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.