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
(Athanasiouet al. , 2010). Here, we have presented evidence that acclimation
to cold is also important in determining fitness and seed yield -
wild-type Col-0 plants are unaffected by changes in temperature, whilefum2.2 , 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 capacity to
both light and temperature involves metabolic signalling, as evidenced
by knockouts of either the glucose-6-phosphate/phosphate translocator
GPT2 or of the cytosolic fumarase FUM2 being deficient in their
acclimation responses
(Athanasiouet al. , 2010; Dyson et al. , 2015; Dyson et al. ,
2016; Miller et al. ,
2017). Recently,
Weise et al. (2019) confirmed that the increase in GTP2
transcripts in response to environmental change is linked to TPT export
and that this link is an important feature of day-time metabolism. Here
we show that cold acclimation involves a reconfiguration of diel carbon
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 the cold acclimation of photosynthesis in Arabidopsis
highlighted the importance of sucrose synthesis and, specifically, the
activity of sucrose phosphate synthase (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 thefum2.2 mutant speaks against a direct role for phosphate in
controlling the acclimation of photosynthetic capacity. Non-acclimatingfum2.2 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 down-stream 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 to plants maintained at 20°C
(Figure 3 c,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 the reduction in 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
metabolism – plants 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 (Zellet al. , 2010). Our data show that stored organic acids are also
mobilised 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
synthesised, either from 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). SS would in theory
represent a lower energy pathway to generate sucrose from hexoses.
However, SS is generally believed to operate in the direction of sucrose
breakdown, releasing glucose for metabolic processes. It is therefore
not obvious why SS would normally be present in mature leaves, which are
net sources for carbon, and which do not store sucrose to a significant
degree. It is possible though that night-time sucrose synthesis may
involve SS.
The synthesis of fumarate has an impact on diurnal carbon export from
the leaf which cannot be explained by a reduction in storage capacity.
At 20°C, fum2.2 plants maintain a similar photosynthetic rate but
store a larger proportion of total carbon in the leaf than do wild-type
Col-0 plants. Although fumarate accumulation is inhibited, this is
largely compensated for by increased accumulation of malate. At the same
time, starch storage is also greater. As in Col-0, short term exposure
to cold increases this effect and following 7 days acclimation, only a
very small proportion of fixed carbon is exported during the day.
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 (TP – glyceraldehyde-3-phosphate and dihydroxy acetone
phosphate). These compounds are all transported by the same translocator
– the 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 3 c,d, 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, 2000). 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 though 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.
Acknowledgements: We would like to thank Drs David Knight,
Ronan O’Cualain and Julian Selley (University of Manchester) for their
help with the proteomic analysis. This work was supported by a grant
from the Biotechnology and Biological Sciences Research Council (BBSRC;
BB/J04103/1). HAH and MAEM were supported BBSRC studentships
(BB/M011208/1).
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