This hydrolytic step is obviously required during R428 starch re-mobilization, but is interestingly also required for normal starch synthesis. Debranching enzymes can be divided into two classes: isoamylase and limit-dextrinase. The isoamylase class is subdivided into proteins designated as ISA1, ISA2 and ISA3. ISA3 and LDA are mainly involved in starch breakdown, whereas ISA1 and ISA2 participate in starch synthesis. Current evidences suggest that ISA1 is an active enzyme, whereas ISA2 proteins are non-catalytic due to changes in 6 out of 8 key amino acids within the active site. In all plant species studied, ISA1 and ISA2 are found in heteromultimeric complexes, in which the ISA2 subunit is proposed to have a regulatory function or confer substrate specificity. In addition, ISA1 homomultimers have been shown to occur in rice and maize endoperm and proposed for C. reinhardtii. Thus far, homomultimer forms of ISA1 have not been reported in leaves. Therefore, it is still unknown whether this reflects a tissuespecific feature or an evolutionary difference between monocot and dicot plants. The role of these isoamylase complexes in starch biosynthesis has been determined through phenotypic analysis in a range of plant species and tissues in which ISA1 or ISA2 gene expression has been reduced or abolished. The loss of ISA1 results in a reduction of granular starch in endosperms of maize, rice and barley, in C. reinhardtii cells, in Arabidopsis leaves and in potato tubers. In all instances, the starch was partially replaced by a water soluble glucose polymer called phytoglycogen, which had shorter chain lengths compared to amylopectin in chain length distribution analyses, and a higher degree of branching. The impact of mutations in ISA2 is more variable. In endosperms of maize and rice, loss of ISA2 had no measurable effect, explained by the fact that these tissues contain still the active ISA1 homomultimeric complexes. In Arabidopsis, loss of ISA2 causes the same phenotype as the loss of ISA1. This observation is explained by the fact that Arabidopsis leaves appears to contain only the heteromultimeric ISA1/ISA2 complex, and the loss of either protein subunit results in a loss of the enzymatic activity destabilization of the remaining subunit. Overall, the differing phenotypic severity caused by the loss of ISA1 and the existence of both homomultimeric ISA1 and heteromultimeric ISA1/ISA2 complexes means that function of isoamylases in amylopectin biosynthesis is still not fully understood. In this study, we wanted to address three hypotheses arising from our current knowledge. First, are differences in starch structure between different plant species due to different catalytic specificities of ISA1 and ISA2 protein complexes? Second, are the functions between the ISA complexes from different plant species conserved? Third, are the subunits of the ISA complexes adequately conserved such that they are interchangeable between species? To answer these questions we replaced the endogenous ISA proteins in the dicot Arabidopsis with the respective monocot rice isoforms.