Organic acid accumulation across temperatures requires no
regulatory mechanisms
To further test whether fumarate acts as a buffer for malate across the
physiological temperature range of Arabidopsis, we constructed a kinetic
model of the primary reactions in leaf carbon metabolism (Fig. 1, Table
S1). To keep the number of reactions in this model to a minimum, only
relevant branch and end points (as shown in red in Fig. 4) were included
in the model. In comparison to starch, malate and fumarate, neither
sugars nor amino acids accumulate substantially in Arabidopsis leaves in
our growth conditions (Dyson et al ., 2016); however, it must be
assumed that there is a significant flux through these pools that
constitutes phloem export (marked as C export in Fig. 1; Lalondeet al ., 2003, Ainsworth et al ., 2011). Rates of
photosynthesis and respiration (Fig. 2b,c; Fig. S1) were used to
constrain the model. The remaining parameters were estimated to fit the
measured malate, fumarate and starch concentrations in control
conditions, and on the first day of cold and warm treatment,
respectively. It is assumed that on the first day of temperature
treatment, plants will not yet have significantly acclimated to the
stress condition; thus, observed changes in metabolism would be mainly
the result of kinetic or regulatory effects rather than changes in
enzyme concentrations.
When fitting separate models for Col-0, fum2 and C24 plants, the
models predict the same kinetic parameters for all reactions except for
the conversion of fumarate to malate and that of triose phosphate to
starch (Table 2). C24 has significantly lower cytosolic fumarase
activity than Col-0 and accumulates only about three quarters of the
amount of fumarate accumulated in Col-0 leaves (Riewe et al .,
2016). The models predict no cytosolic fumarase activity in fum2and a reduced activity in C24 compared to Col-0 plants, confirming
experimental evidence. The results obtained show that increased fumarate
production in response to both warm and cold treatment can result of
temperature-dependent enzyme kinetics rather than post-translational
regulation of enzyme activity. While we cannot exclude that these
regulatory effects are occurring, the model simply shows that a solution
for organic acid production without temperature-dependent regulatory
effects is possible.
In the solutions to the models, most effective Q10 values are estimated to be around 2.0 (Table 2), as is typical for
temperature-dependence of biological reactions (Elias, 2014). The
cytosolic fumarase reaction, however, shows no apparent
temperature-dependence, effective Q10 = 1.0.
Although we are aware that all enzymatic reactions are affected by
temperature, we note that an effective Q10 = 1.0
does not mean that the reaction is not temperature-sensitive but rather
that the corresponding enzyme is not limiting. Thus, our model results
merely demonstrate that differences in the rate constants are enough to
explain the observed difference between genotypes. The conversion of
malate to pyruvate is, according to the models, the most
temperature-sensitive reaction; however, it carries little overall flux.
The conversions of phosphoenolpyruvate to malate and pyruvate, on the
other hand, carry a substantial flux and show a strong
temperature-dependence. The counter-play of these two reactions, along
with the observed changes in rates of photosynthesis and respiration,
are sufficient to account for the experimentally observed changes in
organic acid concentrations under warm and cold temperature treatments.
The rate of export was allowed to adjust freely when fitting the model.
This rate accounts for the total remaining carbon, which is assumed to
be exported from the leaves, primarily in the form of sugars, but will
include other forms of fixed carbon retained in the leaf (Wilkinson and
Douglas, 2003). All genotypes are predicted to decrease their diurnal
rates of export on the first day of cold treatment (Fig. 5). On the
first day of warm treatment, Col-0 and fum2 plants show little
change in the rate of export, compared to control conditions (Fig. 5).
C24 plants, however, are predicted to increase their rates of export
under warm treatment. This is the result of decreased starch
accumulation (Fig. 3f) as well as decreased rates of respiration (Fig.
S1) in response to warm treatment.