Although high-amylose starches are not a recent innovation, their popularity in recent years has been increasing due to their unique functional properties and enhanced nutritional values in food applications. While high-amylose maize, barley, and potato are commercially available, high-amylose variants of other main crops such as wheat and rice have once been developed more recently and will be available commercially in the near future. This review summarizes the development, structure, and nutritional functionality of high-amylose starches developed and reported so far. The range of biotechnological strategies utilized are reviewed, as are the consequent effects on structural properties at different length scales, as well as sensory aspects of foods containing high-amylose starch (HAS). This review identifies the molecular and microstructural features contributing to digestive enzyme resistance not only in native HAS but also in forms of relevance to food processing. During heat treatment, HAS tends to retain or form dense molecular structures that resist amylase degradation through the retention of the granular structure as well as helices (type-2 resistant starch [RS]), reassociation of glucan chains (type-3 RS), and formation of lipid-amylose complexes (type-5 RS). The review also identifies opportunities for food manufacturers and consumers to incorporate HAS in food products and diets for better nutritional outcomes.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/1541-4337.12416

High-Amylose Starches to Bridge the “Fiber Gap”: Development, Structure, and Nutritional Functionality

https://www.mdpi.com/2504-3900/36/1/140/pdf

Wxlv, the Ancestral Allele of Rice Waxy Gene

Starch is a vital energy source for living organisms and is a key raw material and additive in the food and non-food industries. Starch has received continuous attention in multiple research fields. The endosperm of cereals (e.g., rice, corn, wheat, and barley) is the most important site for the synthesis of storage starch. Around 2010, several excellent reviews summarized key progress in various fields of starch research, serving as important references for subsequent research. In the past 10 years, many achievements have been made in the study of starch synthesis and regulation in cereals. The present review provides an update on research progress in starch synthesis of cereal endosperms over the past decade, focusing on new enzymes and non-enzymatic proteins involved in starch synthesis, regulatory networks of starch synthesis, and the use of elite alleles of starch synthesis-related genes in cereal breeding programs. We also provide perspectives on future research directions that will further our understanding of cereal starch biosynthesis and regulation to support the rational design of ideal quality grain.

Starch biosynthesis in cereal endosperms: An updated review over the last decade.

Starch and storage proteins determine the weight and quality of cereal grains. Synthesis of these two grain components has been comprehensively investigated, but the transcription factors responsible for their regulation remain largely unknown. In this study, we investigated the roles of NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20, and OsNAC26 in starch and storage protein synthesis in rice (Oryza sativa) endosperm. OsNAC20 and OsNAC26 showed high levels of amino acid sequence similarity. Both were localized in the aleurone layer, starchy endosperm, and embryo. Mutation of OsNAC20 or OsNAC26 alone had no effect on the grain, while the osnac20/26 double mutant had significantly decreased starch and storage protein content. OsNAC20 and OsNAC26 alone could directly transactivate the expression of starch synthaseI (SSI), pullulanase (Pul), glutelin A1 (GluA1), glutelin B4/5 (GluB4/5), α-globulin, and 16 kD prolamin and indirectly influenced plastidial disproportionating enzyme1 (DPE1) expression to regulate starch and storage protein synthesis. Although they could also bind to the promoters of ADP-Glc pyrophosphorylase small subunit 2b (AGPS2b), ADP-Glc pyrophosphorylase large subunit 2 (AGPL2), and starch branching enzymeI (SBEI), and the expression of the three genes was largely decreased in the osnac20/26 mutant, ADP-Glc pyrophosphorylase and starch branching enzyme activities were unchanged in this double mutant. In addition, OsNAC20 and OsNAC26 are main regulators of Pul, GluB4, α-globulin, and 16 kD prolamin In conclusion, OsNAC20 and OsNAC26 play an essential and redundant role in the regulation of starch and storage protein synthesis.

/pdf/the-nac-transcription-factors-osnac20-and-osnac26-regulate-21zdbnaz6c.pdf

The NAC Transcription Factors OsNAC20 and OsNAC26 Regulate Starch and Storage Protein Synthesis

Reliably generating rice varieties with low glycaemic index (GI) is an important nutritional intervention given the high rates of Type II diabetes incidences in Asia where rice is staple diet. We integrated a genome-wide association study (GWAS) with a transcriptome-wide association study (TWAS) to determine the genetic basis of the GI in rice. GWAS utilized 305 re-sequenced diverse indica panel comprising ~2.4 million single nucleotide polymorphisms (SNPs) enriched in genic regions. A novel association signal was detected at a synonymous SNP in exon 2 of LOC_Os05g03600 for intermediate-to-high GI phenotypic variation. Another major hotspot region was predicted for contributing intermediate-to-high GI variation, involves 26 genes on chromosome 6 (GI6.1). These set of genes included GBSSI, two hydrolase genes, genes involved in signalling and chromatin modification. The TWAS and methylome sequencing data revealed cis-acting functionally relevant genetic variants with differential methylation patterns in the hot spot GI6.1 region, narrowing the target to 13 genes. Conversely, the promoter region of GBSSI and its alternative splicing allele (G allele of Wxa ) explained the intermediate-to-high GI variation. A SNP (C˃T) at exon-10 was also highlighted in the preceding analyses to influence final viscosity (FV), which is independent of amylose content/GI. The low GI line with GC haplotype confirmed soft texture, while other two low GI lines with GT haplotype were characterized as hard and cohesive. The low GI lines were further confirmed through clinical in vivo studies. Gene regulatory network analysis highlighted the role of the non-starch polysaccharide pathway in lowering GI.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13051

Integrating a genome-wide association study with a large-scale transcriptome analysis to predict genetic regions influencing the glycaemic index and texture in rice.

High-amylose cereal starches provide many health benefits for humans. The inhibition or mutation of starch branching enzyme (SBE) genes is an effective method to develop high-amylose cereal crops. This review summarizes the development of high-amylose cereal crops through the inactivation of one or more SBE isoforms or combination with other genes. This review also reveals the causes of increase in amylose content in high-amylose crops. A series of changes, including amylopectin structure, crystalline structure, thermal properties, and hydrolysis properties, occurs as amylose content increases. The different morphological starch granules nominated as heterogeneous starch granules or differently stained starch granules are detected in high-amylose cereal crops. Detailed studies on four heterogeneous starch granules in high-amylose rice, which is developed by antisense RNA inhibition of SBEI/IIb, indicate that granules with different morphologies possess various molecular structures and physicochemical and functional properties. This variation diversifies their applications in food and non-food industries. However, current knowledge regarding how these heterogeneous starch granules form and why they exhibit regional distribution in endosperm remain largely unknown.

/pdf/progress-in-high-amylose-cereal-crops-through-inactivation-18h93va1bx.pdf

Progress in High-Amylose Cereal Crops through Inactivation of Starch Branching Enzymes.

Rice (Oryza sativa) endosperm is mainly occupied by homogeneous polygonal starch from inside to outside. However, morphologically different (heterogeneous) starches have been identified in some rice mutants. How these heterogeneous starches form remains unknown. A high-amylose rice line (TRS) generated through the antisense inhibition of starch branching synthase I (SBEI) and SBEIIb contains four heterogeneous starches: polygonal, aggregate, elongated, and hollow starch; these starches are regionally distributed in the endosperm from inside to outside. Here, we investigated the relationship between SBE dosage and the morphological architecture of heterogeneous starches in TRS endosperm from the view of the molecular structure of starch. The results indicated that their molecular structures underwent regular changes, including gradually increasing true amylose content but decreasing amylopectin content and gradually increasing the ratio of amylopectin long chain but decreasing the ratio of amylopectin short chain. Granule-bound starch synthase I (GBSSI) amounts in the four heterogeneous starches were not significantly different from each other, but SBEI, SBEIIa, and SBEIIb showed a gradually decreasing trend. Further immunostaining analysis revealed that the gradually decreasing SBEs acting on the formation of the four heterogeneous granules were mainly due to the spatial distribution of the three SBEs in the endosperm. It was suggested that the decreased amylopectin in starch might remove steric hindrance and provide extra space for abundant amylose accumulation when the GBSSI amount was not elevated. Furthermore, extra amylose coupled with altered amylopectin structure possibly led to morphological changes in heterogeneous granules.

/pdf/gradually-decreasing-starch-branching-enzyme-expression-is-433it1gyfx.pdf

Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules.

Cereal crops with inhibition expression of starch branching enzyme (SBE) contain highly resistant starch in the endosperm, and have potential health benefits for human. However, seed plumpness is significantly different, with different inhibition effects of SBE expression, resulting in differently shrunken seeds. In this study, a transgenic resistant starch rice line, which has highly resistant starch and is developed through inhibition expression of SBEs, had non-, slightly, and moderately shrunken seeds with plumpness from high to low. The differently shrunken seeds had significantly different seed weight and starch content. Different morphological starch granules were detected in the endosperm and had similar spatial distribution pattern among the non-, slightly, and moderately shrunken seeds. Starches from differently shrunken seeds had similar amylose content and amylopectin structure. The protein amount of amylose/amylopectin synthesis key enzymes in endosperm was no different between differently shrunken seeds. The primary branch of the panicle had a higher percentage of non-shrunken seeds than did the secondary branch at the same part of the panicle. From the upper part to the base of the panicle, non-shrunken seeds gradually decreased but slightly and moderately shrunken seeds gradually increased. The above results indicated that the differently shrunken seeds in transgenic rice line had the same SBE dosage, and the starch morphology and structure had no relationship with seed plumpness. The grain position on the panicle influenced seed plumpness, but had no effect on starch properties.

/pdf/seed-plumpness-of-rice-with-inhibition-expression-of-starch-59h72qedgy.pdf

Zichun Chen

Papers

Progress in High-Amylose Cereal Crops through Inactivation of Starch Branching Enzymes.

The NAC Transcription Factors OsNAC20 and OsNAC26 Regulate Starch and Storage Protein Synthesis

Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules.

Seed Plumpness of Rice with Inhibition Expression of Starch Branching Enzymes and Starch Properties, Grain Position on Panicle