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 cooked rice and cooking rice flour on RS content, RS content of mashed and un-mashed cooked rice as well as raw and gelatinized rice flour were measured using be1 be2b and its parent mutant lines. The RS contents of mashed cooked 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 cooked 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.…”

“…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. Consistent with previous studies, the seed weight of the two ss2a be2b lines analyzed in this study was also increased.…”

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

“…Silencing of both OsSBEII and OsSBEI at same time by antisense RNA leads to signi cant increase in AC from 27.2 to 64.8% and RS from 0.1 to 14.6 with a signi cant decrease in grain weight and size (Zhu et al, 2012). Hybridizing OsSBEI mutant rice (EM557) and OsSBEIIb mutant rice (EM10), both isolated from N-methyl-N-nitrosourea (NMU) mutagenized populations of japonica cultivars Taichung 65 and Kinmaze, respectively, to produce OsSBEI/OsSBEIIb double mutant rice also leads to much higher AAC (51.7%) and RS (35.1%) than the single mutant EM557 or EM10 (Miura et al, 2021). It's contemplated that the two SBE isoform may exhibit a synergistic effect on synthesis of amylopectin.…”

Improving Agricultural Traits While Maintaining High Resistant Starch Content in Rice

“…Absence of BEIIb in japonica rice mutants drastically increases the RS content (Tsuiki et al 2016 ). The RS content of cooked rice grains of wild-type (WT) japonica rice is approximately 1% (Tsuiki et al 2016 ; Miura et al 2021 ), whereas those of be2b , ss3a be2b , ss1 L be2b , and be1 be2b are 21–28% (Tsuiki et al 2016 ; Itoh et al 2017 ; Miura et al 2021 ), 17% (Tsuiki et al 2016 ), 34% (Tsuiki et al 2016 ), and 76% (Miura et al 2021 ), respectively. The RS found in be2b mutant lines is categorized as RS2 since all of these lines display B-type crystallinity because of the high proportion of long amylopectin branches (Nishi et al 2001 ; Wei et al 2010 ; Abe et al 2013 , 2014 ; Asai et al 2014 ; Miura et al 2021 ).…”

“…Absence of BEIIb decreases the number, but increases the length, of amylopectin branches, thus making starch retrogradation easier and degradation more difficult. Residual glucans, obtained after the treatment of starch with digestive enzymes, are enriched with long amylopectin branches (DP > 21) and partially digested amylose, although the residual glucan structure can vary, depending on the processing method, initial amylopectin structure, and amylose content (Miura et al 2021 ). Increase in the RS content of japonica be2b mutant rice is often accompanied by a reduction in seed weight.…”