flux sampling results confirmed two of these pathways to be feasible in the Col-0 and fum220oC models (Figure 6). These pathways differ from one another in terms of their relative consumption of ATP and NADPH. Activity of Rubisco produces 3-phosphoglyceric acid (PGA) from ribulose-1.5-phosphate and CO2. PGA can then be converted to triose phosphate (TP) in reactions requiring ATP and NADPH. There are two forms of TP (glyceraldehyde-3-phosphate and dihydroxy acetone phosphate); when exporting either of the two forms from the chloroplast in our analysis we obtained the same results and therefore refer to the two forms collectively as TP export. TP is exported from the chloroplast in exchange for inorganic phosphate by the triose phosphate translocator (TPT). Conversion of TP to fumarate includes the reconversion of TP to PGA in the cytosol. The PGA is then carboxylated and reduced to form malate. The TPT is also capable of exporting PGA directly, eliminating the reduction reaction in the chloroplast.
Our flux sampling results highlight that the export of PGA versus TP from the chloroplast varies under changing conditions using flux sampling (Figure 7). In models of 20oC conditions, most carbon is exported from the chloroplast in the form of TP, with thefum2 model tending to have higher PGA export (Figure 7 a,e). In the Day 0 cold model, where the rate of photosynthesis is restricted, PGA export is increased and TP export is decreased in Col-0, whilst infum2 both show a tendency to be reduced (Figure 7 b,f). In Col-0 plants acclimated to cold (“Day 7 – 4oC”), where the rate of photosynthesis recovers (Figure 1 b), PGA export is modelled to decrease relative to Day 0, while TP export is largely unaffected (Figure 7 c,g). At the same time, in the fum2 model, which does not consider a recovery of the photosynthetic rate (Figure 1 b). PGA export it largely absent.
Previous experimental data have indicated that the ATP/NADPH ratio increases at low temperature in Arabidopsis (Savitch et al. , 2001), possibly reflecting changes in the ratio of cyclic to linear electron transport (Clarke & Johnson, 2001). To simulate this, we ran the Col-0 and fum2 20oC models with their NADPH production restricted (simulating a restriction in electron transport capacity). When limiting the NADPH production in the cell (by setting minimum NADPH production as an optimisation constraint) similar effects to the initial cold response in Col-0 and fum2 were achieved (Figure 7 d,h). Again, PGA export increased in Col-0, this time even more markedly than in response to cold. By implementing cyclic electron flow in the model, it makes sense that a reduced rate of photosynthesis on the first day of photosynthesis will have a similar effect to limiting NADPH production. Whilst metabolic modelling suggests an increase in PGA:TP export from the chloroplast under cold and NADPH-limited conditions, this effect is not observed in the fum2models. In fact, for fum2, PGA export is potentially highest in 20oC conditions (Figure 7 a).