Plants have evolved strategies to tightly regulate metabolism during acclimation to a changing environment. Low temperature significantly constrains distribution, growth and yield of many temperate plant species. Exposing plants to low but non-freezing temperature induces a multigenic processes termed cold acclimation, which eventually results in an increased freezing tolerance. Cold acclimation comprises reprogramming of the transcriptome, proteome and metabolome and affects communication and signaling between subcellular organelles. Carbohydrates play a central role in this metabolic reprogramming. This review summarizes current knowledge about the role of carbohydrate metabolism in plant cold acclimation with a focus on subcellular metabolic reprogramming, its thermodynamic constraints under low temperature and mathematical modelling of metabolism.

Carbohydrates are direct products of photosynthetic CO₂ assimilation. Within a changing temperature regime, both photosynthesis and carbohydrate metabolism need tight regulation to prevent irreversible damage of plant tissue and to sustain energy metabolism, growth and development. Due to climate change, plants are and will be exposed to both long‐term and short‐term temperature changes with increasing amplitude. Particularly sudden fluctuations, which might comprise a large temperature amplitude from low to high temperature, pose a challenge for plants from the cellular to the ecosystem level. A detailed understanding of fundamental regulatory processes, which link photosynthesis and carbohydrate metabolism under such fluctuating environmental conditions, is essential for an estimate of climate change consequences. Further, understanding these processes is important for biotechnological application, breeding and engineering. Environmental light and temperature regimes are sensed by a molecular network that comprises photoreceptors and molecular components of the circadian clock. Photosynthetic efficiency and plant productivity then critically depend on enzymatic regulation and regulatory circuits connecting plant cells with their environment and re‐stabilising photosynthetic efficiency and carbohydrate metabolism after temperature‐induced deflection. This review summarises and integrates current knowledge about re‐stabilisation of photosynthesis and carbohydrate metabolism after perturbation by changing temperature (heat and cold).

Stabilization of central carbohydrate metabolism plays a key role in plant stress response. Carbohydrates are substrate for numerous metabolic and stress‐responsive reactions and have been shown to be involved in diverse signalling processes on a whole‐plant level. Regulation of enzymatic sucrose synthesis and degradation is well‐known to be central to many stress‐related processes as it significantly impacts stress tolerance. Leaf sucrose metabolism involves sucrose cleavage by invertases and ATP‐consuming resynthesis catalysed by hexokinase and sucrose phosphate synthase. These reactions establish a metabolic cycle. To study the physiological role of sucrose cycling, a kinetic model was developed to simulate dynamics of subcellular sugar concentrations in Arabidopsis thaliana under combined cold and high‐light stress. Model simulation revealed that subcellular reprogramming of invertase‐driven sucrose cleavage varies substantially between natural accessions of Arabidopsis which differ in their cold tolerance levels. A stress‐induced shift of sucrose cleavage from the cytosol into the vacuole could only be observed for the tolerant accession while the susceptible accession increased the cytosolic proportion of sucrose cleavage. Under stress, reduction in vacuolar invertase activity significantly affected maximum quantum yield of photosystem II and CO₂ assimilation rates. While wild‐type plants circumvented a limitation of sucrose cleavage by increasing vacuolar invertase activity, mutant plants were not able to compensate their deficiency of vacuolar by cytosolic activity. Consequently, the capacity for cytosolic hexose generation was lower than for enzymatic hexose phosphorylation suggesting a role of vacuolar invertase activity in preventing a limitation in cytosolic hexose metabolism under stress.Enzymes: Invertase, EC 3.2.1.26; Hexokinase, EC 2.7.1.1.

Summary: Plant cells are characterized by a high degree of compartmentalization and a diverse proteome and metabolome. Only a very limited number of studies has addressed combined subcellular proteomics and metabolomics which strongly limits biochemical and physiological interpretation of large‐scale ’omics data. Our study presents a methodological combination of nonaqueous fractionation, shotgun proteomics, enzyme activities and metabolomics to reveal subcellular diurnal dynamics of plant metabolism. Subcellular marker protein sets were identified and enzymatically validated to resolve metabolism in a four‐compartment model comprising chloroplasts, cytosol, vacuole and mitochondria. These marker sets are now available for future studies that aim to monitor subcellular metabolome and proteome dynamics. Comparing subcellular dynamics in wild type plants and HXK1‐deficient gin2‐1 mutants revealed a strong impact of HXK1 activity on metabolome dynamics in multiple compartments. Glucose accumulation in the cytosol of gin2‐1 was accompanied by diminished vacuolar glucose levels. Subcellular dynamics of pyruvate, succinate and fumarate amounts were significantly affected in gin2‐1 and coincided with differential mitochondrial proteome dynamics. Lowered mitochondrial glycine and serine amounts in gin2‐1 together with reduced abundance of photorespiratory proteins indicated an effect of the gin2‐1 mutation on photorespiratory capacity. Our findings highlight the necessity to resolve plant metabolism to a subcellular level to provide a causal relationship between metabolites, proteins and metabolic pathway regulation.

Although compartmentation is a key feature of eukaryotic cells, biological research is frequently limited by methods allowing for the comprehensive subcellular resolution of the metabolome. It has been widely accepted that such a resolution would be necessary in order to approximate cellular biochemistry and metabolic regulation, yet technical challenges still limit both the reproducible subcellular fractionation and the sample throughput being necessary for a statistically robust analysis. Here, we present a method and a detailed protocol which is based on the non-aqueous fractionation technique enabling the assignment of metabolites to their subcellular localization. The presented benchtop method aims at unravelling subcellular metabolome dynamics in a precise and statistically robust manner using a relatively small amount of tissue material. The method is based on the separation of cellular fractions via density gradients consisting of organic, non-aqueous solvents. By determining the relative distribution of compartment-specific marker enzymes together with metabolite profiles over the density gradient it is possible to estimate compartment-specific metabolite concentrations by correlation. To support this correlation analysis, a spreadsheet is provided executing a calculation algorithm to determine the distribution of metabolites over subcellular compartments. The calculation algorithm performs correlation of marker enzyme activity and metabolite abundance accounting for technical errors, reproducibility and the resulting error propagation. The method was developed, tested and validated in three natural accessions of Arabidopsis thaliana showing different ability to acclimate to low temperature. Particularly, amino acids were strongly shuffled between subcellular compartments in a cold-sensitive accession while a cold-tolerant accession was characterized by a stable subcellular metabolic homeostasis. Finally, we conclude that subcellular metabolome analysis is essential to unambiguously unravel regulatory strategies being involved in plant-environment interactions.

Regulation of intracellular sugar homeostasis is maintained by regulation of activities of sugar import and export proteins residing at the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 (ERDL4) protein, a member of the monosaccharide transporter family, resides in the vacuolar membrane in Arabidopsis (Arabidopsis thaliana). Gene expression and subcellular fractionation studies indicated that ERDL4 participates in fructose allocation across the tonoplast. Overexpression of ERDL4 increased total sugar levels in leaves due to a concomitantly induced stimulation of TONOPLAST SUGAR TRANSPORTER 2 (TST2) expression, coding for the major vacuolar sugar loader. This conclusion is supported by the finding that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. ERDL4 activity contributing to the coordination of cellular sugar homeostasis is also indicated by two further observations. Firstly, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, and secondly, the ERDL4 gene is markedly expressed during cold acclimation, representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger rosettes and roots, a delayed flowering time, and increased total seed yield. Consistently, erdl4 knockout plants show impaired cold acclimation and freezing tolerance along with reduced plant biomass. In summary, we show that modification of cytosolic fructose levels influences plant organ development and stress tolerance.