Arabidopsis accessions differ significantly in carbohydrate compartmentation during cold exposure
Many studies have emphasized the importance of primary carbohydrate metabolism during cold acclimation, and various regulatory instances significantly affecting this process have been identified (Strand et al., 1997, 1999; Klotke et al., 2004; Zuther et al., 2004; Lundmark et al., 2006; Nägele et al., 2011). Yet, due to the complexity of the cellular network of metabolic interactions and compartmentalization, still many aspects of metabolism and its regulation are not well understood. As already stated, compartmentalization of metabolism in eukaryotic cells significantly affects activity and function of enzymes as well as concentrations and regulatory impact of metabolites (Lunn, 2007; Klie et al., 2011). While compartmentalization of enzymes can be predicted computationally based on the presence of sequence motifs (Emanuelsson et al., 2000), the subcellular allocation of metabolites is difficult to assess because of redundant biochemical pathways, as for example the cytosolic and plastidic pathways of carbohydrate oxidation (Masakapalli et al., 2010), and various transport processes enabling rapid exchange of small compounds (Linka & Weber, 2010; Wingenter et al., 2010; Schneider et al., 2012). In the present study, compartmentalization of the central carbohydrate metabolism was studied to gain further insight into regulatory instances involved in the cold acclimation process in Arabidopsis thaliana.
Cold tolerance, expressed as LT50 values, showed significant differences before and during a 1-wk period of cold acclimation in three accessions, C24, Rsch and Te. Dynamics of cold acclimation revealed a fast increase in tolerance during the first 3 d, which is in accordance with previous findings (Nägele et al., 2011) and showed that Te from Scandinavia is the most tolerant accession at any time point of cold acclimation. Substantial redistribution of metabolites during the first 3 d in the cold occurred for sucrose and hexoses, while cytosolic increase of the raffinose concentration was delayed. In vitro experiments performed by Hincha et al. (2003) showed that sucrose and raffinose can protect large unilamellar liposomes from damage during drying, indicating a possible role in protecting the plasma membrane during freeze-induced dehydration. However, a lack of raffinose in a raffinose synthase mutant isolated from the cold-tolerant accession Col-0 did not alter cold tolerance determined as freeze–thaw stability of the plasma membrane (Zuther et al., 2004). In agreement with this, cytosolic raffinose content did not correlate with cold tolerance in the present study, again implying that the plasma membrane is not a target for protection by raffinose, as has recently been demonstrated (Knaupp et al., 2011).
Besides raffinose, cytosolic sucrose concentration was also not correlated with cold tolerance at day 7 of the cold exposure. However, the sucrose concentration had already rapidly increased during the first day of cold exposure, thus accompanying the significant decrease of the LT50 in all accessions. It has been hypothesized that cold acclimation is the result of a sequential accumulation and disappearance of several metabolites (Alberdi et al., 1993). In this sense, sucrose could serve as an early protectant of cellular membranes, but is later substituted by other compounds. In line with this, cytosolic sucrose was highest at day 1 in Rsch, when the increase in cold tolerance was maximal in this accession, while it peaked at day 3 in Te that displayed a larger gain in tolerance between day 1 and 3 than did Rsch. The very high concentrations of cytosolic sucrose at day 1 in Rsch and day 3 in Te, reaching concentrations of 45–50 mM, may also indicate saturation during cold acclimation. It might thus be speculated that cytosolic sucrose accumulates rapidly after cold exposure, serving as a transient cryoprotectant for cellular membranes at early stages of cold exposure, while later it becomes replaced by a metabolically less critical compound, for example, raffinose. At that stage, sucrose in the cytosol would serve as a substrate for the synthesis of other cryoprotectants or has a regulatory role in cold acclimation. In this context, it is interesting that raffinose accumulation follows the rise in cytosolic sucrose. As sucrose and galactinol are the substrates for raffinose synthesis taking place in the cytosol (Peterbauer & Richter, 2001), the increase of sucrose in the cytosol might be considered a trigger for raffinose production. Raffinose could then be transported across the chloroplast envelope, probably by an active transport mechanism as proposed by Schneider & Keller (2009), serving in the plastids as a cryoprotectant of the thylakoids (Knaupp et al., 2011). However, both cytosolic and plastidial sucrose content increased significantly faster than raffinose content, suggesting an additional transitory protective effect of sucrose for the thylakoids. This is difficult to test because of the simultaneous role of sucrose as substrate for raffinose synthesis. A likely reason for a substitution of sucrose by raffinose might be the reduced metabolic reactivity of raffinose and its marginal regulatory influence on primary carbon metabolism.
While an increase of plastidial sucrose and raffinose was also found in cold-acclimated cabbage (Santarius & Milde, 1977) and Arabidopsis, accession Columbia (Col-0) (Knaupp et al., 2011), a decrease of the cytosolic content has only been shown for Arabidopsis, accession Col-0 (Knaupp et al., 2011). The obvious difference in behaviour of Col-0 and the accessions investigated in the present study might be related to the longer time of cold exposure, that is, 14 vs 7 d, used by Knaupp and co-workers. This would imply that cold acclimation is either not saturated after 7 d, or that during a longer cold exposure, leaves develop that are in a metabolic state different from those that were shifted from warm to cold. The latter has clearly been demonstrated by Strand et al. (1999). However, it is difficult to judge whether 14 d in the cold would be long enough to bring about such an effect, because the rosette of the plants is a mixture of shifted and newly developed leaves. Also, a deviation of the laboratory strain Col-0 from the accessions used in our study cannot be excluded.
Steady-state simulations indicate differences in metabolic reprogramming
The rapid accumulation of sucrose and other metabolites observed in all accessions can be considered an immediate consequence of the cold-induced thermodynamic effects on enzyme activities and/or transport rates, which can be deduced from the experimentally determined maximum turnover rates. For example, vacuolar and cytosolic invertase activity dropped approximately five-fold in the cold in all accessions, while the reduction in hexokinase activity was even stronger directly after the temperature shift, and activity came back to only c. 20% of the initial value after 7 d (Fig. 4). However, sugar accumulation would also result from reduced consumption of assimilates in the cold when growth is stalled, or from an at least transiently reduced export of carbohydrates to sink organs, as predicted by modeling the impact of cold acclimation on photosynthesis and assimilate export to sink organs (Nägele et al., 2011). Although thermodynamic and growth effects of low temperature should be similar for all accessions, simulated reaction rates of steady-state models discriminated the cold-sensitive from the cold-tolerant accessions, the latter showing a more pronounced reduction predominantly of cytosolic sucrose cleavage and hexose phosphorylation. Reaction rates in the sensitive accession C24 were also reduced at 4°C, yet the reduction was not as strong as in Rsch and Te. While this proves that the cold response of the tolerant accessions is more distinct, simulations of reaction rates at the whole-cell level did not reveal whether differences in turnover rates between tolerant accessions and C24 would result in substantial differences at the metabolite level that could explain the differential cold tolerance.
This led us to implement intracellular redistribution of metabolites during cold acclimation in our model of steady-state simulations of carbohydrate metabolism in order to investigate whether metabolite dynamics at the compartment level could be more significant than at the whole-cell level. To this end we first identified the minimal rates of metabolite exchange between cellular compartments that would allow establishing a metabolic homeostasis, that is, a steady state, which would be compatible with the reduced enzyme activities as well as the modified metabolite concentrations observed in cold exposed plants. Certainly it would have been preferable to measure transport activities. However, the insufficient characterization of many of the transporters precluded this concept. We thus applied a modeling approach using stepwise increments of upper and lower bounds for the transport rate constants. This was done until a steady state of metabolite concentrations could be simulated within the experimentally determined ranges of enzyme activities given as the standard deviations of the measurements. We then compared the calculated limiting transport rates obtained for cold-sensitive and cold-tolerant accessions.
While simulated rates of metabolite transport did not change significantly during cold exposure in C24, modeling suggested substantially elevated rates of sucrose transport across the chloroplast envelope, as well as hexose transport across the tonoplast, in Rsch and even more so in Te. Additionally, sucrose transport across the tonoplast was modeled to be strongly elevated in cold-acclimated Te. As mentioned above, because knowledge on intracellular sugar transporters is still incomplete, especially regarding sucrose and raffinose import into plastids (Schneider & Keller, 2009), the results of the simulations depend on the assumption that bidirectional transport of hexoses, sucrose and raffinose is possible across the plastid envelope as well as the tonoplast. This provided, our findings indicate that intracellular metabolite transport could make a substantial contribution to sustaining metabolic homeostasis during cold acclimation. Given that only few metabolites, at the whole-cell level, could be correlated to cold tolerance of various accessions of Arabidopsis (Hannah et al., 2006), intracellular re-distribution might in addition also be important to achieve protection of cellular organelles such as plastids against damage during freeze–thaw cycles.
As stated above, raffinose synthesis is delayed when compared with the time course of cold acclimation, and thus sucrose transport into the plastids might be necessary to protect the thylakoids until raffinose accumulation catches up. A very interesting result of the simulations was that, after 3 d of cold exposure, the rate of sucrose transport across the chloroplast envelope and the tonoplast could even discriminate the two cold-tolerant accessions Rsch and Te. The latter, which at day 3 was c. 1.7°C more cold-tolerant than Rsch, not only displayed higher transport rates, but also had significantly higher sucrose concentrations in the cytosol and vacuole, while hexoses predominantly accumulated in Rsch. Our data indicate that an increased transport of sucrose from cytosol into the vacuole allowed for more sucrose to accumulate in mesophyll cells of Te, probably because of the low vacuolar invertase activity. In contrast, high cytosolic hexose concentrations were observed in Rsch at day 3 of the cold exposure, which were again reduced at day 7, when the cold tolerance of Rsch drew near that of Te. It is tempting to speculate that the high cytosolic hexose concentration in Rsch at day 3 could be related to the low gain in cold tolerance between days 1 and 3 in this accession. High hexose concentrations may cause feedback-inhibition of cytosolic invertase, thus inhibiting sucrose cyling, which is important for buffering primary carbohydrate metabolism against environmental disturbances (Nägele et al., 2010). In fact, cytoslic sucrose cleavage was calculated to be higher in Te than in Rsch and, thus, higher transport rates for hexoses into the vacuole must be claimed for Te. In this respect, it is interesting that the tonoplast monosaccharide transporter, TMT1, which re-directs glucose and fructose from the cytsol into the vacuole, was demonstrated to be strongly induced at low temperature in the cold-tolerant accession Col-0 (Wormit et al., 2006). This may, in fact, point to a major difference in the cold-acclimation strategies of Rsch and Te: by shuffling sucrose into the vacuole, Te reduces accumulation of hexoses in the cytosol which in turn allows for a sustained cytosolic sucrose cycling and a stabilization of primary carbohydrate metabolism against environmental changes (Nägele et al., 2010). An exchange of sucrose between cytosol and vacuole may thus be important for supporting regulation of cytosolic reactions of primary carbon fixation.
Of course, a final proof of the concept of limiting transport rates as applied in this work would rely on measuring transport activities in planta. However, under conditions of insufficient knowledge of transporters involved, the approach of mathematically simulating transport processes at limiting transporter activities may help to understand how cold-induced processes taking place at the intracellular membranes could be involved in cold acclimation. The simulations plausibly demonstrated the importance of intracellular re-distribution of carbohydrates and indicated that, besides raffinose transport into the plastids, which has already been demonstrated experimentally (Schneider & Keller, 2009), transport of sucrose into the plastids and the vacuole as well as hexose transport across the tonoplast might become limiting for cold acclimation in Arabidopsis thaliana. Taking into account that the volumes of compartments may change during the process of cold acclimation, particularly the cytosolic and vacuolar volume (Strand et al., 1999), predicted minimum rates of transport, as well as metabolite interconversion for steady-state simulations, would vary to a corresponding extent. Yet, as long as the effect on compartment volumes can be assumed to be systematic across all considered accessions, the inference derived from our model simulations would hold although absolute values of the minimal transport rates may deviate. This would not be the case if volume changes were different for the different accessions. However, investigations by us (Hannah et al., 2006) and others (Cook et al., 2004) have shown that global metabolite changes during cold acclimation, which would result from changes in cellular water content, are very similar for cold-sensitive as well as cold-tolerant accessions.
Taken together, the present study reveals a significant elevation of cytosolic, plastidial and vacuolar sucrose concentrations as an early response to cold exposure, being stronger in the cold-tolerant accessions Rsch and Te as compared to the sensitive accession C24. While C24 did not show any further alterations in subcellular carbohydrate allocation, the tolerant accessions displayed an exchange of raffinose for sucrose especially in the plastids. The substitution of sucrose by raffinose is in agreement with the protective role of raffinose for the thylakoids and may indicate the need for a replacement of sucrose by a metabolically less reactive compound in tolerant accessions. Steady-state simulations of carbohydrate metabolism suggested that intracellular carbohydrate transport processes are indispensable in establishing a low temperature compatible carbohydrate homeostasis in tolerant accessions, while in the cold-sensitive accession C24 this adjustment appears less pronounced.