Production of High Amylose and Resistant Starch Rice Through Targeted Mutagenesis of Starch Branching Enzyme Iib by Crispr/cas9

Rice is the staple food for half of the world’s population. Starch accounts for 80-90% of the total mass of rice seeds, and rice starch is low in resistant starch (RS) with a high glycemic index (GI). RS has gained important since it is beneficial in preventing various diseases. Starch branching enzyme IIb (SBEIIb) plays a key role in the amylopectin synthesis pathway in the endosperm of cereals. Down-regulation of SBEIIb in several important crops has led to high amylose, high RS and low GI starch. In this study, we mutated OsSBEIIb in the japonica rice cultivar TNG82 through CRISPR/Cas9 and investigated the molecular and physicochemical modifications in OsSBEIIb mutant lines, e.g., gene expression, enzyme activity, apparent amylose content (AAC), RS and GI. As expected, the levels of modification in these starch related traits in heterozygous mutant lines were about half as those of homozygous mutant lines. Gene expression and enzyme activity of OsSBEIIb were down-regulated significantly while AAC and RS contents increased progressively from 17.4% and 0.5% in WT, respectively, to as high as 25.0% and 7.5% in heterozygous mutant lines and 36.0% and 12.0% in homozygous mutant lines. Consequently, with increased RS and decreased rate of reducing sugar production, GI progressively decreased in heterozygous and homozygous mutant rice endosperms by 11% and 28%, respectively. Our results demonstrate that it has huge potential for precise and efficient generation of high RS and low GI rice through CRISPR/Cas9 to provide a more suitable source of starch for type II diabetes.

Virus-induced Silencing SBEIIa and SSIIa, Alone or Together, Increased the Amylose and Resistant Starch Contents in Spring Wheat

Foods rich in amylose and resistant starch (RS) have great potential to improve human health and lower the risk of noninfectious diseases. Common wheat (Triticum aestivumL.) is a major staple food crop with low RS content in the grains. The content of RS, preferentially derived from amylose, may be increased by suppressing amylopectin synthesis viasilencing the starch branching enzyme (SBE) II a or/and starch synthase (SS)IIa. In this study, SBEIIaand SSIIawere silenced separately and simultaneously using a barley stripe mosaic virus-virus-induced gene silencing (BSMV-VIGS) system. Compared with grains from control BSMV:00-inoculated spikes, grains from BSMV:SBEIIa-and BSMV:SSIIa-infected spikeshad fewer SBEIIa and SSIIa transcripts,together with increased amylose contents (18.62 and 24.48%, respectively) and RS contents (11.61 and 16.67%, respectively). Infection with BSMV:SBEIIa-SSIIa reduced SBEIIa and SSIIatranscript levels and increased the amylose and RS contents (32.02 and 22.33%, respectively). Thus, BSMV-VIGS is a useful tool for the rapid silencing of single or multiple starch synthase-related genes and BSMV shows great potential to study the functions of genes involved in starch biosynthesis or other processes/traits in developing grains.We showed that the SSIIagene plays an important role in the synthesis of amylose and RS and that the effects of simultaneously silencing SBEIIaand SSIIaon starch synthesis aregreater than those of single gene silencing. Our study lays the foundation for the molecular design-based breeding of high-amylose and high-RS wheat.© 2021 Friends Science Publishers

Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating theOsSBEIIbgene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites.