Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.

SummaryA minimum of four soluble starch synthase families have been documented in all starch-storing green plants. These activities are involved in amylopectin synthesis and are extremely well conserved throughout the plant kingdom. Mutants or transgenic plants defective for SSII and SSIII isoforms have been previously shown to have a large and specific impact on the synthesis of amylopectin while the function of the SSI type of enzymes has remained elusive. We report here that Arabidopsis mutants, lacking a plastidial starch synthase isoform belonging to the SSI family, display a major and novel type of structural alteration within their amylopectin. Comparative analysis of b-limit dextrins for both wild type and mutant amylopectins suggests a specific and crucial function of SSI during the synthesis of transient starch in Arabidopsis leaves. Considering our own characterization of SSI activity and the previously described kinetic properties of maize SSI, our results suggest that the function of SSI is mainly involved in the synthesis of small outer chains during amylopectin cluster synthesis.

Three genes, BE1, BE2, and BE3, which potentially encode isoforms of starch branching enzymes, have been found in the genome of Arabidopsis thaliana. Although no impact on starch structure was observed in null be1 mutants, modifications in amylopectin structure analogous to those of other branching enzyme II mutants were detected in be2 and be3. No impact on starch content was found in any of the single mutant lines. Moreover, three double mutant combinations were produced (be1 be2, be1 be3, and be2 be3), and the impact of the mutations on starch content and structure was analyzed. Our results suggest that BE1 has no apparent function for the synthesis of starch in the leaves, as both be1 be2 and be1 be3 double mutants display the same phenotype as be2 and be3 separately. However, starch synthesis was abolished in be2 be3, while high levels of a-maltose were assayed in the cytosol. This result indicates that the functions of both BE2 and BE3, which belong to class II starch branching enzymes, are largely redundant in Arabidopsis. Moreover, we demonstrate that maltose accumulation depends on the presence of an active ADP-glucose pyrophosphorylase and that the cytosolic transglucosidase DISPROPORTIONATING ENZYME2, required for maltose metabolization, is specific for b-maltose.