Introduction
Through their lifecycle, plants experience environmental conditions that vary on timescales from seconds to seasons. Arabidopsis thalianais typically a winter annual, germinating in the autumn and persisting through the winter, prior to flowering in spring (Grimeet al. , 1988). Across this time-period, mean daily temperatures may vary by 20°C or more. To optimise survival and growth, plants acclimate to changes in temperature, altering their investment in different processes to suit the conditions experienced (Ruelland, Vaultier, Zachowski, & Hurry, 2009; Walters, 2005). The process of acclimation can involve both structural changes, with tissues differing when developed in different conditions, and relatively rapid (days-weeks) dynamic responses, which track changes in the environment (Athanasiou, Dyson, Webster, & Johnson, 2010; Walters, 2005). Changes in light intensity and temperature are both known to trigger acclimation (Athanasiou et al. , 2010; Dyson et al. , 2015; Dysonet al. , 2016; Huner et al. , 1993; Savitch et al. , 2001; Stitt & Hurry, 2002; Strand, Hurry, Gustafsson, & Gardestrom, 1997; Walters & Horton, 1994).
Exposure to low temperature triggers a complex array of acclimation responses. A significant amount of work in plant cold responses has focused on the acquisition of freezing tolerance (see Knight & Knight, 2012 for a review). In addition, it is recognised that plants can optimise their metabolism to suit changing conditions. For example, 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 synthesised 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 cellular concentration of inorganic phosphate (Pi) which in turn limits the export of triose phosphate (TP) from the chloroplast. This inhibits photosynthesis, as the 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. Hurry et al. (2000) proposed, based on the responses of different mutants with altered phosphate content, that Pi depletion provides a signal for cold acclimation of SPS content.
Recently, we re-examined the responses of Arabidopsis to low temperature (Dysonet 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 (Pracharoenwattanaet 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 accumulatedhigher concentrations of phosphorylated intermediates. Crucially, whilst 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 results from metabolic modelling constrained using experimental data, we propose that 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.