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; Fahnenstichet 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 4oC than 20oC on the first day of cold and increased each day, until Day 4 of cold treatment (Figure 2 a). The amount of starch seen at the end-of-day was higher infum2.2 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 mobilised overnight throughout the acclimation period. Infum2.2 , 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 2 b). This was accompanied by an increase in the accumulation of malate (Figure 2 c), 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 fum2.2but 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 from Figures 1-2, S1 and from Dysonet al. (2016) to perform a carbon budget audit (Figure 3). The accumulation of starch, sucrose, fumarate and malate were estimated as the difference in beginning- and end-of-day concentrations (Figure 2, S1). Rates of gas exchange were measured under growth conditions at intervals through the photoperiod (See Figure 1c and Dyson et al. , 2016) and 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 infum2.2 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 3 a, 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 3 d). 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.
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 4 a), 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 andfum2.2 , albeit to a much smaller extent in the latter. We were able to estimate the relative abundance of 2427 polypeptides, based on a minimum of 3 unique peptides per protein. Principal Component Analysis of proteomic data indicates that the proteomes of Col-0 andfum2.2 differ already under 20oC conditions, however there is a clear separation of cold treated plants from their corresponding 20oC controls in both genotypes (Figure 4 b). Cluster analysis was performed using data from the 2015 proteins which showed significantly altered expression in one of more conditions. As expected, given the total increase in protein, the most common response to cold is for proteins to increase in Col-0 but less so or not at all in fum2.2 (Figure 4 c,d,g). A far smaller cluster of proteins increased in both genotypes following cold treatment (Figure 4 e) whilst a few proteins decreased in response to cold in fum2.2(Figure 4 f).
Examination of the relative concentration of proteins involved in the photosynthetic electron transport chain demonstrated that only subtle changes were occurring (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 (Kerenet al. , 2005), however components of the PSII core did not change significantly in response to cold. Amongst the proteins of the photosynthetic electron transport chain, 2 of the 4 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 cytochrome b6f abundance in Col-0, whilst infum2.2 there is no evidence for a change in the abundance of this complex. While plastocyanin showed no change in abundance in either genotype, the only detected ferredoxin isoform increased in both genotypes, as did one of the four detected FNR isoforms. 4 of the detected 8 ATP synthase subunits were upregulated in Col-0, whilst 2 showed a significant change in fum2.2 . Taking these data overall, we conclude that there were no changes in photosystem stoichiometry in response to cold in either genotype and that changes in electron transport proteins in Col-0 were either reduced or absent infum2.2 . There were however consistent and significant differences between the genotypes both in warm and cold conditions, with a greater abundance of subunits of all complexes seen in Col-0.
In contrast to the components of the photosynthetic electron transport chain, changes in the enzymes associated with the Benson Calvin cycle gave a clearer and more consistent pattern of response (Figure 5). The CO2 fixing enzyme, Rubisco, is by far the most abundant protein in the leaf. We were able to quantify the chloroplast-encoded large subunit (RBCL) and 2 isoforms of the nuclear-encoded small subunit, RBCS. All increased significantly in Col-0 in response to cold, with a mean 1.8-fold increase in relative abundance. In fum2.2there was no significant change in RBCL abundance. One isoform of RBCS increased significantly, whilst the other decreased to a similar extent. Combining data from both isoforms, there was no significant change in RBCS abundance. For other reactions associated with the Benson Calvin cycle, we were able to quantify a total of 20 distinct proteins, including isoforms of specific enzymes. In Col-0, 14 of these significantly increase, whilst 5 decreased. In fum2.2 , although 6 enzymes involved in the Benson Calvin cycle came out as significantly increased, the overall extent of this change was lower.
Across other major metabolic pathways – including starch and sucrose synthesis, glycolysis and the tricarboxylic acid cycle, similar 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 summarise, 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 . 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