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.

Summary Resistant starch (RS), a type of dietary fibre, plays an important role in human health; however, the content of RS in most modern processed starchy foods is low. Cereal starch, when structurally manipulated through a modified starch biosynthetic pathway to greatly increase the amylose content, could be an important food source of RS. Transgenic studies have previously revealed the requirement of simultaneous down‐regulation of two starch branching enzyme (SBE) II isoforms both located on the long arm of chromosome 2, namely SBEIIa and SBEIIb, to elevate the amylose content in wheat from ~25% to ~75%. The current study revealed close proximity of genes encoding SBEIIa and SBEIIb isoforms in wheat with a genetic distance of 0.5 cM on chromosome 2B. A series of deletion and single nucleotide polymorphism (SNP) loss of function alleles in SBEIIa, SBEIIb or both was isolated from two different wheat populations. A breeding strategy to combine deletions and SNPs generated wheat genotypes with altered expression levels of SBEIIa and SBEIIb, elevating the amylose content to an unprecedented ~85%, with a marked concomitant increase in RS content. Biochemical assays were used to confirm the complete absence in the grain of expression of SBEIIa from all three genomes in combination with the absence of SBEIIb from one of the genomes.

Background Conventional wheat-based foods contain high concentrations of readily digestible starch that commonly give these foods a high postprandial glycemic response and may contribute to the development of type 2 diabetes and cardiovascular disease. Objectives The aim of this study was to determine if bread made from high-amylose wheat (HAW) and enriched in resistant starch dampens postprandial glycemia compared with bread made from conventional low-amylose wheat (LAW). Methods This single-center, randomized, double-blinded, crossover controlled study involved 7 consecutive weekly visits. On separate mornings, 20 healthy nondiabetic men and women (mean age 30 ± 3 y; body mass index 23 ± 0.7 kg/m2) consumed a glucose beverage or 4 different breads (each 121 g); LAW-R (refined), LAW-W (wholemeal), HAW-R, or HAW-W. The starch contents of the LAW and HAW breads were 24% and 74% amylose, respectively. Venous blood samples were collected at regular intervals before and for 3 h after the breakfast meal to measure plasma glucose, insulin, ghrelin, and incretin hormone concentrations, and the incremental area under the curve (AUC) was calculated (mmol/L × 3 h). Satiety and cravings were also measured at 30-min intervals during the postprandial period. Results HAW breads had a glycemic response (AUC) that was 39% less than that achieved with conventional wheat breads (HAW 39 ± 5 mmol/L × 3 h; LAW 64 ± 5 mmol/L × 3 h; P < 0.0001). Insulinemic and incretin responses were 24–30% less for HAW breads than for LAW breads (P < 0.05). Processing of the flour (wholemeal or refined) did not affect the glycemic, insulinemic, or incretin response. The HAW breads did not influence plasma ghrelin, or subjective measures of satiety or cravings during the postprandial period. Conclusions Replacing LAW with HAW flour may be an effective strategy for lowering postprandial glycemic and insulinemic responses to bread in healthy men and women, but further research is warranted. This trial was registered at the Australian and New Zealand Clinical Trials Registry as ACTRN12616001289404.