Efforts to capture high amylose in rice

Screening of wild and cultivated rice in IRRI germplasm collection revealed that majority have intermediate apparent amylose content. It appears that ancient farmers selected rice based on texture of the lower amylose varieties, considering that the majority of rice consumers today prefer intermediate to soft-textured rice. Furthermore, 30% seems to be the natural upper natural limit of amylose levels in wild-type rice. If this is the case, the rich biodiversity of rice has been subjected to the bottleneck of domestication to select for grains that have superior cooking and eating but not nutritional or satiating qualities considering that the majority of rice consumers today eat rice three times a day. On the other hand, the amylose content of available rice mutants with deficient SBEIIb or an over-expressed GBSSI also revealed amylose levels of around 35% which is significantly lower by comparison with other high amylose cereals, whose amylose content ranges from 70–90%. Hence, to produce the high amylose phenotype in rice, one might need to target different sets of enzymes or regulatory pathways. Since increasing the amylose levels in rice might mean a concomitant increase in its resistant starch content and in its levels of satiety, and a decrease in its glycemic response, developing high amylose rice by biotechnology is imperative. This type of rice will be important not only in addressing the growing obesity epidemic which now also affects the developing countries but also as a basis of novel degradable biopolymers and for further elucidating the mechanisms of starch synthesis in the cereal endosperm. In this paper, we also present the status of our research project which aims to silence the expression of SBEIIa, SBEIIb and SSIIa singly or in combination using microRNA and RNAi silencing technologies with the aim of increasing the amylose levels in rice beyond its natural limits

Association between alleles of the waxy gene and traits of grain quality in Philippine Seed Board rice varieties

The association between alleles of the Waxy gene, defined by the number of CT repeats on exon 1, and traits of grain quality was carried out using 47 Philippine Seed Board rice varieties. The major alleles of the Waxy gene in the set of 47 were (CT), (CT) (CT)and (CT)20. Varieties were divided into four clusters based on the metric of each trait of grain quality. (CT) (27-32% amylose content (AC)) and (CT) (22-30% AC) were mainly in clusters 3 and 4 (hard texture), and (CT) (20-24% AC) and (CT) (18- 27% AC) were exclusively in grain quality cluster 1 (soft texture) and 2 (medium texture). (CT) associated negatively with AC in this set. Only six (CT) and one (CT) rices were in cluster 4 (high-AC low-GT) and had high RVA consistency (final viscosity - trough viscosity) > 200 RVU. (CT) seemed to be the preferred source of low-intermediate AC in the Philippine rice breeding program, followed by (CT)

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed

Rice starch granule amylolysis - Differentiating effects of particle size, morphology, thermal properties and crystalline polymorph

The underlying mechanism of amylolysis of rice starch granules was investigated using isolated starch granules from wild-type, as well as SBEllb mutant and down-regulated lines. Fused granule agglomerates isolated from mutant and transgenic lines were hydrolysed at similar rates by amylases, and had similar crystalline patterns and thermal properties as individual granules. Surface pores, a feature previously only reported for A-polymorphic starch granules, were also observed in B- and C-polymorphic rice starch granules. Although the microscopic patterns of hydrolysis among granules with different crystalline polymorphs were qualitatively similar, the extent and the rate of amylolysis were different, suggesting that B-type crystalline polymorphs are intrinsically more resistant to enzymatic hydrolysis than A-type in rice starch granules. It is proposed that the slightly longer branch lengths of amylopectin which leads to the formation of more stable B-type double helical structures compared to their A-type counterparts is the major parameter, with other factors such as granule size, surface pores and interior channels having secondary roles, in determining the rate of enzymatic hydrolysis of rice starch granules. (C) 2014 Elsevier Ltd. All rights reserved

Paralytic shellfish toxin concentration and cell density changes in Pyrodinium bahamense - Noctiluca scintillans feeding experiments

For the first time the potential of Noctiluca scintillans, a non-toxic mixotrophic dinoflagellate, in bioconverting and/or excreting saxitoxin has been illustrated, thus contributing to the limited knowledge on the aspects of toxin pathways in the food chain/web and predator-prey preferences. Noctiluca growth rate increased with higher Pyrodinium concentration but the ratio of Noctiluca to Pyrodinium should at least be 1:250 cells per mL. Noctiluca fed with Pyrodinium alone was found to decrease in number suggesting that the nutrients from this prey were insufficient. This was confirmed by the improved cell density of Noctiluca upon addition of 0.01% casitone to the Pyrodinium-fed Noctiluca. The alternative prey (Gymnodinium sanguineum) slowed down the grazing impact of Noctiluca on Pyrodinium. Noctiluca depleted Gymnodinium earlier than Pyrodinium showing preference over a prey with less saxitoxin. After the feeding experiments, total saxitoxin levels decreased to 72% in the Noctiluca–Pyrodinium setup whereas no saxitoxin was detected in the Noctiluca culture fed with Pyrodinium and G. sanguineum. It is possible that Gymnodinium can provide some nutrients needed to make Noctiluca more efficient in bioconverting saxitoxin

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