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.