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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