Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population

BACKGROUND: The genetic diversity of six starch biosynthetic genes (Wx, SSI, SSIIa, SBEI, SBEIIa and SBEIIb) in indica and japonica rices opens an opportunity to produce a new variety with more favourable grain starch quality. However, there is limited information about the effects of these six gene allele combinations on starch structure and properties. A recombinant inbred line population from a cross between indica and japonica varieties offers opportunities to combine specific alleles of the six genes. RESULTS: The allelic (indica vs japonica) effects of six starch biosynthetic genes on starch structure, functional properties, and abundance of granule bound proteins in rice grains were investigated in a common genetic background using a recombinant inbred line population. The indica Wx (Wxi) allele played a major role while indica SSI (SSIi), japonica SSIIa (SSIIaj) and indica SBEI (SBEIi) alleles had minor roles on the increase of amylose content. SSIIaj and japonica SBEIIb (SBEIIbj) alleles had a major and a minor role on high ratio of ∑DP ≤ 10 to ∑DP ≤ 24 fractions (RCL10/24), respectively. Both major alleles (Wxi and SSIIaj) reduced peak viscosity (PV), onset, peak and end gelatinization temperatures (GTs) of amylopectin, and increased amylose-lipid complex dissociation enthalpy compared with their counterpart-alleles, respectively. SBEIIai and SBEIIbj decreased PV, whereas SSIi and SBEIIbj decreased FV. SBEIi reduced setback viscosity and gelatinization enthalpy. RCL10/24 of chain length distribution in amylopectin is negatively correlated with PV and BD of paste property and GTs of thermal properties. We also report RILs with superior starch properties combining Wxi, SSIj, SSIIaj, SBEIi and SBEIIbj alleles. Additionally, a clear relation is drawn to starch biosynthetic gene alleles, starch structure, properties, and abundance of granule bound starch biosynthetic enzymes inside starch granules. CONCLUSIONS: Rice Wxi and SSIIaj alleles play major roles, while SSIi, SBEIi, SBEIIai and SBEIIbj alleles have minor roles in the determination of starch properties between indica and japonica rice through starch structural modification. The combination of these alleles is a key factor for starch quality improvement in rice breeding programs. RCL10/24 value is critical for starch structure and property determination.Jixun Luo was supported by CSC (Chinese Scholarship Council) and Australian National University scholarships. This work was funded by CSIRO Food Future National Research Flagship

Understanding the impact of starch synthase IIa on starch structure and function in cereal endosperm

Starch synthase IIa (SSIIa) is one of the major starch biosynthetic enzymes in starch biosynthesis process in cereal endosperms. It catalyzes the synthesis of intermediate chains in amylopectin of the grain starch. During starch deposition it interacts with other starch synthases (SSs) and starch branching enzymes (SBEs) to form functional protein complexes. The defective SSIIa mutants of cereals in common showed significantly altered amylopectin chain length distribution (CLD), and pleiotropic effects inside the starch granules with reduced amount of starch granule bound proteins (GBPs). However, these effects observed in the mutants varied at different degrees in different cereals. In this study, SSIIa was chosen to investigate its impact on starch structure, functional properties and regulatory role in starch biosynthesis in the endosperms of barley, wheat and rice. The rice ssIIa mutants with japonica type SSIIa were obtained from a population of recombinant inbred lines (RILs) derived from an IR64 and Nipponbare hybrid. In addition, five other starch biosynthetic genes, Wx, SSI, SBEI, SBEIIa and SBEIIb, which could have potential allelic effects on starch structure and functional properties, were also investigated to understand the allelic recombination effect. Defective SSIIa hybrid populations of barley and wheat were used for screening ssIIa mutants for the quantitatively comparative analysis. The comparison was done at three levels: gene transcription, posttranslational regulation and starch structure. The allelic effect study showed that SSIIa and Wx had the most significant influences on starch structure and functional properties. The impact of Wxi and SSIIaj on starch functional properties was similar, having lower peak viscosity (PV) and breakdown (BD), higher final viscosity (FV) and setback (SB), lower amylopectin gelatinization temperatures (GTs) and higher amylose-lipid complex dissociation enthalpy compared with Wxj and SSIIai, respectively. Only SSIIaj haplotype produced a novel amylopectin structure with the most pronounced alteration in short chain and intermediate chain fractions. At similar levels of starch AC, the ratio of short chain/ short plus intermediate chain fraction percentage in debranched starch was negatively correlated to PV, BD and GTs. The remarkable changes in starch structure were also related with the abundance of GBPs in starch granules. SSI, SBEI, SBEIIa and SBEIIb had only limited allelic effects on starch structure and functional properties compared with SSIIa and Wx. The comparison analysis in the three cereals indicated that the total amount of SSI, SBEIIa and SBEIIb synthesized in the endosperm of the three ssIIa mutants was not affected by the SSIIa defection. The pleiotropic effects on GBPs in starch granules were due to the changes in protein partitions between amyloplast stroma and starch granules regulated by SSIIa. The order of changes (HvssIIa > TassIIa > OsssIIa) in the ssIIa mutants compared with corresponding wildtypes were consistent in the starch AC, amylopectin CLD, and starch biosynthetic enzyme partitions. These changes were related with the variation of SSI, SSIIa, SBEIIa and SBEIIb abundance inside starch granules and/or the dosage of SSIIa gene. The various SSIIa abundances inside starch granules were due to the differences in SSIIa mutations in different cereal mutants

Down-Regulation of FAD2-1 Gene Expression Alters Lysophospholipid Composition in the Endosperm of Rice Grain and Influences Starch Properties

Small quantities of lipids accumulate in the white rice grains. These are grouped into non-starch lipid and starch lipid fractions that affect starch properties through association with starch. Lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) are two major lipid classes in the two fractions. Using high-oleic rice grains, we investigated the fatty-acid composition in flour and starch by LC-MS and evaluated its impact on starch properties. In the wild-type grain, nearly 50% of fatty acids in LPC and LPE were palmitic acid (C16:0), over 20% linoleic acid (C18:2) and less than 10% oleic acid (C18:1). In the high-oleic rice grain, C18:1 increased at the expense of C18:2 and C16:0. The compositional changes in starch lipids suggest that LPC and LPE are transported to an amyloplast with an origin from endoplasmic reticulum-derived PC and PE during endosperm development. The high-dissociation temperature of the amylose-lipid complex (ALC) and restricted starch swelling power in the high-oleic rice starch indicates that the stability of the ALC involving C18:1 is higher than that of C18:2 and C16:0. This study provides insight into the lipid deposition and starch properties of rice grains with optimized fatty-acid composition

The impact of the indica rice SSIIa allele on the apparent high amylose starch from rice grain with downregulated japonica SBEIIb

Rice (Oryza sativa) genotypes with inactive starch synthase IIa (SSIIa) with recessive variants of starch branching enzyme IIb (SBEIIb) exhibit a range of alterations in grain phenotype, starch granule morphology, starch granule bound proteins, starch structure, and functional properties. However, the interactions between the two enzymes have not been thoroughly investigated yet. We analysed recombinant rice lines having down-regulated SBEIIb expression (SBEIIbDR) with either indica or japonica type SSIIa (SSIIaind or SSIIajap). In SBEIIbDR rice starch granules, the increased abundance of two protein bands (SSI and SSIIa) was found with eight additional protein bands not generally associated with starch granules. The amount of SSIIa was higher in SSIIaindSBEIIbDR than SSIIajapSBEIIbDR, which indicated that indica type SSIIa, possibly in the monomer form, was extensively involved in starch biosynthesis in the SBEIIbDR endosperm. Furthermore, SSIIaindSBEIIbDR grains had higher total starch content and higher starch swelling power than SSIIajapSBEIIbDR lines, but the amylopectin gelatinization temperatures and enthalpy and the apparent amylose content remained similar. In summary, this work suggests that SSIIaind can partly compensate for the alteration of starch synthesis resulting from the SBEIIb down-regulation in japonica background without reducing its amylose content. The study provides insight into the starch structural and textural improvements of high amylose starch

Functional genomic validation of the roles of soluble starch synthase IIa in japonica rice endosperm

The enzyme starch synthase IIa (SSIIa) in cereals has catalytic and regulatory roles during the synthesis of amylopectin that influences the functional properties of the grain. Rice endosperm SSIIa is more active in indica accessions compared to japonica lines due to functional SNP variations in the coding region of the structural gene. In this study, downregulating the expression of japonica-type SSIIa in Nipponbare endosperm resulted in either shrunken or opaque grains with an elevated proportion of A-type starch granules. Shrunken seeds had severely reduced starch content and could not be maintained in succeeding generations. In comparison, the opaque grain morphology was the result of weaker down-regulation of SSIIa which led to an elevated proportion of short-chain amylopectin (DP 6-12) and a concomitant reduction in the proportion of medium-chain amylopectin (DP 13-36). The peak gelatinization temperature of starch and the estimated glycemic score of cooked grain as measured by the starch hydrolysis index were significantly reduced. These results highlight the important role of medium-chain amylopectin in influencing the functional properties of rice grains, including its digestibility. The structural, regulatory and nutritional implications of down-regulated japonica-type SSIIa in rice endosperm are discussed

The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast st

Key message: The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Abstract: Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different. This study compared the levels of transcripts and partitioning of proteins of starch synthase I (SSI) and starch branching enzyme IIb (SBEIIb) inside and outside the starch granules in the developing endosperms of these ssIIa mutants and inactive SSIIa variant. Pleiotropic effects on starch granule-bound proteins suggested that the different effects of SSIIa mutations on endosperm amylose content of barley, wheat and rice are determined by the distribution of SSI and SBEIIb between the starch granule and amyloplast stroma in cereals. Regulation of starch synthesis in ssIIa mutants and inactive SSIIa variant may be at post-translational level or the altered amylopectin structure deprives the affinity of SSI and SBEIIb to amylopectin

Polyphenols from Australian-grown pigmented red and purple rice inhibit adipocyte differentiation

Coloured rice is rich in polyphenols and has been shown to have significant antioxidant and anti-adipogenic potential. The study aimed to investigate the anti-adipogenic properties of polyphenol extract (PE) derived from Australian-grown rice varieties. Eight wholegrain pigmented rice varieties were screened for their polyphenol content and antioxidant activity, of which, Yunlu29 (red), Purple (purple) and Reiziq (brown) had the highest values. The selected varieties were then subjected to in-vitro investigation to determine the effect of rice-derived PE on adipocyte differentiation. Lipid accumulation and peroxisome proliferator-activated receptor gamma (PPARγ) expression were quantified by oil red O staining and RT-PCR respectively. PE from Yunlu29 (red) and Purple rice varieties significantly reduced (p < 0.001) lipid accumulation by 53% and 56% respectively and PPARγ expression in adipocytes by 61.2% and 35.6% respectively. PE from Reiziq (brown), did not inhibit lipid accumulation in adipocytes however, did reduce PPARγ expression (p < 0.001). © 2018 Elsevier Lt