Discussion
Previous work has shown that the ability to acclimate photosynthesis and
metabolism to changes in the abiotic environment plays an important role
in determining plant fitness and seed yield
(Athanasiouet al. , 2010). We have seen that acclimation of photosynthetic
capacity to both light and temperature involves metabolic signalling, as
evidenced by knock outs 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 retrograde 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 thefum2.1 mutant speaks against a direct role for phosphate in
controlling the acclimation of photosynthetic capacity– non-acclimatingfum2.1 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 well play a role in some of the
short-term regulatory responses seen on exposure to cold, however (Hurryet 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). This effect might be explained by a drop in sink strength,
however, if this is the case, then this is not alleviated by acclimation
at the whole plant level. At the end of the acclimation period, the
proportion of carbon retained in the leaf during the day is even lower
than on the first day of acclimation. 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). At the same time, our data show that stored
organic acids are also mobilised. 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. SS is generally believed however 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, fum2.1 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 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 (see methods for details), 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). Comparison of plants
lacking one or other of these exports is therefore not possible via
traditional experimental approaches such as reverse genetics or using
inhibitors.
The main modelling approaches adopted to understand metabolism can be
classified as kinetic or constraint-based models (Herrmann, Schwartz, &
Johnson, 2019). Kinetic models require detailed kinetic information
about enzymes and are computationally expensive, limiting the complexity
of systems which can be analysed. Constraint based models can be much
more complex, however the most widely used approach, flux balance
analysis, has the disadvantage that it requires the assumption of one or
more objective functions – model solutions are established based on a
presumed cellular goal, often maximising a portmanteau function
describing “biomass”. This introduces a researcher bias into the
modelling process. Recently, we highlighted an alternative approach,
flux sampling, which eliminates this bias. Rather than using an
objective function, the entire solution space of the model is explored
and a frequency distribution of different flux solutions considered for
each metabolic reaction. This allows us to define the range and the
likelihood of possible solutions.
Here we have applied flux sampling to gain an understanding of the
impact of fumarate synthesis on wider metabolism. 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 constrained in
proportion to changes in the proteome (Table S1). The broad validity of
this model comes from the observation that carbon export, which was not
constrained, varied in a way consistent with the experimental data
(Figure 3, Figure S3). Based on this, we conclude that 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 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
represents a potential new retrograde signal which signal to the nucleus
the redox state of the chloroplast.
PGA in the cytosol is converted to phospho-enol -pyruvate (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. 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 retrograde signal reflecting the redox poise of the
chloroplast.
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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