Generation and Starch Characterization of Non-Transgenic BEI and BEIIb Double Mutant Rice (Oryza sativa) with Ultra-high Level of Resistant Starch

Abstract

Background Cereals high in resistant starch (RS) are gaining popularity, as their intake is thought to help manage diabetes and prediabetes. Number of patients suffering from diabetes is also increasing in Asian countries where people consume rice as a staple food, hence generation of practically growable high RS rice line has been anticipated. It is known that suppression of starch branching enzyme (BE) IIb increases RS content in cereals. To further increase RS content and for more practical use, we generated a non-transgenic be1 be2b double mutant rice (Oryza sativa) line, which completely lacked both proteins, by crossing a be1 mutant with a be2b mutant. Results The be1 be2b mutant showed a decrease in intermediate amylopectin chains and an increase in long amylopectin chains compared with be2b. The amylose content of be1 be2b mutant (51.7%) was the highest among all pre-existing non-transgenic rice lines. To understand the effects of chewing steamed rice and cooking rice flour on RS content, RS content of mashed and un-mashed steamed rice as well as raw and gelatinized rice flour were measured using be1 be2b and its parent mutant lines. The RS contents of mashed steamed rice and raw rice flour of be1 be2b mutant (28.4% and 35.1%, respectively) were 3-fold higher than those of be2b mutant. Gel-filtration analyses of starch treated with digestive enzymes showed that the RS in be1 be2b mutant was composed of the degradation products of amylose and long amylopectin chains. Seed weight of be1 be2b mutant was approximately 60% of the wild type and rather heavier than that of be2b mutant. Conclusions The endosperm starch in be1 be2b double mutant rice were enriched with long amylopectin chains. This led to a great increase in RS content in steamed rice grains and rice flour in be1 be2b compared with be2b single mutant. be1 be2b generated in this study must serve as a good material for an ultra-high RS rice cultivar.

Structure and Properties of Starch in Rice Double Mutants Lacking Starch Synthase (SS) IIa and Starch Branching Enzyme (BE) IIb

“…Previous studies reported an increase of amylose in some mutant rice lines. For example, the apparent amylose content of ss3a be2b 26) and be1 be2b 29) is 45.1 % and 51.7 %, respectively, which is higher than that of the be2b single mutant 26) (28.1 %). The reason for this is that loss of SS and BE increases GBSSI expression levels.…”

“…14) The increased amylose content in these mutant rice lines can be explained by the reduction of amylopectin biosynthesis resulting in leftover ADP-glucose as a substrate, and GBSSI was able to use ADP-glucose for amylose synthesis. 14) The seed weight of double mutant lines lacking SS and BE isozymes, such as ss3a be2b, 26) ss1 L be2b, 28) and be1 be2b, 29) is greater than that of the be2b single mutant line.…”

Interaction of starch branching enzyme 3 and granule-bound starch synthase 1 alleles increases amylose content and alters physico-chemical properties in japonica rice (Oryza sativa L.)

“…Starch is the main energy-reserve polysaccharide in cereal crops, and is the chief source of carbohydrates in the human diet (Amagliani et al, 2016). Starch also has many technological, medical, and industrial uses (Miura et al, 2021). Rice starch is mainly composed of two biopolymers: amylose and amylopectin.…”

“…Amylopectin is a branched glucose polymer and is mainly synthesized via concerted reactions catalyzed by three types of biosynthetic enzymes: starch synthases (SSs), starch branch enzymes (SBEs), and debranching enzymes (DBEs; Wang et al, 2017). Among these enzymes, mutations in the SBE isozymes showed significant association with amylose content variation (Nishi et al, 2001;Satoh et al, 2003;Butardo et al, 2011;Yang et al, 2012;Kim et al, 2018;Adeva et al, 2020;Miura et al, 2021). In rice, three isoforms of SBE are present: BEI (SBE1 or BE1), BEIIa (SBE4 or BE2a), and BEIIb (SBE3 or BE2b).…”

“…Asai et al (2014) crossed two japonica rice mutants EM10 (be2b) and e1 (ss3a) to generate a ss3a/be2b double null mutant line with 45% AAC. A higher amylose line (be1be2b) with an ultra-high level of RS was developed by crossing BEI and BE2b mutant lines (Miura et al, 2021). The be1be2b mutant had 51.7% AAC and 35.1% RS content.…”