Loss of Starch Synthase IIIa changes starch molecular structure and granule morphology in grains of hexaploid bread wheat

Abstract

Starch Synthase III plays a key role in starch biosynthesis and is highly expressed in developing wheat grains. To understand the contribution of SSIII to starch and grain properties, we developed wheat ssIIIa mutants in the elite cultivar Cadenza using in silico TILLING in a mutagenized population. SSIIIa protein was undetectable by immunoblot analysis in triple ssIIIa mutants carrying mutations in each homoeologous copy of ssIIIa (A, B and D). Loss of SSIIIa in triple mutants led to significant changes in starch phenotype including smaller A-type granules and altered granule morphology. Starch chain-length distributions of double and triple mutants indicated greater levels of amylose than sibling controls (33.8% of starch in triple mutants, and 29.3% in double mutants vs. 25.5% in sibling controls) and fewer long amylopectin chains. Wholemeal flour of triple mutants had more resistant starch (6.0% vs. 2.9% in sibling controls) and greater levels of non-starch polysaccharides; the grains appeared shrunken and weighed ~11% less than the sibling control which was partially explained by loss in starch content. Despite the elevation in resistant starch, the other effects on starch and grain properties may hamper the usefulness of ssIIIa mutants in breeding applications.

Impact of storage on starch digestibility and texture of a high-amylose wheat bread

“…Chain-length distribution of debranched starch was analysed as described by Fahy and colleagues (Fahy B., Gonzalez O., Savva G.A., et al, 2022) using a mixture model where the distribution components were estimated using constrained values of degree of polymerisation (DP) previously described by Hanashiro et al 1996(Hanashiro, et al, 1996 and Vilaplana et al 2012(Vilaplana, et al, 2012. The mixdist (version 0.5-5) R package was used to estimate mixture models (Macdonald P. & Du J., 2018).…”

“…Starch isolated from sbeII and WT wheat flour was used to determine starch chain length distribution by size exclusion chromatography (SEC). Starch was isolated as described in (Corrado M., et al, 2020) and debranched using iso-amylase prior to analysis by HPLC-SEC as described by Fahy et al . 2022 (Fahy B., et al 2022).…”

“…Starch isolated from sbeII and WT wheat flour was used to determine starch chain length distribution by size exclusion chromatography (SEC). Starch was isolated as described in (Corrado M., et al, 2020) and debranched using iso-amylase prior to analysis by HPLC-SEC as described by Fahy et al 2022(Fahy B., et al, 2022. The relative peak heights corresponding to amylopectin and amylose chains were calculated as defined by Hanashiro et al 1996(Hanashiro, Abe, & Hizukuri, 1996 and Vilaplana et al 2012(Vilaplana, Hasjim, & Gilbert, 2012.…”

“…Samples and standards were resolubilised in 4 and 2 mg/mL, respectively with DMSO+0.5% (w/w), and stored at 80°C overnight prior to analysis. The HPLC-SEC system for peak resolution was a Waters Alliance e2695 HPLC (Milford, MA, USA) equipped with refractive index detector (RI), autosampler (40 °C), and column heater (90°C), as described by (Fahy B., Gonzalez O., Savva G. M., et al, 2022).…”

Development of a high-throughput starch digestibility assay reveals wide variation among the A. E. Watkins wheat landrace collection

“…Thus, breeding for wheat with less digestible starch is an important strategy to develop healthier staple foods. So far, reverse genetic studies in wheat have demonstrated potential for increasing resistant starch levels using induced mutations in starch biosynthesis genes (Botticella et al, 2018, Schonhofen et al, 2017, Hazard et al, 2015, Fahy et al, 2022). However, initial analyses of some mutants have shown detrimental effects on yield and on pasta and bread-making quality, thus identifying additional sources of genetic variation for starch digestibility could support the development of improved traits for commercial breeding applications (Schonhofen et al, 2017, Hazard et al, 2015).…”