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Glutinous rice accumulates amylose-free starch and is utilized for rice cakes and crackers, owing to the loss of the Waxy gene which encodes granule-bound starch synthase I (GBSSI). Starch synthase IIa (SSIIa) elongates amylopectin chains with a degree of polymerization (DP) of 6–12 to 13–24 and greatly influences starch properties. To elucidate the relationship between the branch length of amylopectin and the thermal and rheological properties, viscoelasticity, and eating quality of glutinous rice, three allelic near isogenic lines with high, low, or no SSIIa activity were generated (designated as SS2a wx, ss2aL wx, and ss2a wx, respectively). Chain length distribution analyses revealed that ss2a wx exhibited the highest short chain (DP < 12) number and lowest gelatinization temperature, whereas SS2a wx showed the opposite results. Gel filtration chromatography showed that the three lines contained essentially no amylose. Viscoelasticity analyses of rice cakes stored at low temperature for different durations revealed that ss2a wx maintained softness and elasticity for up to 6 days, while SS2a wx hardened within 6 h. Sensory evaluation was consistent with mechanical evaluation. The relationship of amylopectin structure with the thermal and rheological properties, viscoelasticity, and eating quality of glutinous rice is discussed. [ABSTRACT FROM AUTHOR]

Early flowering trait is essential for rice cultivars grown at high latitude since delayed flowering leads to seed development at low temperature, which decreases yield. However, early flowering at high temperature promotes the formation of chalky seeds with low apparent amylose content and high starch gelatinization temperature, thus affecting grain quality. Deletion of starch synthase IIa (SSIIa) shows inverse effects of high temperature, and the ss2a mutant shows higher apparent amylose content and lower gelatinization temperature. Heading date 1 (Hd1) is the major regulator of flowering time, and a nonfunctional hd1 allele is required for early flowering. To understand the relationship among heading date, starch properties, and yield, we generated and characterized near-isogenic rice lines with ss2a Hd1, ss2a Hd1 hd1, and ss2a hd1 genotypes. The ss2a Hd1 line showed the highest plant biomass; however, its grain yield varied by year. The ss2a Hd1 hd1 showed higher total grain weight than ss2a hd1. The ss2a hd1 line produced the lowest number of premature seeds and showed higher gelatinization temperature and lower apparent amylose content than ss2a Hd1. These results highlight Hd1 as the candidate gene for developing high-yielding rice cultivars with the desired starch structure. [ABSTRACT FROM AUTHOR]

Background: Resistant starch (RS) is beneficial for human health. Loss of starch branching enzyme IIb (BEIIb) increases the proportion of amylopectin long chains, which greatly elevates the RS content. Although high RS content cereals are desired, an increase in RS content is often accompanied by a decrease in seed weight. To further increase the RS content, genes encoding active-type starch synthase (SS) IIa, which elongates amylopectin branches, and high expression-type granule-bound SSI (GBSSI), which synthesizes amylose, were introduced into the be2b mutant rice. This attempt increased the RS content, but further improvement of agricultural traits was required because of a mixture of indica and japonica rice phonotype, such as different grain sizes, flowering times, and seed shattering traits. In the present study, the high RS lines were backcrossed with an elite rice cultivar, and the starch properties of the resultant high-yielding RS lines were analyzed. Results: The seed weight of high RS lines was greatly improved after backcrossing, increasing up to 190% compared with the seed weight before backcrossing. Amylopectin structure, gelatinization temperature, and RS content of high RS lines showed almost no change after backcrossing. High RS lines contained longer amylopectin branch chains than the wild type, and lines with active-type SSIIa contained a higher proportion of long amylopectin chains compared with the lines with less active-SSIIa, and thus showed higher gelatinization temperature. Although the RS content of rice varied with the cooking method, those of high RS lines remained high after backcrossing. The RS contents of cooked rice of high RS lines were high (27–35%), whereas that of the elite parental rice was considerably low (< 0.7%). The RS contents of lines with active-type SSIIa and high-level GBSSI expression in be2b or be2b ss3a background were higher than those of lines with less-active SSIIa. Conclusions: The present study revealed that backcrossing high RS rice lines with elite rice cultivars could increase the seed weight, without compromising the RS content. It is likely that backcrossing introduced loci enhancing seed length and width as well as loci promoting early flowering for ensuring an optimum temperature during RS biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Cereals 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. Results: The 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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]

CO2-responsive CCT protein (CRCT) is suggested to be a positive regulator of starch biosynthesis in the leaf sheaths of rice, regulating the expression levels of starch biosynthesis-related genes. In this study, the effects of CRCT expression levels on the expression of starch biosynthesis-related enzymes and the quality of starch were studied. Using native-PAGE/activity staining and immunoblotting, we found that the protein levels of starch synthase I, branching enzyme I, branching enzyme IIa, isoamylase 1 and phosphorylase 1 were largely correlated with the CRCT expression levels in the leaf sheaths of CRCT transgenic lines. In contrast, the CRCT expression levels largely did not affect the expression levels and/or activities of starch biosynthesis-related enzymes in the leaf blades and endosperm tissues. The analysis of the chain-length distribution of starch in the leaf sheaths showed that short chains with a degree of polymerization from 5 to 14 were increased in the overexpression lines but decreased in the knockdown lines. The amylose content of starch in the leaf sheath was greatly increased in the overexpression lines. In contrast, the molecular weight of the amylopectin of starch in the leaf sheath of overexpression lines did not change compared with those of the non-transgenic rice. These results suggest that CRCT can control the quality and the quantity of starch in the leaf sheath by regulating the expression of particular starch biosynthesis-related enzymes. [ABSTRACT FROM AUTHOR]

Summary: The CS8 transgenic rice (Oryza sativa L.) lines expressing an up‐regulated glgC gene produced higher levels of ADPglucose (ADPglc), the substrate for starch synthases. However, the increase in grain weight was much less than the increase in ADPglc levels suggesting one or more downstream rate‐limiting steps. Endosperm starch levels were not further enhanced in double transgenic plants expressing both glgC and the maize brittle‐1 gene, the latter responsible for transport of ADPglc into the amyloplast. These studies demonstrate that critical processes within the amyloplast stroma restrict maximum carbon flow into starch. RNA‐seq analysis showed extensive re‐programming of gene expression in the CS8 with 2073 genes up‐regulated and 140 down‐regulated. One conspicuous gene, up‐regulated ~15‐fold, coded for a biochemically uncharacterized starch binding domain‐containing protein (SBDCP1) possessing a plastid transit peptide. Confocal microscopy and transmission electron microscopy analysis confirmed that SBDCP1 was located in the amyloplasts. Reciprocal immunoprecipitation and pull‐down assays indicated an interaction between SBDCP1 and starch synthase IIIa (SSIIIa), which was down‐regulated at the protein level in the CS8 line. Furthermore, binding by SBDCP1 inhibited SSIIIa starch polymerization activity in a non‐competitive manner. Surprisingly, artificial microRNA gene suppression of SBDCP1 restored protein expression levels of SSIIIa in the CS8 line resulting in starch with lower amylose content and increased amylopectin chains with a higher degree of polymerization. Collectively, our results support the involvement of additional non‐enzymatic factors such as SBDCP in starch biosynthesis. Significance Statement: Transcriptome analysis of a rice CS8 line that expressed an up‐regulated glgC (ADPglucose pyrophosphorylase) gene revealed extensive re‐programming especially of genes associated with starch synthesis including a previously uncharacterized, amyloplast‐localized SBDCP1. This CBM20 domain‐containing protein interacts with SSIIIa and inhibits its catalytic activity and, thereby, restraining maximum carbon flow into starch. [ABSTRACT FROM AUTHOR]

Deficiencies in both starch synthase IIIa and branching enzyme IIb lead to significantly increased amylose content in japonica rice endoseprm due to pleiotropic effects on other starch biosynthetic enzymes.Starch synthase (SS) IIIa has the second highest activity of the total soluble SS activity in developing rice endosperm. Branching enzyme (BE) IIb is the major BE isozyme, and is strongly expressed in developing rice endosperm. A mutant (ss3a/be2b) was generated from wild-type japonica rice which lacks SSIIa activity. The seed weight of ss3a/be2b was 74–94% of that of the wild type, whereas the be2b seed weight was 59–73% of that of the wild type. There were significantly fewer amylopectin short chains [degree of polymerization (DP) ≤13] in ss3a/be2b compared with the wild type. In contrast, the amount of long chains (DP ≥25) connecting clusters of amylopectin in ss3a/be2b was higher than in the wild type and lower than in be2b. The apparent amylose content of ss3a/be2b was 45%, which was >1.5 times greater than that of either ss3a or be2b. Both SSIIIa and BEIIb deficiencies led to higher activity of ADP-glucose pyrophosphorylase (AGPase) and granule-bound starch synthase I (GBSSI), which partly explains the high amylose content in the ss3a/be2b endosperm. The percentage apparent amylose content of ss3a and ss3a/be2b at 10 days after flowering (DAF) was higher than that of the wild type and be2b. At 20 DAF, amylopectin biosynthesis in be2b and ss3a/be2b was not observed, whereas amylose biosynthesis in these lines was accelerated at 30 DAF. These data suggest that the high amylose content in the ss3a/be2b mutant results from higher amylose biosynthesis at two stages, up to 20 DAF and from 30 DAF to maturity. [ABSTRACT FROM PUBLISHER]

RNAs for the storage proteins, glutelins and prolamines, contain zipcode sequences, which target them to specific subdomains of the cortical endoplasmic reticulum in developing rice (Oryza sativa) seeds. Fifteen RNA binding proteins (RBPs) specifically bind to the prolamine zipcode sequences and are likely to play an important role in the transport and localization of this storage protein RNA. To understand the underlying basis for the binding of multiple protein species to the prolamine zipcode sequences, the relationship of five of these RBPs, RBP-A, RBP-I, RBP-J, RBP-K, and RBP-Q, were studied. These five RBPs, which belong to the heterogeneous nuclear ribonucleoprotein class, bind specifically to the 5' coding regions as well as to the 3' untranslated region zipcode RNAs but not to a control RNA sequence. Coimmunoprecipitation-immunoblot analyses in the presence or absence of ribonuclease showed that these five RBPs are assembled into three multiprotein complexes to form at least two zipcode RNA-protein assemblies. One cytoplasmicqocalized zipcode assembly contained two multiprotein complexes sharing a common core consisting of RBP-J and RBP-K and either RBP-A (A-J-K) or RBP-I (I-J-K). A second zipcode assembly of possibly nuclear origin consists of a multiprotein complex containing RBP-Q and modified forms of the other protein complexes. These results suggest that prolamine RNA transport is initiated in the nucleus to form a zipcode-protein assembly, which is remodeled in the cytoplasm to target the RNA to its proper location on the cortical endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Abstract: Rice endosperm starch is composed of 0–30% linear amylose, which is entirely synthesized by granule-bound starch synthase I (GBSSI: encoded by Waxy, Wx). The remainder consists of branched amylopectin and is elongated by multiple starch synthases (SS) including SSI, IIa and IIIa. Typical japonica rice lacks active SSIIa and contains a low expressing Wx b causing a low amylose content (ca. 20%). WAB2-3 (SS3a/Wx a ) lines generated by the introduction of a dominant indica Wx a into a japonica waxy mutant (SS3a/wx) exhibit elevated GBSSI and amylose content (ca. 25%). The japonica ss3a mutant (ss3a/Wx b ) shows a high amylose content (ca. 30%), decreased long chains of amylopectin and increased GBSSI levels. To investigate the functional relationship between the ss3a and Wx a genes, the ss3a/Wx a line was generated by crossing ss3a/Wx b with SS3a/Wx a , and the starch properties of this line were examined. The results show that the apparent amylose content of the ss3a/Wx a line was increased (41.3%) compared to the parental lines. However, the GBSSI quantity did not increase compared to the SS3a/Wx a line. The amylopectin branch structures were similar to the ss3a/Wx b mutant. Therefore, Wx a and ss3a synergistically increase the apparent amylose content in rice endosperm, and the possible reasons for this increase are discussed. [Copyright &y& Elsevier]

The rice esp2 mutation was previously characterized by the abnormal accumulation of elevated levels of proglutelin and the absence of an endosperm-specific protein disulfide isomerase like (PDIL1-1). Here we show that Esp2 is the structural gene for PDIL1-1 and that this lumenal chaperone is asymmetrically distributed within the cortical endoplasmic reticulum (ER) and largely restricted to the cisternal ER. Temporal studies indicate that PDIL1-1 is essential for the maturation of proglutelin only when its rate of synthesis significantly exceeds its export from the ER, a condition resulting in its build up in the ER lumen and the induction of ER quality control processes which lower glutelin levels as well as those of the other storage proteins. As proglutelin is initially synthesized on the cisternal ER, its deposition within prolamine protein bodies in esp2 suggests that PDIL1-1 helps retain proglutelin in the cisternal ER lumen until it attains competence for ER export and, thereby, indirectly preventing heterotypic interactions with prolamine polypeptides. [ABSTRACT FROM PUBLISHER]

The RNAs for the storage proteins of rice ( Oryza sativa), prolamines and glutelins, which are stored as inclusions in the lumen of the endoplasmic reticulum (ER) and storage vacuoles, respectively, are targeted by specific cis-localization elements to distinct subdomains of the cortical ER. Glutelin RNA has one or more cis-localization elements (zip codes) at the 3′ end of the RNA, whereas prolamine has two cis-elements; one located in the 5′ end of the coding sequence and a second residing in the 3′-untranslated region (UTR). We had earlier demonstrated that the RNAs for the maize zeins (‘prolamine’ class) are localized to the spherical protein body ER (PB-ER) in developing maize endosperm. As the PB-ER localization of the 10-kDa δ-zein RNA is maintained in developing rice seeds, we determined the number and proximate location of their cis-localization elements by expressing GFP fusions containing various zein RNA sequences in transgenic rice and analyzing their spatial distribution on the cortical ER by in situ RT-PCR and confocal microscopy. Four putative cis-localization elements were identified; three in the coding sequences and one in the 3′-UTR. Two of these zip codes are required for restricted localization to the PB-ER. Using RNA targeting determinants we show, by mis-targeting the storage protein RNAs from their normal destination on the cortical ER, that the coded proteins are redirected from their normal site of deposition. Targeting of RNA to distinct cortical ER subdomains may be the underlying basis for the variable use of the ER lumen or storage vacuole as the final storage deposition site of storage proteins among flowering plant species. [ABSTRACT FROM AUTHOR]

Rice glutelin RNAs are localized to the cisternal endoplasmic reticulum (ER) by a regulated RNA transport process requiring specific cis-localization elements. We set out to identify these glutelin sequences by their dominant character of being able to re-direct the normal protein body ER localization of a maize 10 kDa δ-zein RNA to the cisternal ER. In situ RNA localization analysis showed that the glutelin RNA contains multiple cis-localization elements; two located at the 5′ and 3′ ends of the coding sequences and a third located within the 3′-untranslated region. These three regions contain two conserved sequences, suggesting that these RNA recognition signals may be sequence based. [ABSTRACT FROM PUBLISHER]

Rice is unique among plants in that it accumulates major amounts of two types of seed storage proteins, prolamines and glutelins, which along with starch, serve as sources of carbon and nitrogen for the post- germinative seedling. Here, we investigate the role of the small GTPase Rab5 in the biosynthesis of storage proteins through characterization of the loss-of-function mutant glup4. In situ RT-PCR and fluorescence microscopy revealed the glup4 mutant is impaired in glutelin mRNA localization to the cortical endoplasmic reticulum in developing endosperm cells. Furthermore, microarray and two-dimensional difference in gel electrophoresis analysis of transcript and protein abundance, respectively, identified a number of genes whose expression is affected by the loss of Rab5, including starch biosynthetic enzymes. Our results indicate Rab5 is important for RNA and protein sorting in rice seed and supports previous observations that the biosynthesis of starch and storage proteins may be interrelated. [ABSTRACT FROM AUTHOR]