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).