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Low temperature is one of the most severe factors that limit plant growth and yield. Temperate plants develop enhanced tolerance to freezing temperature after exposure to a period of non-freezing temperature, known as cold acclimation. Rapid and complex changes in gene expression and metabolites were observed during cold acclimation, but discrepancies between gene expression and metabolites were poorly characterized.In this study, two-dimensional difference gel electrophoresis （2-D DIGE） analysis of sub-fractions of Arabidopsis proteome coupled with spot identification by tandem mass spectrometry were used to identify early （within 2-hr） cold-responsive proteins in Araidopsis. A number of cold-responsive proteins were identified, which include those involved in starch degradation, the tricarboxylic acid cycle, sucrose metabolism, and stress response.Four proteins involved in starch degradation, including DPE2, GWD1 （SEX1）, GWD3, PHS2, were regulated during early cold response. DPE2, which showed rapid increase in the microsomal faction in as early as 5 minutes, exhibited reduced enzymatic activity, solubility and was aggregated after cold treatment. Its T-DNA knockout line showed increased freezing and drought tolerance, correlated with increased maltose accumulation previously reported. This data indicate that rapid increase of maltose content during cold acclimation could be partially explained by reduced maltose degradation because of lower DPE2 solubility.In summary, we identified a number of early cold-responsive proteins form different sub-fraction of Arabidopsis by 2D-DIGE and tandem mass spectrometry technology, these proteins are involved in starch degradation, the tricarboxylic acid cycle, sucrose metabolism, and stress response. Furthermore, we analyzed DPE2 protein function in the plant cold stress and demonstrated that DPE2 is a novel early cold responsive protein in Arabidopsis. Our data provide evidence that posttranscriptional modification of carbohydrate metabolizing proteins, such as protein aggregation or changes in subcellular localization, is a crucial factor in early cold response.