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 a variety of forms. In Arabidopsis, the principal leaf carbon stores are starch and organic acids, especially fumarate and malate (Chia, Yoder, Reiter, & Gibson, 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). We measured the beginning and end of photoperiod concentrations of starch, fumarate and malate in Col-0 and fum2.1 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. Starch accumulation was greater than 20oC on the first day of cold and continued to increase each day, until Day 4 of cold treatment, in both wild type and mutant plants (Figure 2a). The amount of starch seen at the end of day was higher in fum2.1 than in Col-0 throughout the experiment, with the absolute difference between genotypes being approximately constant. In Col-0, essentially all starch accumulated during the day was mobilised overnight throughout the acclimation period, however in fum2.1 , 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 fum2.1 , 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 fum2.1but then fell the following day, before rising again towards the end of the treatment period in response to cold treatment.
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, as plants acclimated to cold, this cycle was lost, with beginning of day sucrose content increasing and end of day content decreasing progressively through the week. At the end of the cold treatment,fum2.1 contained slightly more sucrose than Col-0 but in neither case was a significant diel variation seen.
To understand better how the partitioning of carbon between different pools varies in response to acclimation, 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). The rate of photosynthesis was measured under growth conditions at intervals through the photoperiod (Dysonet al. , 2016) and used to calculate the integrated daily photosynthesis. Diurnal respiration was estimated based on gas exchange measurements during short interruptions in irradiance (Figure 1c) and assumed to be constant through the photoperiod.
In Col-0 at 20°C, stored carbon accounted for approx. 1/3 of total fixed carbon. When combined with the estimated rate of daytime respiration, slightly over half of the fixed carbon could be accounted for. The remaining carbon is assumed to be exported from the leaf or remain in the form of other organic compounds not measured here. It is assumed that the bulk of this carbon will be exported, consistent with estimates by Lundmarket al. (2006). Plants of fum2.1 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, we conclude that total unaccounted carbon, primarily diurnal export, is lower infum2.1 than in Col-0.
During the first day of exposure to cold, there were notable changes in carbon distribution between different sinks (Figure 3b,e). 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 fum2.1 . When plants were cold-acclimated for 7 days, this effect became more marked (Figure 3c,f). From this, we conclude that there is a substantial inhibition of diurnal carbon export from the leaf in cold acclimated plants of both genotypes. Given that all metabolite pools retain a diel turnover, we conclude that acclimation to cold involves a shift from diurnal carbon export to nocturnal processes.