Three Starch Synthase IIa (SSIIa) Alleles Reveal the Effect of SSIIa on the Thermal and Rheological Properties, Viscoelasticity, and Eating Quality of Glutinous Rice.

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.

Can EUS-FNA cytology reliably classify gastric submucosal spindle cell tumors?

BACKGROUND: Cytological diagnosis using endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for gastric submucosal spindle cell tumors, such as gastrointestinal stromal tumors (GISTs), leiomyomas, and schwannomas, is challenging because of their similar morphological characteristics. OBJECTIVE AND MATERIALS: To clarify the cytological differential points, we reviewed the EUS-FNA cytology specimens of GISTs (37 cases), leiomyomas (11 cases), and schwannomas (4 cases). METHOD: Twelve cytomorphological features were evaluated: lymphocytes, crushed nuclei, naked spindle nuclei, mast cell, length of the streaming arrangement, cellularity, nuclei at the cluster margin (nuclei located at the periphery of the cell cluster), peripheral feathering (loosely aggregated cells at the margin of a cell cluster tended to taper like feathers), metachromasia, wavy nuclei, fishhook-type nuclei, and anisonucleosis. RESULTS: Among these features, lymphocytes, naked spindle nuclei, length of the streaming arrangement, cellularity, nuclei at the cluster margins, peripheral feathering, and anisonucleosis were statistically significant for differentiation. Based on these findings, we developed an algorithm for cytodiagnosis. The algorithm was taught to four cytologists, and the interobserver agreement and correct diagnosis rates were compared before and after education, which showed a significant improvement. DISCUSSION: The histological types of gastric submucosal spindle cell tumors can be estimated using this algorithm for EUS-FNA cytology. Furthermore, this algorithm can be applied for cytological diagnosis at bedside during rapid on-site evaluation.

Effect of Heading Date on the Starch Structure and Grain Yield of Rice Lines with Low Gelatinization Temperature.

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.

Improving Agricultural Traits While Maintaining High Resistant Starch Content in Rice.

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.

Mutation in BEIIb mitigates the negative effect of the mutation in ISA1 on grain filling and amyloplast formation in rice.

KEY MESSAGE: Mutation of the BEIIb gene in an isa1 mutant background mitigates the negative effect of the ISA1 mutation on grain filling, and facilitates recovery of amyloplast formation in rice endosperm. In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the BEIIb and ISA1 genes and showed no BEIIb and ISA1 activity, implying that both lines are beIIb isa1 double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to beIIb mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to isa1 mutants. Furthermore, beIIb isa1 double mutant F2 lines derived from a cross between Chikushi-kona 85 and Nishihomare (wild-type cultivar) showed significantly heavier seed weight than the beIIb and isa1 single mutant lines. These results suggest that co-occurrence of beIIb and isa1 mutant alleles in a single genetic background mitigates the negative effect of the isa1 allele on grain filling, and contributes to recovery of the amyloplast formation defect in the isa1 single mutant.

Active-type starch synthase (SS) IIa from indica rice partially complements the sugary-1 phenotype in japonica rice endosperm.

KEY MESSAGE: Introduction of higher SSIIa activity to mild-type isa1 mutant by crossing results in restoration of crystallinity, starch granule structure, and production of plump seeds. Isoamylase 1 (ISA1) removes improper α-1, 6 glycosidic branches of amylopectin generated by starch branching enzymes and is essential for the formation of proper amylopectin structure. Rice isa1 (sug-1) mutants in japonica cultivar with less-active starch synthase IIa (SSIIa) and low granule-bound SSI (GBSSI) expression display wrinkled seed phenotype by accumulating water-soluble phytoglycogen instead of insoluble amylopectin. Expression of active SSIIa in transgenic rice produced with a severe-type isa1 mutant accumulated some insoluble glucan with weak B-type crystallinity at the periphery of seeds but their seeds remained wrinkled. To see whether introduction of high levels of SSIIa and/or GBSSI can restore the grain filling of the mild-type sug-1 mutant (EM653), new rice lines (SS2a gbss1L isa1, ss2aL GBSS1 isa1, and SS2a GBSS1 isa1) were generated by crossing japonica isa1 mutant (ss2aL gbss1L isa1) with wild type indica rice (SS2a GBSS1 ISA1). The results showed that SS2a gbss1L isa1 and SS2a GBSS1 isa1 lines generated chalky plump seeds accumulating insoluble amylopectin-like glucans with an increase in DP 13-35, while ss2aL GBSS1 isa1 generated wrinkly seeds and accumulated soluble glucans enriched with DP < 13. Scanning electron microscopic observation of cross-section of the seeds showed that SS2a gbss1L isa1 and SS2a GBSS1 isa1 produced wild type-like polygonal starch granules. These starches showed the A-type crystallinity comparable to the wild type, while the japonica isa1 mutant and the transgenic rice do not show any or little crystallinity, respectively. These results indicate that introduction of higher SSIIa activity can mostly complements the mild-type sug-1 phenotype.

Starch synthases SSIIa and GBSSI control starch structure but do not determine starch granule morphology in the absence of SSIIIa and SSIVb.

KEY MESSAGE: High 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.

Generation and Starch Characterization of Non-Transgenic BEI and BEIIb Double Mutant Rice (Oryza sativa) with Ultra-High Level of Resistant Starch.

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.

CO2-Responsive CCT Protein Stimulates the Ectopic Expression of Particular Starch Biosynthesis-Related Enzymes, Which Markedly Change the Structure of Starch in the Leaf Sheaths of Rice.

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.

Starch Synthase IIa-Deficient Mutant Rice Line Produces Endosperm Starch With Lower Gelatinization Temperature Than Japonica Rice Cultivars.

The gelatinization temperature of endosperm starch in most japonica rice cultivars is significantly lower than that in most indica rice cultivars. This is because three single nucleotide polymorphisms in the Starch synthase (SS) IIa gene in japonica rice cultivars (SSIIaJ ) significantly reduce SSIIa activity, resulting in an increase in amylopectin short chains with degree of polymerization (DP) ≤ 12 compared to indica rice cultivars (SSIIaI ). SSIIa forms a trimeric complex with SSI and starch branching enzyme (BE) IIb in maize and japonica rice, which is likely important for the biosynthesis of short and intermediate amylopectin chains (DP ≤ 24) within the amylopectin cluster. It was unknown whether the complete absence of SSIIa further increases amylopectin short chains and reduces gelatinization temperature and/or forms altered protein complexes due to the lack of a suitable mutant. Here, we identify the SSIIa-deficient mutant rice line EM204 (ss2a) from a screen of ca. 1,500 plants of the rice cultivar Kinmaze (japonica) that were subjected to N-methyl-N-nitrosourea mutagenesis. The SSIIa gene in EM204 was mutated at the boundary between intron 5 and exon 6, which generated a guanine to adenine mutation and resulted in deletion of exon 6 in the mRNA transcript. SSIIa activity and SSIIa protein in developing endosperm of EM204 were not detected by native-PAGE/SS activity staining and native-PAGE/immunoblotting, respectively. SSIIa protein was completely absent in mature seeds. Gel filtration chromatography of soluble protein extracted from developing seeds showed that the SSI elution pattern in EM204 was altered and more SSI was eluted around 300 kDa, which corresponds with the molecular weight of trimeric complexes in wild type. The apparent amylose content of EM204 rice grains was higher than that in its parent Kinmaze. EM204 also had higher content of amylopectin short chains (DP ≤ 12) than Kinmaze, which reduced the gelatinization temperature of EM204 starch by 5.6°C compared to Kinmaze. These results indicate that EM204 starch will be suitable for making foods and food additives that easily gelatinize and slowly retrograde.

Characterization of the endosperm starch and the pleiotropic effects of biosynthetic enzymes on their properties in novel mutant rice lines with high resistant starch and amylose content.

Resistant starch (RS) is beneficial to human health. In order to reduce the current prevalence of diabetes and obesity, several transgenic and mutant crops containing high RS content are being developed. RS content of steamed rice with starch-branching enzyme (BE)IIb-deficient mutant endosperms is considerably high. To understand the mechanisms of RS synthesis and to increase RS content, we developed novel mutant rice lines by introducing the gene encoding starch synthase (SS)IIa and/or granule-bound starch synthase (GBSS)I from an indica rice cultivar into a japonica rice-based BEIIb-deficient mutant line, be2b. Introduction of SSIIa from an indica rice cultivar produced higher levels of amylopectin chains with degree of polymerization (DP) 11-18 than those in be2b; the extent of the change was slight due to the shortage of donor chains for SSIIa (DP 6-12) owing to BEIIb deficiency. The introduction of GBSSI from an indica rice cultivar significantly increased amylose content (by approximately 10%) in the endosperm starch. RS content of the new mutant lines was the same as or slightly higher than that of the be2b parent line. The relationship linking starch structure, RS content, and starch biosynthetic enzymes in the new mutant lines has also been discussed.

Analysis of the Rice ADP-Glucose Transporter (OsBT1) Indicates the Presence of Regulatory Processes in the Amyloplast Stroma That Control ADP-Glucose Flux into Starch.

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[(14)C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.

Mutation of the plastidial alpha-glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm.

Plastidial phosphorylase (Pho1) accounts for approximately 96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30 degrees C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20 degrees C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures.

Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between japonica and indica rice varieties.

Four amino acids were variable between the 'active' indica-type and 'inactive' japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the 'active' indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B(1) chains by elongating short A and B(1) chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.

The structure of starch can be manipulated by changing the expression levels of starch branching enzyme IIb in rice endosperm.

When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications.

Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm.

We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) > or = 37 and short chains with DP 12 to 21, marked increase in short chains with DP < or = 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2-3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.