Starch Synthase II Research Articles

Multi-omics analyses reveal mechanism for high resistant starch formation in an indica rice SSIIIa mutant

Soluble starch synthase IIIa (SSIIIa) is a key enzyme involved in amylopectin biosynthesis in rice, and deficiency of SSIIIa results in high content of resistant starch, which is benefit to human health. However, little is known about metabolic differences and carbon re-allocation in the seeds of the indica rice ss3a mutant. We found that SSIIIa deficiency impaired the storage of starch, but increased the soluble sugars, free amino acids and lipids. By multi-omic analyses, we found inactivation of SSIIIa triggered carbon repartitioning by downregulating sucrose synthase, grain incomplete filling 1, fructokinase and hexokinase (HK), and promoted the accumulation of soluble sugars. Meanwhile, the downregulation of HK and upregulation of plastidic phosphoglucomutase reduced the carbon flow through glycolysis and promoted glycogenesis. The downregulation of OsbZIP58 and the deleterious effect on ribosome formation might result in the reduction of storage protein synthesis and increased free amino acids content in ss3a. The higher levels of amylose and lipids could form more amylose-lipid complexes (starch phospholipids), resulting in a higher resistant starch content. Taken together, our study unraveled a functional cross talk between starch, protein and lipids in rice endosperm during seed development of ss3a, providing new insights for formation of high resistant starch in rice.

Starch structural reasons for the effects of SSIIIa deficiency on the textural and digestive properties of cooked rice

Due to the urgent requirements for development of healthy diets, foods with slow digestibility combined with superior eating quality attract increasingly more attention. A high amylose indica rice mutant with starch synthase IIIa (SSIIIa) deficiency was reported to have higher resistant starch content, while how the starch structure affects the textural and digestive properties of the cooked rice have not been clarified. The ss3a starch had a significantly higher amylose content (AC), longer amylose intermediate chains but shorter amylose long chains, and also displayed a higher proportion of amylopectin A, B1, and B2 chains but a lower amount of B3 chains and shorter chain length of amylopectin molecules. The gelatinization temperatures and paste viscosity were decreased because of the increased AC and more amylopectin short chains, which further contributed to the harder texture of cooked rice. In addition, more amylose intermediate to long chains and longer amylose intermediate chains of the mutant resulted in the slow digestion rate of the fast-digestible fraction, and less amylopectin intermediate to long chains caused the complete digestion of cooked rice. These findings explained the nutritional benefits of the ss3a rice and its importance to the production of slow-digestive food products with acceptable texture.

Identification of a new allele of soluble starch synthase IIIa involved in the elongation of amylopectin long chains in a chalky rice mutant

A chalky endosperm mutant (GM03) induced from an indica rice GLA4 was used to investigate the functional gene in starch biosynthesis. Bulked segregant analysis and sanger sequencing determined that a novel mutation in soluble starch synthase IIIa (SSIIIa) is responsible for the chalky phenotype in GM03. Complementary test by transforming the active SSIIIa gene driven by its native promoter to GM03 recovered the phenotype to its wildtype. The expression of SSIIIa was significantly decreased, while SSIIIa protein was not detected in GM03. The mutation of SSIIIa led to increased expression of most of starch synthesis related genes and elevated the levels of most of proteins in GM03. The CRISPR/Cas9 technology was used for targeted disruption of SSIIIa, and the mutant lines exhibited chalky endosperm which phenocopied the GM03. Additionally, the starch fine structure in the knockout mutant lines ss3a-1 and ss3a-2 was similar with the GM03, which showed increased amylose content, higher proportions of B1 and B2 chains, much lower proportions of B3 chains and decreased degree of crystallinity, leading to altered thermal properties with lower gelatinization temperature and enthalpy. Collectively, these results suggested that SSIIIa plays an important role in starch synthesis by elongating amylopectin long chains in rice.

Loss of starch synthase IIIa changes starch molecular structure and granule morphology in grains of hexaploid bread wheat

Starch synthase III plays a key role in starch biosynthesis and is highly expressed in developing wheat grains. To understand the contribution of SSIII to starch and grain properties, we developed wheat ssIIIa mutants in the elite cultivar Cadenza using in silico TILLING in a mutagenized population. SSIIIa protein was undetectable by immunoblot analysis in triple ssIIIa mutants carrying mutations in each homoeologous copy of ssIIIa (A, B and D). Loss of SSIIIa in triple mutants led to significant changes in starch phenotype including smaller A-type granules and altered granule morphology. Starch chain-length distributions of double and triple mutants indicated greater levels of amylose than sibling controls (33.8% of starch in triple mutants, and 29.3% in double mutants vs. 25.5% in sibling controls) and fewer long amylopectin chains. Wholemeal flour of triple mutants had more resistant starch (6.0% vs. 2.9% in sibling controls) and greater levels of non-starch polysaccharides; the grains appeared shrunken and weighed ~ 11% less than the sibling control which was partially explained by loss in starch content. Interestingly, our study revealed gene dosage effects which could be useful for fine-tuning starch properties in wheat breeding applications while minimizing impact on grain weight and quality.

A Pilot Study to Assess Glucose, Insulin, and Incretin Responses Following Novel High Resistant Starch Rice Ingestion in Healthy Men.

IntroductionA newly developed resistant starch (RS) rice line with double mutation of starch synthase IIIa and branching enzyme IIb (ss3a/be2b) exhibits a tenfold greater percentage RS value than the wild-type rice line. Currently, the effects of cooked rice with such high RS content on secretion and action of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are unclear. Therefore, we conducted a pilot study to assess postprandial responses of GLP-1 and GIP along with glucose and insulin and also gastric emptying after ingestion of the high-RS cooked rice with ss3a/be2b in healthy subjects.MethodsIn a non-randomized crossover design, five healthy men ingested two test foods, control (low-RS) and high-RS cooked rice, with at least 1-week washout period between testing days. Plasma glucose, serum insulin, plasma total GLP-1, plasma total GIP, and also gastric emptying rate were measured after ingestion of each test food, and the incremental area under the curves (iAUC) was calculated for each biochemical parameter using the values from 0 to 180 min after ingestion.ResultsThe high-RS cooked rice ingestion tended to reduce iAUC-glucose (p = 0.06) and significantly reduced iAUC-insulin (p < 0.01) and iAUC-GLP-1 (p < 0.05) but not iAUC-GIP (p = 0.21) relative to control cooked rice ingestion. In addition, the high-RS cooked rice ingestion did not affect gastric emptying.ConclusionsThe present results indicate that the suppressive effects of the high-RS cooked rice ingestion on postprandial responses of glucose and insulin may be provided through attenuation in GLP-1 secretion along with its low digestibility into glucose. We suggest that the high-RS rice with ss3a/be2b may serve as a better carbohydrate source and also as a novel functional food for dietary interventions to improve postprandial hyperglycemia and hyperinsulinemia without both enhancing GLP-1 secretion and affecting gastric emptying in patients with diabetes.

Effects of starch synthase IIIa (&lt;i&gt;ssIIIa&lt;/i&gt;) genotype and grain hardness on the barley β-glucan content and grain morphology traits

オオムギの水溶性食物繊維β-グルカンが示す食後血糖値の上昇抑制などの健康機能性が注目されており，高β-グルカン品種の育成が求められている．本研究では，β-グルカン含有率の高いオオムギの育種において，簡易的な選抜技術の開発を目標として，これまでβ-グルカン含有率への強い関連性が示されている高アミロース性遺伝子amo1の極近傍にあるstarch synthase IIIa（ssIIIa）の遺伝子型とβ-グルカン含有率および穀粒形質との関係を解析した．解析には2組合せの交配に由来する後代系統群を用いた．「北陸皮54号」（後の「ゆきはな六条」）とamo1を有するもち性系統「四R系3755」との交配に由来するもち性の94の後代系統（F4–F5），「四R系3755」ともち性系統「はねうまもち」の交配に由来する165の後代系統（F4–F5）について，ssIIIa遺伝子型を識別するdCAPSマーカーで遺伝子型を判別し，β-グルカン含有率，SKCS穀粒硬度，硝子率，千粒重および空洞粒率を測定した．その結果，両組合せともssIIIa遺伝子型によってβ-グルカン含有率，SKCS穀粒硬度および硝子率に統計的有意差が認められ，「北陸皮54号」×「四R系3755」および「四R系3755」×「はねうまもち」の交配後代共に「四R系3755型」では「野生型」に比べ，β-グルカン含有率は約1.6倍多く，SKCS穀粒硬度は約23ポイント高く，硝子率は約7%高くなった．また，β-グルカン含有率はSKCS穀粒硬度と0.75から0.84の高い相関を示し，高β-グルカン化によって穀粒が硬くなる傾向が明らかとなった．これらのことから，amo1系統を母本とした高β-グルカン化を育種目標とする育種計画においては，ssIIIa遺伝子型やSKCS穀粒硬度を指標とした選抜が有効であると考えられた．

Identification of SNPs in crucial starch biosynthesis genes in rice

Rice is the foremost crop catering to the major calorific requirement of the human population but has the disadvantage of having high glycaemic index (GI). The fine quality rice varieties, BPT and RNR have been recently identified as having low GI in nature and are grown mostly in southern parts of India. Starch (80%) is the major component of rice endosperm attributing to GI. The study aimed to unravel the molecular basis of low GI through targeted pooled amplicon sequencing of major starch biosynthetic genes. A total of 13 candidate genes involved in starch synthesis were amplified and pooled in equimolar proportion for sequencing. Single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) were detected in both coding and noncoding regions. Among the genes that are under study, the highest number of variations were identified in starch synthase I (SSI) followed by starch synthase IIIA (SSIIIA) genes. Nonsynonymous SNPs with high probability of effecting gene function were validated by Sanger sequencing and molecular docking. Identified causative SNPs were mapped on 3000 rice genome database and their allele frequencies were obtained. The outcome of this study has a potential to be applied in breeding programmes to obtain low GI rice varieties with added beneficial traits.

The relationship between the expression pattern of starch biosynthesis enzymes and molecular structure of high amylose maize starch

Two high amylose (HAM) inbred lines with apparent amylose contents of 55 % and 62 %, respectively, were selected to explore the relationship between molecular structure and gene expression of starch-synthase involved enzymes. GPC analysis of debranched starches showed that the HAM starches (HAMSs) had shorter amylose chains and longer amylopectin chains than normal maize starch (NMS). FACE analysis showed that these HAMSs had a higher content of amylopectin chains of DP > 21. Quantitative Real-Time PCR analysis showed that the HAM lines had specifically low expression of the starch branching enzyme IIb (SBEIIb), and the starch synthase IIIa (SSIIIa) homologue, and high expression of the isoamylase 2 (ISA2), potentially suppressing the generation of amylopectin molecules through deficient branching and excessive debranching process, thereby increasing the relative amylose content. A high expression of GBSS1 was potentially associated with increased short amylose chain lengths in HAMSs.

Effects of single ingestion of rice cracker and cooked rice with high resistant starch on postprandial glucose and insulin responses in healthy adults: two randomized, single-blind, cross-over trials.

Rice with double mutation of starch synthase IIIa and branching enzyme IIb (ss3a/be2b) has much higher amounts of apparent amylose and resistant starch (RS) than usual varieties. In this study, we conducted two randomized, single-blind, crossover trials to investigate the effect of single ingestion of two processed foods composed of ss3a/be2b mutant rice on postprandial blood glucose and insulin response in healthy adults, compared to those of usual cultivar. In trial 1, of ingestion of rice crackers, the incremental area under the curves of glucose (IAUCglc) and insulin (IAUCins) in RS group was significantly lower than in the control group. In trial 2, of the ingestion of cooked rice, IAUCglc in the RS group was significantly lower than in the control group. These results showed that the ss3a/be2b-mutant rice cracker and cooked rice having high RS can attenuate postprandial blood glucose and insulin response.

Re-programming of gene expression in the CS8 rice line over-expressing ADPglucose pyrophosphorylase induces a suppressor of starch biosynthesis.

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.

Effects of Different Nitrogen Application Rates on Starch Accumulation, Starch Synthase Gene Expression and Enzyme Activity in Two Distinctive Potato Cultivars

Nitrogen (N) is the most important nutrient for potato growth. N fertilizer has an important effect on the tuber yield and starch content in potatoes. In this study, taking the high-starch cultivar Kexin 22 and the low-starch cultivar Kexin 19 as experimental materials, three N fertilizer application rates—0, 150 and 300 kg/ha—were used to investigate the effects of different N application rates on starch accumulation and the expression of starch synthase genes in potato tubers with different starch contents. In the cultivar Kexin 22, the accumulations of amylose, amylopectin and total starch showed a ranking of N150 > N300 > N0 for the entire growth period. The cultivar Kexin 19 showed an accumulation pattern of N0 > N150 > N300 in the early growth period, N150 > N300 > N0 in the middle growth period and N300 > N150 > N0 in the late growth period. Compared with those in Kexin 19, the expressions of the glucose-1-phosphate adenyltransferase (AGPP-L), granule-bound starch synthase (GBSSI), starch-branching enzyme I (SBEI) and soluble starch synthase III (SSIII) genes in Kexin 22 were upregulated, whereas no obvious difference existed in the expression of the alkylglycerone phosphate synthase (AGPP-S) and soluble starch synthase II (SSII) genes between the two cultivars. In Kexin 22, the different N application rates had a significant effect on the peak expression levels of AGPP-L, GBSSI, SBEI and SSIII but had a small effect on the peak expression time for these genes. Among these genes, most showed a ranking of N150 > N300 > N0 in the early and middle growth periods, and all showed a ranking of N300 > N150 > N0 in the late growth period; most showed a peak expression time on the 65th day after emergence (DAE 65). In Kexin 19, the different N application rates had a significant effect on the peak expression levels and peak expression times of the AGPP-L, GBSSI, SBEI and SSIII genes. Among these genes, all showed a ranking of N0 > N150 > N300 in the early growth period, whereas most showed a ranking of N150 > N300 > N0 in the middle growth period, and all showed a ranking of N300 > N150 > N0 in the late growth period. In Kexin 19, the peak gene expression was shifted to an earlier date under the low N levels, and it was delayed under the high N levels. The effects of the N application rate on the activities of starch synthases AGPP, GBSS, SSS and SBE showed largely the same trends as those in the expression levels of the related genes. Therefore, to obtain a high harvest of starch yield, different N application rates should be recommended for different cultivars.

Dissection of gene loci underlying pasting temperature in cassava

ABSTRACTIn this study, a fine genetic map within the quantitative trait loci (QTL) underlying pasting temperature (PT) of cassava (Manihot esculenta Crantz) was constructed using newly developed simple sequence repeat (SSR) markers. The SSRs were designed on the basis of two scaffolds (S11341 and S4043) of the cassava genome, which covered previously identified QTL regions of the PT trait. A total of 55 and 61 SSR markers derived from S11341 and S4043, respectively, representing 0.29% of the cassava genome, were generated; of which 23 and 19 showed informative polymorphic patterns. Consequently, all identified informative polymorphic markers were used to genotype 200 F1 progeny plants. The genotypic data were then analyzed, and the results showed that 480 markers were distributed across 23 linkage groups (LGs) with total length of 1,334 centimorgans (cM). An analysis of QTL underlying the PT trait revealed that marker EME81 on LG 1 had significant associations (P < 0.0001) in all environments evaluated. Four candidate genes were identified and selected for gene expression analysis in the parents, and among F1 lines with high and low PT values. Significant differences were observed in relative expression of carbohydrate phosphorylase (CP) and starch synthase II (SSII) between high and low PT in 6-month-old cassava. We found CP and SSII genes to potentially control the PT trait. In addition, the marker EME81 was found to be a promising marker for specific PT trait selection in cassava populations, which should facilitate marker-assisted selection for desired PT traits.

Starch synthase IIIa and starch branching enzyme IIb-deficient mutant rice line ameliorates pancreatic insulin secretion in rats: screening and evaluating mutant rice lines with antidiabetic functionalities.

Diabetes mellitus is a metabolic disease spreading worldwide that has been reported to worsen the development and progression of other diseases (cancer, vascular diseases and dementia). To establish functional rice lines with anti-postprandial hyperglycaemic effects, we developed mutant rice lines, which lack one or two gene(s) related to starch synthesis, and evaluated their effects. Powder of mutant rice lines or other grains was loaded to rats fasted overnight (oral grain powder loading test). Incremental area under time-concentration curves (iAUC) were calculated with monitored blood glucose levels. Rice lines with anti-postprandial hyperglycaemic effects were separated by cluster analysis with calculated iAUC. A double mutant rice #4019 (starch synthase IIIa (ss3a)/branching enzyme IIb (be2b)), one of the screened mutant rice lines, was fed to Goto-Kakizaki (GK) rats, an animal model for type 2 diabetes, for 5 weeks. Plasma levels of C-peptide, a marker of pancreatic insulin secretion, were measured with ELISA. For in vitro study, a rat pancreatic cell line was cultured with a medium containing rat serum which was sampled from rats fed #4019 diet for 2 d. After 24-h of incubation, an insulin secretion test was performed. Through the oral rice powder loading test, seven rice lines were identified as antidiabetic rice lines. The intake of #4019 diet increased plasma C-peptide levels of GK rats. This result was also observed in vitro. In rat serum added to cell medium, ornithine was significantly increased by the intake of #4019. In conclusion, the mutant rice #4019 promoted pancreatic insulin secretion via elevation of serum ornithine levels.

Induced splice site mutation generates alternative intron splicing in starch synthase II (SSII) gene in rice

ABSTRACTThe evolution of starch synthase (SS) has been extensively analysed in the model plant rice, but there have been few detailed studies directed towards exploring the intron splicing variant effect on the divergence of the SS gene. We report here an example that the alternative splicing of a GC-AG intron induced a new splicing isoform of SSII-1 expressed in Oryza sativa japonica cultivar Zhonghua-15. In addition, a GT-AG intron flanking the alternative splicing GC-AG intron was simultaneously spliced at an incorrect position, inducing another intron fragment gained in mature SSII-1 RNA. Moreover, when a GT-AG intron from SSII-3 was artificially mutated into the GC-AG intron, a 28 bp fragment from the intron added to the mature RNA could be observed in transgenic rice. In this study, the alternative intron splicing of the GC-AG intron might offer more variants for genetic divergence of the starch synthase gene family in rice.

Self-preserving lognormal volume-size distributions of starch granules in developing sweetpotatoes and modulation of their scale parameters by a starch synthase II (SSII)

Starch granule size distributions in plant tissues, when determined in high resolution and specified properly as a frequency function, could provide useful information on the granule formation and growth. To better understand genetic control of physical properties of starch granules, we attempted a new approach to analyze developmental and genotypic effects on morphology and size distributions of starch granules in sweetpotato storage roots. Starch granules in sweetpotatoes exhibited low sphericity, many shapes that appeared to be independent of genotypes or developmental stages, and non-randomly distributed sizes. Granule size distributions of sweetpotato starches were determined in high resolution as differential volume-percentage distributions of volume-equivalent spherical diameters, rigorously curve-fitted to be lognormal, and specified using their geometric means $$\bar{x}^{*}$$ and multiplicative standard deviations $$s^{*}$$ in a $$\bar{x}^{*} \times /({\text{multiply/divide}})s^{*}$$ form. The scale ( $$\bar{x}^{*}$$ ) and shape ( $$\bar{s}^{*}$$ ) of these distributions were independently variable, ranging from 14.02 to 19.36 μm and 1.403 to 1.567, respectively, among 22 cultivars/clones. The shape ( $$s^{*}$$ ) of granule lognormal volume-size distributions of sweetpotato starch were found to be highly significantly and inversely correlated with their apparent amylose contents. More importantly, granule lognormal volume-size distributions of starches in developing sweetpotatoes displayed the same self-preserving kinetics, i.e., preserving the shape but shifting upward the scale, as those of particles undergoing agglomeration, which strongly indicated involvement of agglomeration in the formation and growth of starch granules. Furthermore, QTL analysis of a segregating null allele at one of three homoeologous starch synthase II loci in a reciprocal-cross population, which was identified through profiling starch granule-bound proteins in sweetpotatoes of diverse genotypes, showed that the locus is a QTL modulating the scale of granule volume-size distributions of starch in sweetpotatoes.

Allelic variations in the soluble starch synthase II gene family result in changes of grain quality and starch properties in rice (Oryza sativa L.)

SUMMARYSoluble starch synthase II (SSII) plays an important role in the biosynthesis of starch and in rice it consists of three isoforms encoded by SSII-1, SSII-2 and SSII-3. However, the genetic effects of various SSII alleles on grain quality have not been systematically characterized. In the present study, the japonica alleles on SSII-1, SSII-2 and SSII-3 (SSIIa) loci from a japonica cultivar, Suyunuo, were respectively introgressed by molecular marker-assisted selection into a typical indica cultivar, Guichao2, through successive backcrossing, generating three sets of near-isogenic lines (NILs). Grain quality and starch property analysis showed that NIL-SSII-3j exhibited significant decreases in the following parameters: amylose content, average granule size, and setback viscosity and consistency; but increases in peak viscosity, hot paste viscosity, gelatinization temperature and relative crystallinity. Moreover, the proportion of short amylopectin chains and branching degree also increased when compared with those of NIL-SSII-3i (Guochao2). Similar effects were observed in NIL-SSII-1j, and certain alterations in the fine structure of starch (granule size) were revealed. However, NIL-SSII-2j did not exert significant effect on grain quality and starch properties. In brief, among the SSII gene family, the functional diversity occurred on SSII-1 and SSII-3, and not on SSII-2. Therefore, it appears that more attention should be directed to SSII-1 and SSII-3 loci for improving the eating and cooking quality of rice.

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm.

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.

Deficiencies in both starch synthase IIIa and branching enzyme IIb lead to a significant increase in amylose in SSIIa-inactive japonica rice seeds.

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.

Characterization of shrunken endosperm mutants in barley

Despite numerous studies on shrunken endosperm mutants caused by either maternal tissues (seg) or kernel per se (sex) in barley, the molecular mechanism for all of the eight seg mutants (seg1–seg8) and some sex mutants is yet to be uncovered. In this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm (seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9% (seg4a) to 25.8% (seg1). SDS-PAGE analysis revealed that 87kDa proteins corresponding to the starch branching enzyme II (SBEII) and starch synthase II (SSII) were not present in seg1, seg3, seg6, and seg7 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of seg1, seg3, seg6, and seg7 mutants decreased in varying degrees to lower levels until 27days after anthesis (DAA) after reaching the peak at 15–21DAA, which differed from the pattern of normal barley accessions. Further characterization of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sex1 had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg7 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.

Lack of starch synthase IIIa and high expression of granule-bound starch synthase I synergistically increase the apparent amylose content in rice endosperm

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 Wxb causing a low amylose content (ca. 20%).WAB2-3 (SS3a/Wxa) lines generated by the introduction of a dominant indica Wxa into a japonica waxy mutant (SS3a/wx) exhibit elevated GBSSI and amylose content (ca. 25%). The japonica ss3a mutant (ss3a/Wxb) 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 Wxa genes, the ss3a/Wxa line was generated by crossing ss3a/Wxb with SS3a/Wxa, and the starch properties of this line were examined. The results show that the apparent amylose content of the ss3a/Wxa line was increased (41.3%) compared to the parental lines. However, the GBSSI quantity did not increase compared to the SS3a/Wxa line. The amylopectin branch structures were similar to the ss3a/Wxb mutant. Therefore, Wxa and ss3a synergistically increase the apparent amylose content in rice endosperm, and the possible reasons for this increase are discussed.