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In this study, a range of barley allelic mutants lost ADPG binding structure of starch synthase IIa (SSIIa) were created through targeted mutagenesis of SSIIa by RNA-guided Cas9. The transcriptomic and qRT-PCR results showed the increased mRNA expression of HvGBSSI and the decreased HvSSIIa and HvSBEI levels in ssIIa mutant grains, which were consistent with the expressions of GBSSI, SSS and SBE enzymatic activities, respectively. However, the increased expressions of HvSSI cannot effectively compensate for the loss of HvSSIIa. The metabolic pathway analysis showed that the mutation of SSIIa led to increased ADP-glucose synthesis in barley grains. The ssIIa mutant grains had two and six times amylose, and RS contents in control grains, respectively, and significantly changed starch structure and functions compared to the controls. No metabolite changes could compensate for the decrease of starch biosynthesis in the ssIIa null mutant., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Starch biosynthesis in cereal crops is a complex pathway regulated by multiple starch synthetic enzymes. Starch synthase IIa (SSIIa) is well-known to be one of the major starch synthases and is very important in amylopectin biosynthesis. It has significant effects on grain composition and kernel traits. However, there are few reports on the association of natural variation of SSIIa in barley and grain composition and characteristics. In this work, two SSIIa isoforms were first identified as SSIIa H and SSIIa L by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and Western blotting. Sequence analysis of the SSIIa gene demonstrated that a 33 bp insertion coding a peptide of APPSSVVPAKK caused different SSIIa, e.g., SSIIa H and SSIIa L . Based on this molecular difference, a polymerase chain reaction marker was developed, which could be used to screen different SSIIa genotypes easily. Kernel hardness of SSIIa L genotypes was significantly higher than that of SSIIa H Chinese barley cultivars. The proportion of SSIIa L genotypes was extremely low in Australian barley cultivars (5/24) and much higher in Tibetan hull-less barley cultivars (46/74), consistent with the end-use requirements of barley grain. This study provided new information in barley endosperm starch synthesis and indicated that it is valuable for choosing the preferred SSIIa genotype according to the end-use requirements.

Key Message: Catalytically active indica SSIIa allele in high amylose rice with down-regulated japonica SBEIIb can increase starch content and modify the starch structure and properties without changing its amylose content. 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 (SBEIIb DR ) with either indica or japonica type SSIIa (SSIIa ind or SSIIa jap ). In SBEIIb DR 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 SSIIa ind SBEIIb DR than SSIIa jap SBEIIb DR , which indicated that indica type SSIIa, possibly in the monomer form, was extensively involved in starch biosynthesis in the SBEIIb DR endosperm. Furthermore, SSIIa ind SBEIIb DR grains had higher total starch content and higher starch swelling power than SSIIa jap SBEIIb DR lines, but the amylopectin gelatinization temperatures and enthalpy and the apparent amylose content remained similar. In summary, this work suggests that SSIIa ind 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.

Key Message: An EMS-induced single-base mutation at a splice site caused abnormal RNA splicing and resulted in the gene inactivation and the lack of Wx-A1 protein in a wheat EMS mutant line. An EMS-mutagenized population was generated using common wheat cv. SM126 consisting of 10,600 M2 plants. One Wx-A1 null mutant was identified through analyses of 390 grains produced from 130 M2 plants using electrophoresis analyses. The Wx-A1 sequences of parental line SM126 and M2-31 mutant were determined as 2781 bp, and there was only one SNP mutation between them. The SNP was a mutation from G to A at nucleotide sequence position 2168 bp (G2168A) downstream of the start codon which was located at the splicing site within the eighth intron. All 52 cDNA transcripts were found to be incorrectly spliced and can be summarized as five types of variations. The deletion of the exon and the exclusion of intron were structural features in abnormal splicing RNA. Together with the prediction of potential splice regulatory motifs, the mutation G2168A happened within the 5' splice site of the eighth intron and destroyed the splice donor site from GU to AU, which may have brought about a barrier against correct RNA splice, and generated abnormal mRNA, which was the mechanism of the inactivation of Wx-A1 in M2-31. The lack of Wx-A1 has resulted in changes in starch properties in the M2-31 mutant, with the reduction in amylose and starch contents. The increased grains hardness was observed in M2-31, which may be related to the lower expression level of Pinb-D1 gene. As the waxy wheat foods have a lot of advantages, the null waxy genes will be widely applied in breeding waxy wheat for varied amylose contents.

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. 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.

Granule-bound starch synthase I (GBSS I) plays an important role in the synthesis of amylose and in the determination of starch properties in barley grains. Genomic DNAs for the Waxy gene encoding GBSS I protein were sequenced from 34 barley accessions or lines from Qinghai-Tibet plateau in China, to identify Waxy gene nucleotide variations and study the roles of polymorphic sites of the Waxy gene on expression levels of Waxy transcripts and GBSS I proteins and on resulting starch properties. A total of 116 DNA polymorphic sites were identified within the barley Waxy gene, which divided the studied accessions into 11 haplotypes. Among 33 nucleotide polymorphic sites in coding regions, 5 SNPs in three exons were found to play different roles on the expression level of the Waxy transcript and the GBSS I protein and on the amylose content and starch properties. One SNP G(3935)-to-T substitution in the 10th exon in the accession Z999 (HP II-2) caused a high expression level of the Waxy transcript and the GBSS I protein and the amylose free phenotype. The other SNP alteration was a C(2453)-to-T in the fifth exon in the accession Z1191 (HP I-5), which drastically reduced the expression level of the Waxy transcript and the GBSS I protein and, finally, produced the amylose free phenotype. Three SNPs in the seventh exon in the accession Z1337 (HP I-6) did not significantly change the level of Waxy transcript, the GBSS I protein, and starch properties, except obviously reducing the breakdown value of starch viscosity and extending the peak time. A total of 84 DNA polymorphic sites were found in the noncoding regions. A 403 bp deletion at 5'UTR in the accession Z1979 (HP I-3) had low transcript level, low GBSS I protein level, and low amylose content due to the deletion of cis-acting DNA regulatory elements. A 191 bp insertion and a 15 bp insertion in the first intron and second exons, respectively, may be closely related to a higher transcript level of the Waxy gene and significant differences in some starch properties of the Waxy I DNA group as compared to the Waxy II DNA group. This study indicates the specific variations of the Waxy gene have a great effect on amylose synthesis and starch properties of hull-less barley, which could be very useful to produce new barley with variable starch properties.

Studies in Arabidopsis and rice suggest that manipulation of starch synthase I (SSI) expression in wheat may lead to the production of wheat grains with novel starch structure and properties. This work describes the suppression of SSI expression in wheat grains using RNAi technology, which leads to a low level of enzymatic activity for SSI in the developing endosperm, and a low abundance of SSI protein inside the starch granules of mature grains. The amylopectin fraction of starch from the SSI suppressed lines showed an increased frequency of very short chains (degree of polymerization, dp 6 and 7), a lower proportion of short chains (dp 8-12), and more intermediate chains (dp 13-20) than in the grain from their negative segregant lines. In the most severely affected line, amylose content was significantly increased, the morphology of starch granules was changed, and the proportion of B starch granules was significantly reduced. The change of the fine structure of the starch in the SSI-RNAi suppression lines alters the gelatinization temperature, swelling power, and viscosity of the starch. This work demonstrates that the roles of SSI in the determination of starch structure and properties are similar among different cereals and Arabidopsis.

In this study of barley starch synthesis, the interaction between mutations at the sex6 locus and the amo1 locus has been characterized. Four barley genotypes, the wild type, sex6, amo1, and the amo1sex6 double mutant, were generated by backcrossing the sex6 mutation present in Himalaya292 into the amo1 'high amylose Glacier'. The wild type, amo1, and sex6 genotypes gave starch phenotypes consistent with previous studies. However, the amo1sex6 double mutant yielded an unexpected phenotype, a significant increase in starch content relative to the sex6 phenotype. Amylose content (as a percentage of starch) was not increased above the level observed for the sex6 mutation alone; however, on a per seed basis, grain from lines containing the amo1 mutation (amo1 mutants and amo1sex6 double mutants) synthesize significantly more amylose than the wild-type lines and sex6 mutants. The level of granule-bound starch synthase I (GBSSI) protein in starch granules is increased in lines containing the amo1 mutation (amo1 and amo1sex6). In the amo1 genotype, starch synthase I (SSI), SSIIa, starch branching enzyme IIa (SBEIIa), and SBEIIb also markedly increased in the starch granules. Genetic mapping studies indicate that the ssIIIa gene is tightly linked to the amo1 locus, and the SSIIIa protein from the amo1 mutant has a leucine to arginine residue substitution in a conserved domain. Zymogram analysis indicates that the amo1 phenotype is not a consequence of total loss of enzymatic activity although it remains possible that the amo1 phenotype is underpinned by a more subtle change. It is therefore proposed that amo1 may be a negative regulator of other genes of starch synthesis.

Starch synthases (SS) are responsible for elongating the alpha-1,4 glucan chains of starch. A doubled haploid population was generated by crossing a line of wheat, which lacks functional ssIIa genes on each genome (abd), and an Australian wheat cultivar, Sunco, with wild type ssIIa alleles on each genome (ABD). Evidence has been presented previously indicating that the SGP-1 (starch granule protein-1) proteins present in the starch granule in wheat are products of the ssIIa genes. Analysis of 100 progeny lines demonstrated co-segregation of the ssIIa alleles from the three genomes with the SGP-1 proteins, providing further evidence that the SGP-1 proteins are the products of the ssIIa genes. From the progeny lines, 40 doubled haploid lines representing the eight possible genotypes for SSIIa (ABD, aBD, AbD, ABd, abD, aBd, Abd, abd) were characterized for their grain weight, protein content, total starch content and starch properties. For some properties (chain length distribution, pasting properties, swelling power, and gelatinization properties), a progressive change was observed across the four classes of genotypes (wild type, single nulls, double nulls and triple nulls). However, for other grain properties (seed weight and protein content) and starch properties (total starch content, granule morphology and crystallinity, granule size distribution, amylose content, amylose-lipid dissociation properties), a statistically significant change only occurred for the triple nulls, indicating that all three genes had to be missing or inactive for a change to occur. These results illustrate the importance of SSIIa in controlling grain and starch properties and the importance of amylopectin fine structure in controlling starch granule properties in wheat.

Chlamydomonas reinhardtii displays a diurnal rhythm of starch content that peaks in the middle of the night phase if the algae are provided with acetate and CO(2) as a carbon source. We show that this rhythm is controlled by the circadian clock and is tightly correlated to ADP-glucose pyrophosphorylase activity. Persistence of this rhythm depends on the presence of either soluble starch synthase III or granule-bound starch synthase I (GBSSI). We show that both enzymes play a similar function in synthesizing the long glucan fraction that interconnects the amylopectin clusters. We demonstrate that in log phase-oscillating cultures, GBSSI is required to obtain maximal polysaccharide content and fully compensates for the loss of soluble starch synthase III. A point mutation in the GBSSI gene that prevents extension of amylopectin chains, but retains the enzyme's normal ability to extend maltooligosaccharides, abolishes the function of GBSSI both in amylopectin and amylose synthesis and leads to a decrease in starch content in oscillating cultures. We propose that GBSSI has evolved as a major enzyme of amylopectin synthesis and that amylose synthesis comes as a secondary consequence of prolonged synthesis by GBSSI in arrhythmic systems. Maintenance in higher plant leaves of circadian clock control of GBSSI transcription is discussed.

Analysis of barley shrunken grain mutants has identified lines with a novel high amylose starch phenotype. The causal mutation is located at the sex6 locus on chromosome 7H, suggesting the starch synthase IIa (ssIIa) gene as a candidate gene altered by the mutation. Consistent with this hypothesis, no evidence of SSIIa protein expression in either the starch granule or soluble fractions of the endosperm was found. Sequences of the starch synthase IIa gene, ssIIa, from three independent sex6 lines showed the presence of a stop codon preventing translation of the ssIIa transcript in each line. Perfect segregation of the starch phenotype with the presence of stop codons in the ssIIa gene was obtained, providing strong evidence for the lesion in the ssIIa gene being the causal mutation for the sex6 phenotype. The loss of SSIIa activity in barley leads to novel and informative phenotypes. First, a decrease in amylopectin synthesis to less than 20% of the wild-type levels indicates that SSIIa accounts for the majority of the amylopectin polymer elongation activity in barley. Secondly, in contrast to high amylose starches resulting from branching enzyme downregulation, the sex6 starches have a shortened amylopectin chain length distribution and a reduced gelatinisation temperature. Thirdly, the mutation leads to pleiotropic effects on other enzymes of the starch biosynthesis pathway, abolishing the binding of SSI, branching enzyme IIa and branching enzyme IIb to the starch granules of sex6 mutants, while not significantly altering their expression levels in the soluble fraction.

Wheat and barley contain at least four classes of starch synthases in the endosperm, granule bound starch synthase I (GBSSI) and starch synthases I, II and III (SSI, SSII, SSIII). In this work, SSII in barley is shown to be associated with the starch granule by using antibodies. A cDNA from barley encoding SSII and the genes for SSII from barley and Aegilops tauschii ( A. tauschii, the D genome donor to wheat) are characterised. Fluorescent in situ hybridisation (FISH) and PCR were used to localise the wheat SSII gene to the short arm of chromosome 7, showing synteny with the location of the rice SSII gene to the short arm of chromosome 6. Comparison of the genes encoding SSII of A. tauschii, barley and Arabidopsis showed a conserved exon-intron structure although the size of the introns varied considerably. Extending such comparison between the genes encoding starch synthases (GBSSI, SSI, SSII and SSIII) from A. tauschii and Arabidopsis showed that the exon-intron structures are essentially conserved. Separate and distinct genes for the individual starch synthases therefore existed before the separation of monocotyledons and dicotyledons.

A study published in the Plant Biotechnology Journal explores the use of genome editing to increase the resistant starch (RS) and dietary fiber content in barley grains. The researchers targeted five starch synthesis genes using CRISPR/Cas9 technology and successfully generated barley mutants with mutations in one to three of the targeted genes. These mutants exhibited significantly higher levels of amylose, RS, and dietary fiber compared to the control group. The study demonstrates the feasibility of multiplex gene editing for modifying starch and provides a basis for breeding barley with improved health benefits. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Eight types of novel wheat lines with different null alleles for starch synthase IIa, presenting different starch contents and amylose/amylopectin ratios, was processed on a lab-scale with capillary rheometer under 2 conditions (28% moisture content at 140°C and 28% moisture content at 110°C) in order to obtain different degrees of starch transformation. The thermomechanical properties measured by DMA were explained by the composite structure of the products, i.e. gluten dispersed in starch matrix, as observed by fluorescence microscopy. To explain the storage modulus variation with the flour composition, isostress–isostrain model was applied as a function of starch content. The discrepancies with this model are probably related to the variation of amylose/amylopectin ratio. This study finally pointed to the impact of the phase volume of starchy matrix and fillers on the material functionality. [Copyright &y& Elsevier]

Cereal grains are the major source of fructan intake with potential health benefits for human. Measurement of fructan levels in cereal breeding populations is a great challenge and time-consuming. In this study, K-FRUCHK and K-FRUC kits commercialized by Megazyme International Limited (Bray, Ireland) were modified and optimized in a 96 deep well plate and a thermal block (BioShake iQ, Q.Instruments, Jena, Germany). The modified assays are able to measure up to 91 samples per day with a range of fructan concentrations (0.4–10.8% in this experiment). Of the two assays, the modified K-FRUC assay is more desirable for measuring fructan levels in cereal grains with high starch content. This assay was successfully used to screen three wheat starch synthase IIa ( SSIIa ) mutation populations with a total of 466 lines. All 21 SSIIa triple null mutants showed high fructan levels (3.1–10.8%) in wholemeal flours; while all single and double nulls were similar to wild types with ∼1% of fructans. This result indicates that lack of entire SSIIa activity changes carbon flux from starch synthesis into fructan pathway. The identified 21 triple null wheat lines may have potential use for the production of high fructan staple foods. [ABSTRACT FROM AUTHOR]