Metabolic flux from the chloroplast provides signals controlling photosynthetic acclimation to cold in Arabidopsis thaliana

Photosynthesis is especially sensitive to environmental conditions, and the composition of the photosynthetic apparatus can be modulated in response to environmental change, a process termed photosynthetic acclimation. Previously, we identified a role for a cytosolic fumarase, FUM2 in acclimation to low temperature in Arabidopsis thaliana . Mutant lines lacking FUM2 were unable to acclimate their photosynthetic apparatus to cold. Here, using gas exchange measurements and metabolite assays of acclimating and non-acclimating plants, we show that acclimation to low temperature results in a change in the distribution of photosynthetically fixed carbon to different storage pools during the day. Proteomic analysis of wild-type Col-0 Arabidopsis and of a fum2 mutant, which was unable to acclimate to cold, indicates that extensive changes occurring in response to cold are affected in the mutant. Metabolic and proteomic data were used to parameterize metabolic models. Using an approach called flux sampling, we show how the relative export of triose phosphate and 3-phosphoglycerate provides a signal of the chloroplast redox state that could under-lie photosynthetic acclimation to cold.

Exposure to low temperature triggers a complex array of acclimation responses. A significant amount of work in cold responses in plant has focused on the acquisition of freezing tolerance (see Knight & Knight, 2012 for a review). In addition, it is recognized that plants can optimize their metabolism to suit changing conditions. Both photosynthesis (Huner et al., 1993;Strand et al., 1997;Strand et al., 1999) and respiration (Armstrong, Logan, Tobin, O'Toole, & Atkin, 2006;Talts, Pärnik, Gardeström, & Keerberg, 2004) can readily be measured in intact leaves, so it is easy to follow acclimation of these processes in vivo. A prominent feature of metabolic acclimation is an increase in the capacity of metabolism, with enzyme and metabolite concentrations increasing to compensate for the loss of activity at low temperature (Savitch et al., 2001;Savitch et al., 2002;Stitt & Hurry, 2002). This requires the coordination of gene expression across multiple cellular compartments, including retrograde and/or anterograde signals between the nucleus, chloroplast and mitochondrion (Fey, Wagner, Brautigam, & Pfannschmidt, 2005). To date, little is known about either the sensing or the signalling pathways involved in metabolic acclimation.
Acclimation of photosynthesis to low temperature has previously been studied in a range of species, including Arabidopsis. In the short term, low temperature decreases the rate at which sucrose is synthesized and exported from the leaf. Limitations in flux through sucrose phosphate synthase (SPS) result in the accumulation of phosphorylated metabolites (Hurry, Strand, Furbank, & Stitt, 2000). This is thought to lead to depletion of the cytosolic concentration of inorganic phosphate (Pi), which, in turn, limits the export of triose phosphate (TP) from the chloroplast. This inhibits photosynthesis, as regeneration of Pi in the chloroplast is necessary for ATP synthesis. Over relatively short periods (days) increased expression of SPS removes the limitation in sucrose synthesis. Based on the responses of different mutants with altered phosphate content, Hurry et al. (2000) proposed that Pi depletion provides a signal for cold acclimation of SPS content.
Recently, we re-examined the responses of Arabidopsis to low temperature (Dyson et al., 2016). Using non-targeted metabolomics, we identified the diel accumulation of the organic acid, fumaric acid (predominantly present in the anionic form, fumarate), as important in the response to cold. We showed that the accumulation of fumarate, which requires the presence of a cytosolic isoform of the enzyme fumarase, FUM2 (Pracharoenwattana et al., 2010), is a specific response to temperature. Plants of two independent mutant lines lacking this enzyme not only failed to accumulate fumarate over the photoperiod, they also accumulated higher concentrations of phosphorylated intermediates. Crucially, while wild-type Col-0 Arabidopsis plants increase photosynthetic capacity in response to low temperature, fum2 mutant plants do not. This suggests that Pi deficiency alone is not sufficient to trigger photosynthetic acclimation.
Here, we have used a combined metabolic and proteomic approach, together with physiological analyses to dissect the acclimation processes occurring in Arabidopsis in response to low temperature. We focus on dynamic acclimation responses to cold in leaves developed at higher temperature. Our results show that fumarate accumulation is important for a wide range of metabolic acclimation responses to cold. Our beginning-and end-of-day measurements of carbon pools indicate that there is a shift from day-to night-time carbon consumption upon cold acclimation. Furthermore, based on the results from metabolic modelling, constrained using experimental data, we propose that the changes in the pathway of carbohydrate export from the chloroplast link to fumarate accumulation at low temperature and provide a mechanism controlling photosynthetic acclimation to cold.

| Plant material and growth
Wild-type Col-0 seeds were obtained from the Nottingham Arabidopsis Stock Centre. Seeds possessing insertions in the gene, At5g50950 (fum2.1 and fum2.2), encoding cytosolic FUM2 protein (Pracharoenwattana et al., 2010) were kindly provided by Professor Steven Smith (University of Tasmania, Australia). Plants were grown in 3 00 pots containing peat-based multipurpose compost, in an SGC120 growth cabinet (Weiss Technik) with an 8-hr photoperiod, to supress flowering, at a temperature of 20 C day/16 C night, and irradiance of 100 μmol m −2 s −1 provided by warm white LED tubes (colour temperature 2,800-3,200 K) for 8 weeks. After 8 weeks, plants had reached growth stage 1.10 (Boyes et al., 2001), with more than 10 leaves >1 mm and persisted without development of flowers until 10-12 weeks. Plants for cold treatment were transferred to a similar cabinet at 4 C day/night 1 hr prior to the start of the photoperiod.
Leaves for metabolite measurements were harvested by flash freezing in liquid nitrogen under growth conditions.

| Estimation of seed yield
Eight-week-old wild-type Col-0 and fum2.2 plants were kept in control conditions or were cold treated for 1 week as described above.
Plants were then moved to a growth chamber with a 16-hour day length, 20 C, to trigger flowering. After a further 8 weeks, the number of siliques per plant was counted on six plants for each treatment group, and the number of seeds silique pod was estimated for 10 individual siliques per plant. Total seed yield was estimated as the product of silique number and mean seeds per silique.

| Measurements of photosynthesis and respiration
Measurements of photosynthetic capacity were made using a LiCor LI-6400 infra-red gas analyser, at a temperature of 20 C in an atmosphere of 2,000 μll −1 CO 2 , flow rate 200 cm −3 min −1 , and an irradiance of 2,000 μmol m −2 s −1 provided by a warm white LED (colour temperature 2,800-3,200 K). Leaves were illuminated for 30 mins to fully induce photosynthesis before a measurement was recorded. The light was then switched off to allow an estimate of the dark rate of gas exchange. Measurements of photosynthesis and respiration under growth conditions were carried out using the same gas analyser in the growth cabinet where plants were grown, using ambient air conditions. The gas analyser was placed in the cabinet and allowed to equilibrate for at least 1 hr prior to any measurements. The analyser chamber was placed to give a light intensity at the leaf surface equivalent to that of plants during growth. Respiration was measured by interrupting illumination, covering the plant and analyser chamber with aluminium foil. All measurements were performed between the fifth and sixth hour of the photoperiod. Data were analysed using an analysis of variance (ANOVA), followed by a Tukey's post hoc tests in SPSS (IBM).

| Enzyme-linked assays of metabolites
Enzymatic assays were carried out on extracts from fully expanded leaves as described previously (Dyson et al., 2016). Starch, sucrose and glucose, and malate were measured using total starch (Method E), sucrose D-glucose, and l-malic acid assay kits (Megazyme), respectively. To measure fumarate, the malate kit was modified to include an extra independent reaction step, using 2 units of fumarate hydratase enzyme (Sigma). Data were analysed using an analysis of variance, followed by a Tukey's post hoc test, in SPSS (IBM).

| Proteomic analysis of plant material
Extraction of leaf proteins and analysis of protein content using gel-free LC-MS/MS was carried out as described by Miller et al. (2017) were then re-imported into Progenesis to allow for assignment of proteins from peptide data. Raw protein intensities were then exported from Progenesis and normalized to the sample with the median total protein content for that treatment, as described previously (Miller et al., 2017). Total protein for each sample was calculated by summing the intensities of all the quantified proteins.
A principal component analysis (PCA) was performed in the R software package using log2 scaled protein intensities using the pcaMethods R package. The "svd" function was used, and 10 principal components were included in the calculation. Proteins were considered to have significantly changed in abundance when a p value of <.05 was reached, with a fold change of 1.2 or greater. For hierarchical clustering analysis, log 2 scaled protein values were used.
Hierarchical clustering was performed using Euclidean distance and the complete linkages method. For heatmap/cluster analysis, fold change data were calculated relative to the wild-type Col-0 at LL and log2 scaled. A heatmap was then generated using the heatmap.2 package in R software, using the Euclidean distance algorithm for dendrogram creation. The dendrogram was cut into six clusters.

| Metabolic modelling
We used a metabolic model modified from that of Arnold and Nikoloski (2014), as previously published (Herrmann, Dyson, Vass, Johnson, & Schwartz, 2019). Specifically, the model was modified to ensure that cytosolic fumarate could be produced from cytosolic malate (inclusion of reversible cytosolic FUM reaction) and we added "export reactions" to the model (describing diurnal storage pools) for malate, fumarate and starch, in addition to the already existing sucrose export. In addition, we added a cyclic electron transport reaction to the model which was previously missing. We generated four models for each genotype (Col-0 and fum2): 20 C; 4 C on Day 0; 4 C on Day 7 of treatment; and one with NADPH-limiting conditions. We constrained the models using metabolite assays such that the beginning-of-day concentrations of fumarate, malate and starch subtracted from their respective end-of-day concentrations were used as an estimate of the diurnal flux over the eight-hour photoperiod, consistent with the approximately constant rate of accumulation of these metabolites seen experimentally (Dyson et al., 2016). We assumed a constant rate of photosynthesis through the day (Dyson et al., 2016) and converted the measured rates of photosynthesis to cumulative diurnal fluxes of carbon intake (mmol gFW −1 Day −1 ), as previously described by Herrmann et al. (2019). Flux to sucrose was not constrained but was used to estimate the remaining carbon, which is exported from the leaf during the day. We then used proteomics data to further constrain the upper bounds of the flux reactions (Ramon, Gollub, & Stelling, 2018). For each metabolic reaction, we checked whether all of the corresponding proteins were available in the data set; if so, then those reactions were given an upper bound of the additive values of all of the identified proteins, in case multiple isoforms exist. The proteomic constraints of the Col-0 and fum2 20 C models were also applied to the respective 4 C Day 0 models, assuming that during the first day of cold, changes in metabolic enzyme content are negligible (consistent with measured total protein and photosynthetic capacity). Given that the proteomics data are relative and not quantitative, we scaled all the proteomics constraints to the lowest possible values for which we were able to obtain model solutions across all models. In total, we constrained the upper bounds of 101 reactions in each model. Proteomic constraints were calculated as the averages of four biological replicates for each treatment plus the standard error of those measurements, thus accounting for measurement error.
Because protein presence does not necessarily equate to enzymatic activity, we set the lower bounds for these reactions to zero for irreversible reactions and to the negative value of the upper bound for reversible reactions. Proteomic constraints were applied only to the "inner" model reactions, whereas the metabolite and photosynthesis data were used to set boundary conditions (i.e., influxes and effluxes).
We the used flux solutions, from a flux balance analysis maximizing carbon assimilation via Rubisco (reaction id: RBC_h) within feasible model constraints, in order to eliminate non-essential reactions that generate loops within the model, using the loopless function in the cobra (version 0.10.1) package (Desouki, Jarre, Gelius-Dietrich, & Lercher, 2015). The loopless method removes thermodynamically infeasible loops from the network according to the following criteria: that the objective and exchange fluxes remain unchanged, that all non-exchange fluxes proceed in the same direction as in the original model, and that the sum of absolute non-exchange fluxes is minimized. Based on these criteria, the "least-loopy" solution is identified.
The objective function is irrelevant to the results presented in this paper and was only applied to ensure that none of the pathways required for carbon metabolism contained thermodynamically infeasible solutions. We then conducted flux sampling on the loopless models using the CHRR algorithm in the MATLAB toolbox as outlined in Herrmann et al. (2019). In order to minimize observer bias, the flux sampling was performed without an optimisation constraint for the control, Day 0 and Day 7 models. The NADPH-limited models are the same as the control models, but here, in addition to the experimental constraints, we set minimum NADPH production via linear electron transport as an objective function (reaction id: Fd-NADPR_h).

| Network analysis
We converted the metabolic model to a metabolite-metabolite graph of primary carbon metabolism using the method of Ranganathan and Maranas (2010). A full list of the nodes and edges is available on Zenodo (DOI: 10.5281/zenodo.3366934). We then used the networkx (version 1.10) package in Python (Version 3.6.9) in order to iteratively identify pathways with fewer than 20 nodes connecting the Rubisco and fumarate nodes. We checked the 17 identified pathways against the flux sampling solutions of the Col-0 models and identified two pathways that carried a flux substantial enough to account for fumarate accumulation in the model. All other pathways had at least one reaction for which the sampling solutions were either zero or close to zero. For fluxes to be considered close to zero, we used a cut-off of 0.4, which is three times lower than the carbon flux to fumarate (i.e., not substantial enough to explain our observed accumulation of fumarate in wild-type Col-0 plants).

| Acclimation of both photosynthesis and respiration to cold are impaired in plants lacking FUM2
Plants of the wild-type Arabidopsis, accession Col-0 and a mutant in the same background, fum2.2, were grown for 8 weeks at a daytime temperature of 20 C. Plants were then transferred to a growth cabinet with the same light conditions, but with a temperature of 4 C.
Photosynthetic capacity (P max ; measured at 20 C in saturating light and CO 2 ) of these plants was measured over the following 9 days ( Figure 1a). Prior to transfer to low temperature, P max of Col-0 was slightly higher than that of fum2.2. After 1 day at low temperature, the capacity for photosynthesis (P max ) in Col-0 increased. P max continued to increase over the following days, rising to a new steady-state approximately 50% higher than the starting value by the sixth day of cold treatment. This indicates that, under our experimental conditions, dynamic acclimation of photosynthesis occurs in response to cold and that a new steady-state is reached within 7 days. In contrast, the P max of fum2.2 did not vary significantly over the course of the experiment, confirming previous evidence that both fum2.1 and fum2.2 mutants lacking FUM2 are unable to acclimate their photosynthetic capacity in response to cold (Dyson et al., 2016).
Measurements of the rate of gas exchange achieved by plants in growth cabinets, with ambient CO 2 , light and temperature, were performed in the last hour of the first day of cold. Transfer to cold resulted in a small but significant inhibition of both photosynthesis and respiration at the end of the first day of exposure to cold (ANOVA, p < .05; Figure 1b,c). In Col-0, acclimation of both parameters had occurred after 7 days, such that in situ rates of gas exchange recovered and did not differ significantly from those recorded at 20 C prior to acclimation. In contrast, in fum2.2,n o recovery occurred in either photosynthesis or respiration. This suggests that either fumarate accumulation or FUM2 protein is essential for the acclimation of both photosynthesis and respiration to cold.

| Acclimation to cold changes partitioning of metabolites between sinks
In plants, diurnally produced organic carbon can be directly exported from the leaf, used in cellular respiration, or stored in the leaf in a variety of forms (Chia, Yoder, Reiter, & Gibson, 2000;Fahnenstich et al., 2007;Zell et al., 2010). In Arabidopsis, the principal leaf carbon stores are starch and organic acids, especially fumarate and malate (Chia et al., 2000;Zell et al., 2010). When plants are exposed to cold for a single photoperiod, the accumulation of both starch and organic acids increases (Dyson et al., 2016). Here, we measured the beginning-and end-of-photoperiod concentrations of starch, fumarate and malate in Col-0 and fum2.2 on each of the 7 days following transfer to cold (Figure 2). The diurnal accumulation of these metabolites shows clear evidence of acclimation in both genotypes. In both wild-type Col-0 and fum2.2, starch accumulation was greater at 4 C than 20 C on the first day of cold and increased each day, until Day 4 of cold treatment (Figure 2a). The amount of starch seen at the endof-day was higher in fum2.2 than in Col-0 throughout the experiment, with the absolute difference between genotypes remaining approximately constant throughout the treatment. In Col-0, essentially all starch accumulated during the day was mobilized overnight throughout the acclimation period. In fum2.2, a small amount of starch was retained in the leaf at dawn after the third day of cold treatment.
In Col-0, there was an increase in the amount of fumarate accumulated each day through the acclimation period ( Figure 2b). This was accompanied by an increase in the accumulation of malate (Figure 2c), such that these acids represented an increased proportion of total stored carbon. In fum2.2, the amount of fumarate was always substantially lower than in Col-0 and at no point in the experiment was there evidence of a diurnal accumulation of fumarate. End-of-day malate concentrations were increased on the first day of cold in fum2.2 but then fell the following day, before rising again towards the end of the treatment period.
Arabidopsis does not accumulate substantial amounts of sucrose in its leaves under most conditions, but sucrose is known to play an important role in freezing tolerance (Stitt & Hurry, 2002). We assayed leaf sucrose and glucose content in response to cold treatment ( Figure S1). At 20 C, there is a significant diel cycle in sucrose content; however, in response to cold treatment, this cycle was lost in both genotypes, with beginning-of-day sucrose content increasing and end-of-day content decreasing progressively through the week. At the end of the cold treatment, fum2.2 contained slightly more sucrose than wild-type Col-0.
To understand better how carbon is partitioned between different diurnal storage pools in different conditions, we combined data were used to calculate the daily carbon uptake and respiratory consumption. The difference between carbon fixed through photosynthesis and the measured carbon fluxes to respiration and known carbon stores was assumed to be largely accounted for by carbon export from the leaf in the form of sucrose, consistent with estimates from Lundmark, Cavaco, Trevanion, and Hurry (2006), although other compounds may accumulate or be exported from the leaf and are included in this category.
Plants of fum2.2 at 20 C showed similar photosynthesis and respiration to Col-0. Although they do not accumulate fumarate, the proportion of total carbon stored as organic acid was similar to that seen in Col-0, with an increased accumulation of malate. Combining this with the increase in starch accumulation, which is larger in fum2.2 than in Col-0, we conclude that the total unaccounted carbon, primarily diurnal export, is lower in fum2.2 than in Col-0.
During the first day of exposure to cold, there were notable changes in carbon distribution between different sinks (Figure 3a,b).
Total fixed carbon was lower on Day 0 (first day) of cold due to the lower rate of photosynthesis (Figure 1). In both genotypes, the total amount of fixed carbon we were able to account for increased, implying that diurnal carbon export is probably inhibited. Unaccounted carbon was still greater in Col-0 than in fum2.2. When plants were cold treated for 7 days, this effect became even more marked (Figure 3d).
From this, we conclude that there is a substantial inhibition of diurnal carbon export from the leaf in cold-treated plants of both genotypes, but that this effect becomes more pronounced in fum2.2 over the course of the week. Given that all metabolite pools retain a diel turnover, we conclude that metabolic acclimation to cold involves a shift from diurnal carbon export to nocturnal processes.
3.3 | Acclimation to cold involves changes in the proteome of both Col-0 and fum2.2 In a previous study of dynamic acclimation, we saw that photosynthetic acclimation to increased light entails an increase in enzyme concentrations involved in multiple metabolic processes (Miller et al., 2017). In Col-0, cold exposure for 7 days resulted in a significant increase in leaf protein content (Figure 4a), with an approximately 30% increase in protein content per unit fresh weight of leaf. In fum2.2, protein content did not change significantly. Nevertheless, analysis of the proteome shows that there were changes occurring in both Col-0 and fum2.2, albeit to a much smaller extent in the latter.
We were able to estimate the relative abundance  (Table S1). There were increases in the relative abundance of various peripheral PSII proteins, including isoforms of PSBS, and in PSB29, which has been implicated in PSII assembly (Keren, Ohkawa, Welsh, Liberton, & Pakrasi, 2005), however, components of the PSII core did not change significantly in response to cold. Among the proteins of the photosynthetic electron transport chain, two of the four detected cytochrome b6f complex subunits increased significantly in Col-0; measurements for the other subunit were too variable to allow a confident assessment of changes in abundance. Overall, this suggests a tendency to increase Across other major metabolic pathwaysincluding starch and sucrose synthesis, glycolysis and the tricarboxylic acid cyclesimilar patterns of acclimation were observed, with most proteins increasing in the cold in Col-0 and fum2.2 but to a lesser extent in the latter case (Table S1). In the sucrose synthesis pathway, most enzymes increased their concentration in response to cold in both genotypes, but with the relative abundance of these tending to be lower in fum2.2 ( Figure S2). A notable exception to this was sucrose phosphate phosphatase, which did not increase in fum2.2.
To summarize, acclimation of photosynthesis to low temperature in Col-0 involves an increase in the abundance of some electron transfer proteins, though not reaction centres, and substantial changes in the amount of a broad range of Benson-Calvin cycle enzymes, consistent with increases seen in enzyme activities in response to cold observed previously (Strand et al., 1999). These changes are largely or completely absent in fum2.2. The changes in the proteome, across metabolism, show a similar tendency but to a different extent in other metabolic pathways. These data indicate that fumarate accumulation or FUM2 protein or activity plays a central role in high-level processes, regulating acclimation of a wide range of metabolic enzymes.
Metabolic modelling shows that cold induces an alteration in carbon export from the chloroplast, which is perturbed in fum2.2.   (Figure 7). In models of 20 C conditions, most carbon is exported from the chloroplast in the form of TP, with the fum2 model tending to have higher PGA export (Figure 7a,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, while in fum2 both show a tendency to be reduced (Figure 7b,f). In Col-0 plants acclimated to cold ("Day 7 -4 C"), 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 7c,g).

Results
A tt h es a m et i m e ,i nt h efum2 model, which does not consider a recovery of the photosynthetic rate (Figure 1b), PGA export is largely absent. FIGURE 7 Flux sampling results obtained from the Col-0 (black) and fum2.2 (red) models for the export of PGA (3-phosphoglyceric acid; a-d) and the export of TP (triose phosphate; e-h) from the chloroplast. Models were constrained according to cold conditions (a, e), to control conditions but with the rate of photosynthesis on the first day of 4 C treatment (b, f), to cold conditions on Day 7 of 4 C treatment (c, g) and to control conditions with the production of NADPH set to lowest feasible value (d, h). Each panel shows a frequency diagram, representing the frequency with which each solution value was achieved over repeated iterations of the modelling [Colour figure can be viewed at wileyonlinelibrary.com] 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 20 C 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 7d, 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. While 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 fum2 models. In fact, for fum2, PGA export is potentially highest in 20 C conditions (Figure 7a). were transferred into a growth cabinet with a 16-hr photoperiod to induced flowering. Plants were allowed to set seed, and seed yield was estimated (Figure 8). No significant difference was seen between the seed yield of Col-0 plants maintained at 20 C and those that experienced a cold treatment. In contrast, when fum2.2 plants were exposed to cold for 7 days before being transferred to long photoperiods, the final seed yield was significantly reduced. This reduction was due to a lower number of siliques being formed, rather than a reduction in the number of seeds per silique.

| DISCUSSION
Previous work has shown that the ability to acclimate photosynthesis and metabolism to changes in light plays an important role in determining plant fitness and seed yield (Athanasiou et al., 2010). Here, we have presented evidence that acclimation to cold is also important in determining fitness and seed yieldwild-type Col-0 plants are unaffected by changes in temperature, while fum2.2, which is unable to acclimate to cold, is negatively affected by even short cold periods.
Ability to acclimate photosynthesis to environmental change is, therefore, clearly an important process that will have major impacts on crop yields and may be a target for crop breeding.
Previously, we have seen that acclimation of photosynthetic metabolism of the leaf, with a major shift in the ratio of diurnal carbon leaf storage to export. Plants acclimated to cold retain more carbon in the leaf during the day and, therefore, must export more overnight.
Furthermore, we provide evidence from metabolic modelling that acclimation responses may depend on the form of carbon export from the chloroplast. Specifically, we propose that the PGA:TP chloroplast export ratio provides a novel potential signal, which may drive aspects of acclimation responses both in the chloroplast and the wider cell.
Earlier studies on cold acclimation of photosynthesis in Arabidopsis highlighted the importance of sucrose synthesis and, specifically, the activity of SPS (Stitt & Hurry, 2002;Strand, Foyer, Gustafsson, Gardestrom, & Hurry, 2003). It was suggested that phosphate recycling is impaired at low temperature, due to the accumulation of sugar phosphates, such as glucose-6-phosphate, fructose-1,6-bisphosphate and fructose-6-phosphate. Evidence from the fum2.2 mutant speaks against a direct role for phosphate in controlling the acclimation of photosynthetic capacity. Non-acclimating fum2.2 plants show higher levels of sugar phosphates on the first day of cold than do Col-0 plants, and should, therefore, have a stronger photosynthetic acclimation signal (Dyson et al., 2016). If phosphate is a signal for acclimation, fumarate accumulation must play a role downstream of this, preventing acclimation despite the signal. This conclusion is further supported here. Measurements of the major sugar phosphates involved in sucrose synthesis ( Figure S2) shows that these tend to increase as a result of acclimation. There is, however, no persistent significant difference in the concentrations of these in the different genotypes. Phosphate may still play a role in the short-term regulatory responses seen on exposure to cold (Hurry et al., 2000).
Regardless of the role of phosphate in cold sensing, diurnal flux to sucrose is clearly an important part of the cold response. On the first day of exposure to cold, the estimated maximum possible flux to sugar export dropped significantly, compared with plants maintained at 20 C (Figure 3c,d). This effect might be explained by a drop in sink strength, however, if this is the case, then it is not alleviated by acclimation at the whole plant level. If daytime export is indeed sink limited, it is unlikely to be a consequence of the overall capacity of sinks since, over the diel cycle, there was no evidence of progressive accumulation of fixed carbon in the leaf. Thus, nocturnal processes, including export from the leaf or increased nocturnal respiration, compensate for diurnal export.
Nocturnal metabolism of leaves remains poorly understood. At night, there is a highly controlled mobilisation of starch, which is maintained at an approximately constant rate through the night (Graf & Smith, 2011;Smith & Stitt, 2007). It has also been shown that organic acids (malate and fumarate) make an important contribution to nocturnal metabolismplants with reduced organic acid storage due to over-expression of plastidic malate dehydrogenase, are less fit under short day growth conditions and show a carbon starvation response, metabolising fatty acids and proteins to replace organic acids (Zell et al., 2010). Our data show that stored organic acids are also mobilized overnight both under warm and cold conditions (Dyson et al., 2016). Carbon export in Arabidopsis is thought to largely be in the form of sucrose, however, it is not clear, in detail, how this is synthesized from either starch or organic acids. Starch breakdown involves the formation of maltose (di-glucose) and glucose molecules, which are exported from the chloroplast. If synthesis of sucrose follows the same pathway as in the daytime, the glucose would need to be phosphorylated, by hexokinase, before being incorporated into sucrose. Sucrose phosphate synthase (SPS) is the major enzyme responsible for the diurnal synthesis of sucrose (Huber & Huber, 1996). It is not clear why this pathway would operate more efficiently at night than it does during the day. It may, therefore, be that an alternative pathway for sucrose synthesis at night exists. We did observe a substantial increase in the concentration of the main isoform of sucrose synthase (SS) following cold acclimation (Table S1).
SS produces sucrose from the reaction of UDP-glucose with fructose, in contrast to SPS, which reacts UDP-glucose with fructose-6-phosphate (Stein & Granot, 2019 The role of fumarate accumulation in Arabidopsis leaves is not, we conclude, a simple carbon sink effect; it is affecting the overall distribution of carbon between different storage pools in ways that cannot simply be explained by a loss of storage capacity. In order to better understand the possible processes affected by fumarate accumulation, we adopted a modelling approach. Using a network analysis of a metabolite-metabolite graph, we identified several potential pathways for fumarate synthesis. When modelling potential flux solutions for these pathways, only two of the identified pathways carried a significant flux. These involve export of fixed carbon from the chloroplast in the form of either phosphoglyceric acid (PGA) or triose phosphate (TPglyceraldehyde-3-phosphate and dihydroxy acetone phosphate). These compounds are all transported by the same translocatorthe triose phosphate translocator (TPT) -which is reported to have very similar transport properties for these different compounds (Knappe, Flugge, & Fischer, 2003). A comparison of plants lacking one or the other of these exports is, therefore, not possible via traditional experimental approaches, such as reverse genetics or using inhibitors.
Here, we have applied flux sampling (Herrmann et al., 2019) to gain an understanding of the impact of fumarate synthesis on wider metabolism. Flux sampling is a novel constraint-based modelling approach that has the advantage over flux balance analysis and flux variability analysis that the entire solution space can be captured in the form of a frequency distribution and hence it allows for a more precise comparison of different sets of constraints (Herrmann et al., 2019). Building on a published model (Arnold & Nikoloski, 2014), we show that export of carbon from the chloroplast can occur either as PGA or TP. The model was constrained using experimental data: carbon input and fluxes to major storage sinks were set according to measured physiological parameters, and the relative capacity of individual reactions were constrained in proportion to changes in the proteome (Table S1). The broad validity of this model comes from the observation that carbon export from the leaf, which was not constrained, varied in a way that is consistent with the experimental data (Figure 3c,d and Figure S3). Based on this, we conclude that an increase in the proportion of carbon exported as PGA is an initial response to cold in Col-0 plants. Furthermore, we were able to demonstrate that, in the Col-0 model, the ratio of PGA:TP export varies as a function of NADPH supply from the photosynthetic electron transport chain. Limitation in NADPH is known to be an early response to low temperature, as flux through the linear electron transport chain decreases (Clarke & Johnson, 2001). At the same time, cyclic electron flow at low temperature will tend to increase the ATP: NADPH ratio. NADPH in the chloroplast is essential for the conversion of PGA into TP. Limited NADPH supply will tend to favour PGA export. Thus, the relative export of PGA and TP from the chloroplast encodes information about the redox state of the chloroplast and, as a result, has the potential to act as a signal to the nucleus, controlling acclimation to changing conditions. PGA in the cytosol is converted to phosphoenolpyruvate (PEP) and then carboxylated by PEP carboxylase for form oxaloacetate (OAA). OAA is, in turn, reduced by malate dehydrogenase to form malate. In our modelling, the net accumulation of malate and fumarate was constrained to experimental levels; nevertheless, it remains unclear why flux to malate would be biologically different to flux to fumarate, given that these acids exist in equilibrium, catalysed by fumarase. One possible explanation although lies in the regulation of PEP carboxylase, which is subject to feedback inhibition by malate (Wedding, Black, & Meyer, 1990). If malate accumulates, this is liable to feedback to inhibit its own synthesis. Removing malate, converting it to fumarate, ensures that this pathway does not become limiting.
This may be essential to ensure that fluxes away from PGA are not sink limited, so ensuring the PGA concentrations in the cytosol reflect the rate of export and do not accumulate over the photoperiod.
In conclusion, we have shown that the ability to accumulate fumarate in Arabidopsis leaves has wide-ranging impacts on diurnal carbon partitioning in the leaf. Lack of fumarate synthesis results in widespread differences being seen across the proteome and prevents the acclimation of photosynthetic capacity to low temperature. Fumarate accumulation is important in facilitating diurnal carbon export from the leaf. Low temperatures inhibit diurnal sucrose export and this effect is exacerbated in plants lacking fumarate accumulation.
Modelling of leaf metabolism suggests that the relative export of PGA and TP may be an important signal reflecting the redox poise of the chloroplast. As such, it has the potential to act as a signal controlling the expression of nuclear genes, which underlies photosynthetic acclimation to environmental change.