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Accessions of Arabidopsis thaliana originating from climatically different habitats show different levels of cold acclimation when exposed to low temperatures. The central carbohydrate metabolism plays a crucial role during this acclimation. Subcellular distribution of carbohydrates over the compartments cytosol, vacuole and plastids, and putative interactions of the compartments, are analyzed in three differentially cold-tolerant accessions of Arabidopsis thaliana, originating from the Iberian Peninsula (C24), Russia (Rschew) and Scandinavia (Tenela), respectively. Subcellular carbohydrate concentrations were determined by applying the nonaqueous fractionation technique. Mathematical modeling and steady-state simulation was used to analyse the metabolic homeostasis during cold exposure. In all accessions, the initial response to cold exposure was a significant increase of plastidial and cytosolic sucrose concentrations. Raffinose accumulated in all cellular compartments of cold-tolerant accessions with a delay of 3 d, indicating that raffinose accumulation is a long-term component of cold acclimation. Minimal rates of metabolite transport permitting steady-state simulations of metabolite concentrations correlated with cold tolerance, indicating an important role of subcellular re-distribution of metabolites during cold acclimation. A highly regulated interplay of enzymatic reactions and intracellular transport processes appears to be a prerequisite for maintaining carbohydrate homeostasis during cold exposure and allowing cold acclimation in Arabidopsis thaliana., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)