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

Satoko Miura,Naoko Crofts,Nana Koyama,Naoko Fujita,Misato Abe,Yuko Hosaka

doi:10.1186/s12284-020-00441-0

Abstract

BackgroundCereals 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.ResultsThe 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.ConclusionsThe 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.

Highlights

Introduction of the riceBEIIb gene into the be2b null mutant of rice produced a variety of lines with different BEIIb expression levels and diverse endosperm starch properties (Tanaka et al 2004)
Absence of BEI and BEIIb Total proteins were extracted from the be1 be2b double mutant (#1403), its parental single mutants be1 (EM557) and be2b (EM10), and WT cultivars (Taichung 65 and Kinmaze), and BEI and BEIIb proteins were detected by western blotting (Fig. 1)
When BEI antibody was used, BEI signal was detected in WT cultivars and be2b single mutant, but not in be1 single mutant and be1 be2b double mutant (Fig. 1)

Summary

Introduction

Introduction of the riceBEIIb gene into the be2b null mutant of rice produced a variety of lines with different BEIIb expression levels and diverse endosperm starch properties (Tanaka et al 2004). Overexpression of BEIIb leads to the accumulation of excessively branched, water-soluble glucans enriched in amylopectin short chains (DP < 16) (Tanaka et al 2004) Taken together, these data show that the loss of BEIIb greatly affects starch properties, and the function of BEIIb cannot be compensated for by other BEs (BEI and BEIIa), indicating that BEIIb is indispensable. The loss of BEIIb drastically elevates RS content to much higher orders of magnitude than the RS content of high amylose rice cultivars such as indica rice and the ss3a mutant (Zhou et al 2016) This indicates that the increase in amylopectin long chains leads to a greater increase in the RS content than that caused by the increase in amylose content (Tsuiki et al 2016).

Methods

Results

Conclusion