Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1.

The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

Introduction of glucan synthase into the cytosol in wheat endosperm causes massive maltose accumulation and represses starch synthesis.

We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic ß-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of ß-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan.

STARCH SYNTHASE 4 is required for normal starch granule initiation in amyloplasts of wheat endosperm.

Starch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. We generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild-type initiated single 'A-type' granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation.

Final grain weight is not limited by the activity of key starch-synthesising enzymes during grain filling in wheat.

Since starch is by far the major component of the mature wheat grain, it has been assumed that variation in the capacity for starch synthesis during grain filling can influence final grain weight. We investigated this assumption by studying a total of 54 wheat genotypes including elite varieties and landraces that were grown in two successive years in fields in the east of England. The weight, water content, sugars, starch, and maximum catalytic activities of two enzymes of starch biosynthesis, ADP-glucose pyrophosphorylase and soluble starch synthase, were measured during grain filling. The relationships between these variables and the weights and starch contents of mature grains were analysed. Final grain weight showed few or no significant correlations with enzyme activities, sugar levels, or starch content during grain filling, or with starch content at maturity. We conclude that neither sugar availability nor enzymatic capacity for starch synthesis during grain filling significantly influenced final grain weight in our field conditions. We suggest that final grain weight may be largely determined by developmental processes prior to grain filling. Starch accumulation then fills the grain to a physical limit set by developmental processes. This conclusion is in accord with those from previous studies in which source or sink strength has been artificially manipulated.

Barley mutants with low rates of endosperm starch synthesis have low grain dormancy and high susceptibility to preharvest sprouting.

• Studies of embryo dormancy in relation to preharvest sprouting (PHS) in cereals have focused on ABA and other hormones. The relationship between these phenomena and the rate of grain filling has not been investigated. • A collection of barley mutants impaired in starch synthesis was assessed for preharvest sprouting in the field. In subsequent glasshouse experiments, developing grains were assayed for germination index, sugars, abscisic acid (ABA) and the effects of temperature and exogenous ABA on germination. • Mutant lines displayed greater preharvest sprouting in the field than parental lines. In the glasshouse, nondeep physiological dormancy was reduced in developing grains of five lines with mutations affecting proteins involved in endosperm starch synthesis. Inhibition of germination by exogenous ABA and elevated temperature was decreased in developing mutant grains. Sugar concentrations were high but embryo and endosperm ABA contents were unaltered. • We reveal a direct connection between grain filling and the extent of grain dormancy. Impaired endosperm starch synthesis directly influences the acquisition of embryo dormancy, perhaps because endosperm sugar concentrations modulate the ABA responsiveness of the embryo. Thus environmental or genetic factors that reduce grain filling are likely to reduce dormancy and enhance susceptibility to PHS.

The role of alpha-glucosidase in germinating barley grains.

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.

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.

Effect of high-amylose starch branching enzyme II wheat mutants on starch digestibility in bread, product quality, postprandial satiety and glycaemic response.

High-amylose starch branching enzyme II (sbeII) mutant wheat has potential to be low-glycaemic compared to conventional wheat; however, the effects of bread made from sbeII wheat flour on glycaemic response and product quality require investigation. We report the impact of white bread made from sbeII wheat flour on in vitro starch digestibility and product quality, and on postprandial glycaemia in vivo, compared to an isoglucidic wild-type (WT) control white bread. Starch in sbeII bread was ∼20% less susceptible to in vitro amylolysis leading to ∼15% lower glycaemic response measured in vivo, compared to the WT control bread, without major effects on bread appearance or texture, measured instrumentally. Despite the early termination of the in vivo intervention study due to the COVID-19 outbreak (n = 8 out of 19), results from this study indicate that sbeII wheat produces bread with lower starch digestibility than conventional white bread.

Wheat Grain Filling Is Limited by Grain Filling Capacity rather than the Duration of Flag Leaf Photosynthesis: A Case Study Using NAM RNAi Plants.

It has been proposed that delayed leaf senescence can extend grain filling duration and thus increase yields in cereal crops. We found that wheat (Triticum aestivum) NAM RNAi plants with delayed senescence carried out 40% more flag leaf photosynthesis after anthesis than control plants, but had the same rate and duration of starch accumulation during grain filling and the same final grain weight. The additional photosynthate available in NAM RNAi plants was in part stored as fructans in the stems, whereas stem fructans were remobilised during grain filling in control plants. In both genotypes, activity of starch synthase was limiting for starch synthesis in the later stages of grain filling. We suggest that in order to realise the potential yield gains offered by delayed leaf senescence, this trait should be combined with increased grain filling capacity.

Use of advanced recombinant lines to study the impact and potential of mutations affecting starch synthesis in barley.

The effects on barley starch and grain properties of four starch synthesis mutations were studied during the introgression of the mutations from diverse backgrounds into an elite variety. The lys5f (ADPglucose transporter), wax (granule-bound starch synthase), isa1 (debranching enzyme isoamylase 1) and sex6 (starch synthase IIa) mutations were introgressed into NFC Tipple to give mutant and wild-type BC2F4 families with different genomic contributions of the donor parent. Comparison of starch and grain properties between the donor parents, the BC2F4 families and NFC Tipple allowed the effects of the mutations to be distinguished from genetic background effects. The wax and sex6 mutations had marked effects on starch properties regardless of genetic background. The sex6 mutation conditioned low grain weight and starch content, but the wax mutation did not. The lys5 mutation conditioned low grain weight and starch content, but exceptionally high ß-glucan contents. The isa1 mutation promotes synthesis of soluble α-glucan (phytoglycogen). Its introgression into NFC Tipple increased grain weight and total α-glucan content relative to the donor parent, but reduced the ratio of phytoglycogen to starch. This study shows that introgression of mutations into a common, commercial background provides new insights that could not be gained from the donor parent.

A rice mutant lacking a large subunit of ADP-glucose pyrophosphorylase has drastically reduced starch content in the culm but normal plant morphology and yield.

A mutant of rice (Oryza sativa L.) was identified with a Tos17 insertion in Os05g50380, a gene encoding a plastidial large subunit (LSU) of ADP-glucose pyrophosphorylase (AGPase) that was previously called OsAPL3 or OsAGPL1. The insertion prevents the production of a normal transcript. Characterisation of the mutant showed that this LSU is required for 97% of the starch synthesised in the flowering stem (culm), approximately half of the AGPase activity in developing embryos and that it contributes to AGPase activity in the endosperm. Despite the near absence of starch in the culms and reduced starch content in the embryos, the mutant rice plants grow and develop normally, and show no reduction in productivity. The starch content of leaves is increased in the mutant, revealing plasticity in the distribution of photosynthates among different temporary carbohydrate storage pools within the plant.

Induction and identification of a small-granule, high-amylose mutant in cassava (Manihot esculenta Crantz).

Only two mutations have been described in the literature, so far, regarding starch and root quality traits in cassava. This article reports on an induced mutation in this crop, first identified in 2006. Botanical seed from five different cassava families were irradiated with gamma rays. Seed was germinated, transplanted to the field (M1 plants), and self-pollinated to produce the M2 generation. Abnormal types regarding starch granule morphology were identified during the single plant evaluation of M2 genotypes. To confirm these characteristics, selected genotypes were cloned and a second evaluation, based on cloned plants obtained from vegetative multiplication, was completed in September 2007. Two M2 genotypes presented small starch granules, but only one could be fully characterized, presenting a granule size of 5.80 +/- 0.33 microm compared with three commercial clones with granule sizes ranging from 13.97 +/- 0.12 to 18.73 +/- 0.10 microm and higher-than-normal amylose content (up to 30.1% in cloned plants harvested in 2007, as compared with the typical values for "normal" cassava starch of around 19.8%). The gels produced by the starch of these plants did not show any viscosity when analyzed with the rapid viscoanalyzers (5% suspension), and the gels had low clarity. Low viscosity could be observed at higher concentrations (8 or 10% suspensions). Preliminary results suggest that the mutation may be due to a lesion in a gene encoding one of the isoforms of isoamylase (probably isa1 or isa2).

A mutant of rice lacking the leaf large subunit of ADP-glucose pyrophosphorylase has drastically reduced leaf starch content but grows normally.

A mutant of rice was identified with a Tos17 insertion in OsAPL1, a gene encoding a large subunit (LSU) of ADP-glucose pyrophosphorylase (AGPase). The insertion prevents production of a normal transcript from OsAPL1. Characterisation of the mutant (apl1) showed that the LSU encoded by OsAPL1 is required for AGPase activity in rice leaf blades. In mutant leaf blades, the AGPase small subunit protein is not detectable and the AGPase activity and starch content are reduced to <1 and <5% of that in wild type blades, respectively. The mutation also leads to a reduction in starch content in the leaf sheaths but does not significantly affect AGPase activity or starch synthesis in other parts of the plant. The sucrose, glucose and fructose contents of the leaves are not affected by the mutation. Despite the near absence of starch in the leaf blades, apl1 mutant rice plants grow and develop normally under controlled environmental conditions and show no reduction in productivity.

The gene encoding the cytosolic small subunit of ADP-glucose pyrophosphorylase in barley endosperm also encodes the major plastidial small subunit in the leaves.

The barley (Hordeum vulgare) gene Hv.AGP.S.1 produces two different transcripts encoding small subunits (SSUs) of ADP-glucose pyrophosphorylase (AGPase). It was shown previously that one of these transcripts, Hv.1a, encodes the cytosolic SSU in the endosperm. It is shown here that the other transcript produced from Hv.AGP.S.1, Hv.1b, encodes a plastidial SSU that is required for >90% of the AGPase activity in the leaves. Thus, both of the alternative transcripts encoded by Hv.AGP.S.1 are physiologically relevant: One is important for starch synthesis in the endosperm and the other for starch synthesis in the leaves. Although the Hv.1b transcript is abundant in embryos and present in endosperm, there is no evidence that a protein is produced from this transcript in these organs. This suggests that some, as yet unidentified, post-transcriptional control mechanism prevents the accumulation of the protein encoded by Hv.1b in embryos and endosperm but not in leaves. There is one other known gene in barley, Hv.AGP.S.2, encoding a SSU of AGPase. This gene has been shown to be responsible for the plastidial SSU in the endosperm. It is shown here that Hv.AGP.S.2 probably also makes some contribution to the SSU of AGPase in the leaves and may be responsible for most or all of the plastidial SSU in a range of non-photosynthetic plant organs including the embryo.

The lys5 mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters for starch synthesis in cereal endosperm.

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.

A low-starch barley mutant, risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase, reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit.

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Risø 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Risø 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.

Starch granule initiation is controlled by a heteromultimeric isoamylase in potato tubers.

Starch granule initiation is not understood, but recent evidence implicates a starch debranching enzyme, isoamylase, in the control of this process. Potato tubers contain isoamylase activity attributable to a heteromultimeric protein containing Stisa1 and Stisa2, the products of two of the three isoamylase genes of potato. To discover whether this enzyme is involved in starch granule initiation, activity was reduced by expression of antisense RNA for Stisa1 or Stisa2. Transgenic tubers accumulated a small amount of a soluble glucan, similar in structure to the phytoglycogen of cereal, Arabidopsis, and Chlamydomonas mutants lacking isoamylase. The major effect, however, was on the number of starch granules. Transgenic tubers accumulated large numbers of tiny granules not seen in normal tubers. These data indicate that the heteromultimeric isoamylase functions during starch synthesis to suppress the initiation of glucan molecules in the plastid stroma that would otherwise crystallize to nucleate new starch granules.

The altered pattern of amylose accumulation in the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5'-non-coding region.

Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5'-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxy ancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity.

Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity.

Two mutant lines of barley, Risø 17 and Notch-2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen-accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A-type or B-type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild-type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild-type endosperm plays a role in determining the number, and hence the form, of starch granules.