Each of four starch debranching enzymes (DBE) is distinct and highly conserved across the plant kingdom; however, the specific functions of these proteins in carbohydrate metabolism are not well understood. DBEs function in both biosynthesis and degradation of starch, and two have been shown to function as multimers in various quarternary structures that can contain one or more DBE proteins, i.e. ISA1 homomultimers and ISA1/ISA2 heteromultimers. This study characterizes potential functional relationships between the three isoamylase-type DBE proteins (ISA) of Arabidopsis using a comprehensive bioinformatics analysis and promoter fusion approach to determine tissue-, subcellular-, and temporal specificity of gene expression. The results reveal complementary sets of expression patterns, in particular that AtISA1 (known to be involved in starch biosynthesis) and AtISA2 (a non-catalytic polypeptide) are co-expressed in some conditions in the absence of AtISA3 (known to be involved in starch degradation), whereas in other conditions AtISA2 is co-expressed with AtISA3 in the absence of AtISA1 (AtISA2and AtISA3, but not AtISA1, are co-expressed specially in root columella cells and leaf hydathodes). Thus, AtISA2 may function in starch degradation, in addition to its role in starch biosynthesis. AtISA3 and several other potential regulatory genes, starch metabolic genes, and transcription factors, are specifically induced during cold acclimation; these transcription factors are candidates for involvement of cold-induced changes in starch metabolism. Finally, bioinformatics analysis using MetaOmGraph (http://www.metnetdb.org/MetNet_MetaOmGraph.htm) identifies Arabidopsisgenes of unknown function that might be involved in starch metabolism in the cold.