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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 the OsSBEIIb gene 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.

Starch granules are composed of two distinct glucose polymers – amylose and amylopectin. Amylose constitutes 5–35% of most natural starches and has a major influence over starch properties in foods. Its synthesis and storage occurs within the semicrystalline amylopectin matrix of starch granules, this poses a great challenge for biochemical and structural analyses. However, the last two decades have seen vast progress in understanding amylose synthesis, including new insights into the action of GRANULE BOUND STARCH SYNTHASE (GBSS), the major glucosyltransferase that synthesises amylose, and the discovery of PROTEIN TARGETING TO STARCH1 (PTST1) that targets GBSS to starch granules. Advances in analytical techniques have resolved the fine structure of amylose, raising new questions on how structure is determined during biosynthesis. Furthermore, the discovery of wild plants that do not produce amylose revives a long-standing question of why starch granules contain amylose, rather than amylopectin alone. Overall, these findings contribute towards a full understanding of amylose biosynthesis, structure and function that will be essential for future approaches to improve starch quality in crops.

Summary Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF‐Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF‐Y heterotrimer complex NF‐YB1‐YC12‐bHLH144, which is formed by the binding of NF‐YB1 to NF‐YC12 and then bHLH144 in a sequential order. Knock‐out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf‐yb1 and crnf‐yc12. RNA‐seq analysis identified 1496 genes that were commonly regulated by NF‐YB1 and NF‐YC12, including the key granule‐bound starch synthase gene Wx. NF‐YC12 and bHLH144 maintain NF‐YB1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF‐YB1 directly binds to the ‘G‐box’ domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF‐YB1‐YC12‐bHLH144 complex, which has great potential for rice genetic improvement.

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

Amylose fraction of grain starch is correlated with a type of resistant starch with better nutritional quality. Granule-bound starch synthase I (GBSSI) is the known starch synthase, responsible for elongation of linear amylose chains. GBSSI expression, activity, and binding to starch and other proteins are the key factors that can affect amylose content. Previously, a QTL, qhams7A.1 carrying GBSSI mutant allele, was identified through QTL mapping using F2 population of the high amylose mutant line, ‘TAC 75’. This high amylose mutant line has >2-fold higher amylose content than wild variety ‘C 306’. In this study, we characterized this novel mutant allele, GBSSI.L539P. In vitro starch synthase activity of GBSSI.L539P showed improved activity than the wild type (GBSSI-wt). When expressed in yeast glycogen synthase mutants (Δgsy1gsy2), GBSSI-wt and GBSSI.L539P partially complemented the glycogen synthase (gsy1gsy2) activity in yeast. Structural analysis by circular dichroism (CD) and homology modelling showed no significant structural distortion in the mutant enzyme. Molecular docking studies suggested that the residue Leu539 is distant from the catalytic active site (ADP binding pocket) and had no detectable conformational changes in active site. Both wild and mutant enzymes were assayed for starch binding in vitro, and demonstrating higher affinity of the GBSSI.L539P mutant for starch than the wild type. The present study indicated that distant residue (L539P) influenced GBSSI activity by affecting its starch-binding ability. Therefore, it may be a potential molecular target for enhanced amylose content in grain.

Changes in human lifestyle and food consumption have resulted in a large increase in the incidence of type-2 diabetes, obesity, and colon disease, especially in Asia. These conditions are a growing threat to human health, but consumption of foods high in resistant starch (RS) can potentially reduce their incidence. Strategies to increase RS in rice are limited by a lack of knowledge of its molecular basis. Through map-based cloning of a RS locus in indica rice, we have identified a defective soluble starch synthase gene (SSIIIa) responsible for RS production and further showed that RS production is dependent on the high expression of the Waxya (Wxa ) allele, which is prevalent in indica varieties. The resulting RS has modified granule structure; high amylose, lipid, and amylose–lipid complex; and altered physicochemical properties. This discovery provides an opportunity to increase RS content of cooked rice, especially in the indica varieties, which predominates in southern Asia.

Key messageHigh levels of two major starch synthases, SSIIa and GBSSI, in ss3a ss4b double mutant rice alter the starch structure but fail to recover the polygonal starch granule morphology.The endosperm starch granule is polygonal in wild-type rice but spherical in double mutant japonica rice lacking genes encoding two of the five major Starch synthase (SS) isozymes expressed in endosperm, SSIIIa and SSIVb. Japonica rice naturally has low levels of SSIIa and Granule-bound SSI (GBSSI). Therefore, introduction of active SSIIa allele and/or high-expressing GBSSI allele from indica rice into the japonica rice mutant lacking SS isozymes can help elucidate the compensatory roles of SS isozymes in starch biosynthesis. In this study, we crossed the ss3a ss4a double mutant japonica rice with the indica rice to generate three new rice lines with high and/or low SSIIa and GBSSI levels, and examined their starch structure, physicochemical properties, and levels of other starch biosynthetic enzymes. Lines with high SSIIa levels showed more SSI and SSIIa bound to starch granule, reduced levels of short amylopectin chains (7 ≤ DP ≤ 12), increased levels of amylopectin chains with DP > 13, and consequently higher gelatinization temperature. Lines with high GBSSI levels showed an increase in amylose content. The ADP-glucose content of the crude extract was high in lines with low or high SSIIa and low GBSSI levels, but was low in lines with high GBSSI. Addition of high SSIIa and GBSSI altered the starch structure and physicochemical properties but did not affect the starch granule morphology, confirming that SSIIIa and SSIVb are key enzymes affecting starch granule morphology in rice. The relationship among SS isozymes and its effect on the amount of substrate (ADP-glucose) is discussed.

Morphology, composition and molecular structure of starch directly affect the functional properties. This study investigated the morphological, compositional, and molecular structure properties of starch from starch branching enzyme gene ( SBE ) and granule-bound starch synthase gene ( GBSS ) mutated potato, and their associations with thermal, pasting, and film-making properties. SBE mutations were induced in native variety Desiree while GBSS mutations were herestacked to a selected SBE mutated parental line. Mutations in SBE resulted in smaller starch granules and higher amylose content, while GBSS mutations in the SBE background reduced amylose content. Mutations in SBE , particularly with GBSS mutations, significantly increased total phosphorus content. 31 P NMR spectroscopy revealed higher proportions of C6-bound phosphate than of C3-bound phosphate in all studied lines. Amylopectin unit chain and internal chain distributions showed higher proportions of long chains in mutated lines compared with Desiree. These amylopectin long-chains were positively correlated with gelatinizationand, pasting temperatures, and temperature at peak viscosity. Short amylopectin chains showed positive correlations with breakdown viscosity, but negative correlations with the crystal melting temperature of retrograded starch. Total phosphorus content was positively correlated with the crystal melting temperature of retrograded starch. Starch from different lines was used to produce a series of potato starch films that differed in morphology and functional properties. A negative correlation was observed between Young’s modulus of films and the long amylopectin-chain fraction. Thermal gravimetric analysis revealed highest thermal stability of Desiree starch films, followed by films from SBE -mutated high-amylose lines. Oxygen transmission rate and oxygen permeability analyses showed that films made with starch from selected GBSS and SBE s mutated line maintained comparable oxygen barrier properties to Desiree film. These insights on the impact of genetic mutations on starch properties indicate potential applications of in-planta starch modification for specific end-uses including packaging.

Key messageInduced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery.The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61–71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8–12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.

Key messageFLO6 is involved in starch synthesis by interacting with SSIVb and GBSS in rice.Starch synthesized and stored in plastids including chloroplasts and amyloplasts plays a vital role in plant growth and provides the major energy for human diet. However, the molecular mechanisms by which regulate starch synthesis remain largely unknown. In this study, we identified and characterized a rice floury endosperm mutant M39, which exhibited defective starch granule formation in pericarp and endosperm, accompanied by the decreased starch content and amylose content. The abnormal starch accumulation in M39 pollen grains caused a significant decrease in plant fertility. Chloroplasts in M39 leaves contained no or only one large starch granule. Positional cloning combined with complementary experiment demonstrated that the mutant phenotypes were restored by the FLOURY ENDOSPERM6 (FLO6). FLO6 was generally expressed in various tissues, including leaf, anther and developing endosperm. FLO6 is a chloroplast and amyloplast-localized protein that is able to bind to starch by its carbohydrate-binding module 48 (CBM48) domain. Interestingly, we found that FLO6 interacted with starch synthase IVb (SSIVb) and granule-bound starch synthase (GBSSI and GBSSII). Together, our results suggested that FLO6 plays a critical role in starch synthesis through cooperating with several starch synthesis enzymes throughout plant growth and development.