Metabolic modelling shows that cold induces an alteration in
carbon export from the chloroplast which is perturbed in fum2.1
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 fum2.1 a similar shift occurs, but
with a different distribution of carbon between pools. Plants offum2.1 carry out significantly less photosynthesis in the cold
but retain a greater proportion of fixed carbon in the leaf. Although
the protein changes in fum2.1 are less marked than in Col-0,
there is nevertheless evidence of metabolic changes over the week,
implying that a form of acclimation is occurring. To better understand
the factors underlying these changes in the two genotypes, we adopted a
modelling approach.
Modelling was performed using flux sampling (Herrmann, Dyson, et
al. , 2019) based on a modified version of the model of
Arnold
and Nikoloski (2014; see Methods for details). Previous measurements
have shown that the accumulation of carbon storage pools is
approximately linear across the photoperiod
(Smith
& Stitt, 2007; Dyson et al. , 2016), and so the model was
constrained according to the rates of photosynthesis and the mean
estimated rates for starch, fumarate and malate accumulation. Flux to
export was not constrained. Although the proteomics data obtained do not
provide absolute quantitation, observed changes are relative to one
another and were used to impose further flux constraints onto the model
(Ramon et al. , 2018). We scaled the proteomic data to set
feasible upper bounds on all reactions with corresponding proteins.
Upper bounds for reactions associated with more than one gene product
were set to the total sum of all associated proteins only if data for
all those proteins was available. In total we constrained the upper
bounds of 101 reactions. Because protein presence does not necessarily
equate to enzymatic activity we set the lower bounds of reversible
reactions to the negative value of the upper bounds and the lower bounds
of non-reversible reactions were set to 0. We generated four different
models of different constraint types. This included two models of Col-0,
one constrained with the experimental results obtained from plants grown
in 20oC conditions and one constrained with the
experimental results of 7-day cold-acclimated plants. The same was done
for the fum2.1 genotype, with the addition that the cytosolic
fumarase reaction was deleted in those models to simulate the knockout.
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 the shortest feasible pathways by which
assimilated carbon can be converted to cytosolic fumarate, we
iteratively applied the min-path method (Ranganathan & Maranas, 2010)
and validated all pathways with 20 or fewer metabolites against the flux
sampling results 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 andfum2 20oC models (Figure 6). These pathways
differ 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.
We assessed how 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 the fum2 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 decreased in Col-0, whilst in fum2 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 1b), 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 thefum2 model, 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
Col-0 and fum2 versions of the model in which we restricted NADPH
production (simulating a restriction in electron transport capacity).
When limiting the NADPH production in the cell (by setting the flux
value of the NADPH producing reaction to the minimum feasible value) a
similar effect to the initial cold response was achieved: PGA export
increases (Figure 7d,h). By implementing cyclic electron flow in the
model, it makes sense that reducing the rate of photosynthesis will have
a similar effect to limiting the NADPH production. Whilst metabolic
modelling suggests an increase in PGA:TP export from the chloroplast
under cold and NADPH-limited conditions, this effect disappears in thefum2 models. In fact for fum2 PGA export is potentially
highest in 20oC conditions (Figure 7a).