Characterization of wheat lacking B-type starch granules

ABSTRACTThe physicochemical and agronomical properties of a new form of bread wheat, lacking B-type starch granules (BlessT) was assessed. Three BlessT mutant lines, made by combining homoeologous deletions of BGC1, a gene responsible for the control of B-granule content were compared with two sibling lines with normal starch phenotype and the parent line, cv. Paragon. Quantification of starch granule size and number in developing grain confirmed the lack of small, B-type starch granules throughout development in BlessT. Most starch, flour, grain and loaf characteristics did not vary between BlessT and the wild type sibling controls. However, BlessT starches had higher water absorption, reduced grain hardness and higher protein content, and dough made from BlessT flour required more water and had increased elasticity. Despite the lack of B-granules, BlessT lines do not display a significant decrease in total starch content suggesting that it should be possible to produce commercial wheat varieties that lack B-type starch granules without compromising yield. These findings support the potential utility of this novel type of wheat as a specialist crop in applications ranging from bread making and alcohol production to improved industrial starch products.

A carbohydrate-binding protein, B-GRANULE CONTENT 1, influences starch granule size distribution in a dose-dependent manner in polyploid wheat

In Triticeae endosperm (e.g. wheat and barley), starch granules have a bimodal size distribution (with A- and B-type granules) whereas in other grasses the endosperm contains starch granules with a unimodal size distribution. Here, we identify the gene, BGC1 (B-GRANULE CONTENT 1), responsible for B-type starch granule content in Aegilops and wheat. Orthologues of this gene are known to influence starch synthesis in diploids such as rice, Arabidopsis, and barley. However, using polyploid Triticeae species, we uncovered a more complex biological role for BGC1 in starch granule initiation: BGC1 represses the initiation of A-granules in early grain development but promotes the initiation of B-granules in mid grain development. We provide evidence that the influence of BGC1 on starch synthesis is dose dependent and show that three very different starch phenotypes are conditioned by the gene dose of BGC1 in polyploid wheat: normal bimodal starch granule morphology; A-granules with few or no B-granules; or polymorphous starch with few normal A- or B-granules. We conclude from this work that BGC1 participates in controlling B-type starch granule initiation in Triticeae endosperm and that its precise effect on granule size and number varies with gene dose and stage of development.

A carbohydrate-binding protein, FLOURY ENDOSPERM 6 influences the initiation of A- and B-type starch granules in wheat

Previously, we identified a quantitative trait locus on the group 4 chromosomes of Aegilops and bread wheat that controls B-type starch-granule content. Here, we identify a candidate gene by fine-mapping in Aegilops and confirm its function using wheat TILLING mutants. This gene is orthologous to the FLOURY ENDOSPERM 6 (FLO6) gene of rice and barley and the PTST2 gene of Arabidopsis. In Triticeae endosperm, reduction in the gene dose of functional FLO6 alleles results in reduction, or loss, of B-granules. This is due to repression of granule initiation in late-grain development, but has no deleterious impact on the synthesis of A-granules. The complete absence of functional FLO6, however, results in reduced numbers of normal A-type and B-type granules and the production of highly-abnormal granules that vary in size and shape. This polymorphous starch seen in a wheat flo6 triple mutant is similar to that observed in the barley mutant Franubet. Analysis of Franubet (fractured Nubet) starch suggests that the mutant A-granules are not fractured but compound, due to stimulation of granule initiation in plastids during early-grain development. Thus, in different situations in Triticeae, FLO6 either stimulates or represses granule initiation.

Genetics of starch granule size distribution in tetraploid and hexaploid wheats

Wheat endosperm starch is deposited in large, A-type granules and smaller B-type and C-type granules. The quantitative genetics of starch granule size distribution was investigated in 2 ways. Complete diallel crosses, with 5 parents each, were prepared in tetraploid wheat, Triticum turgidum, and hexaploid wheat, Triticum aestivum. Parent and F1 plants were grown in controlled environment growth chambers with 18�C days and 13�C nights to provide parent and F2 grains from uniform conditions. In the same conditions, the basic generations of parent, F1, F2, and backcross of 6 other individual crosses were grown and F1 and backcross grains were freshly generated on these plants. Starch granule size distribution was determined in parent and F2 grains in the diallels and all possible generations in the other crosses. Granules of <10 μm diameter were considered 'B granules' (thus including C granules), and B-granule content was expressed as a percentage of total starch volume. The modal A-granule diameter was also determined.B-granule content varied widely in both species, whereas modal A-granule diameter was much more variable in tetraploids than in hexaploids. Additive gene action was more important than dominance in determining B-granule content in both species and A-granule diameter in tetraploids, whereas dominance was more important for A-granule diameter in hexaploids. Dominance acted to increase or to decrease B-granule content, depending on the cross. According to variance–covariance analysis, the line with the most dominant alleles in the hexaploids was the one lowest in B granules, but in the tetraploids it was the one highest in B granules. Digenic interactions affected B-granule content and A-granule diameter in all of the analyses of the basic generations, and nucleocytoplasmic interactions affected these traits in most of the crosses. Diallel analyses of the F2 generations, in contrast, showed a limited importance of epistasis. Cytoplasmic effects made small but significant contributions to the variation in B-granule content in some of the crosses. Variation in B-granule content and A-granule diameter appeared to be affected by different gene actions and were therefore likely to be susceptible to independent manipulations in a breeding program.

Analytical subcellular fractionation of alveolar macrophages from normal and BCG-vaccinated rabbits with particular reference to heterogeneity of hydrolase-containing granules

Macrophages were obtained by pulmonary lavage from normal rabbits or rabbits that had developed pulmonary granulomas after receiving intravenous BCG vaccine 2-3 weeks earlier. The cells were disrupted in iso-osmotic sucrose and a low-speed supernatant was fractionated by isopycnic centrifugation on a linear sucrose density gradient. Three populations of hydrolase-containing granules (putative lysosomes) were found in both normal and BCG-induced macrophages. They were distinguished by their different distributions in the gradient and different sensitivities to disruption by digitonin and were termed:type A, containing lysozyme; type B, containing N-acetyl-beta-glucosaminidase, beta-glactosidase, beta-glucuronidase and possibly some lysozyme; type C, containing cathepsin D. Acid phosphatase appeared to be about equally distributed between type B and C granules. Type A and B granules from BCG-induced macrophages showed markedly greater equilibrium density than did those from normal macrophages. Beta-glucuronidase and acid phosphatase had greater specific activity in the induced cells.