Table 1.
List of TILLING mutant lines carrying either missense or nonsense mutations in five genes related to starch metabolism in barley grains that have been isolated as described in Bovina et al. [22] and phenotypically characterized in this work.
Figure 1.
Seed morphology and transverse section of TILLING mutant line 1517-SSIIa (Starch Synthase IIa) (right) showing a shrunken phenotype, compared with cv. Morex wild type (left).
From top to bottom: adaxial and abaxial seed views, and seed cross section. White bars = 2 mm.
Table 2.
Content of starch and amylose in seeds of TILLING mutant lines.
Figure 2.
Electrophoretic separation (SDS–PAGE) of starch granule proteins extract from barley wild type cv. Morex (1) and barley mutants 1284-SSI (2) and 1517-SSIIa (3).
The bands corresponding to starch synthase II and starch branching enzyme II (SSII+SBEII), starch synthase I (SSI) and granule-bound starch synthase (GBSSI) are indicated. In lane 3, the high molecular weight band marked with an asterisk is probably due to impurities present in the starch preparation obtained from the shrunken seeds of line 1517-SSIIa. Molecular weight standard is schematically reported on the right.
Figure 3.
Scanning Electron Microscopy (SEM) analysis of starch granules from barley cv. Morex wild-type (A) and mutants 2253-BMY1 (B), 2682-BMY1 (C), 1090-GBSSI (D), 905-LDA1 (E), 1132-SSI (F), 1284-SSI (G), 5850-SSI (H), 1039-SSIIa (I), 1517-SSIIa (L).
Scale bar: 10 µm.
Figure 4.
Percentage of B-type granules (diameter <8 µm) in grain starch of barley wild-type cv. Morex and mutant lines.
Granules size distribution was determined on 10 couples of SEM images randomly collected for each genotype. Data shown are means ±SD (n = 10). Statistically significant differences between mutants and wild type mean values were estimated by Student's t-test (P<0.01) and are highlighted by a double asterisk (**).
Table 3.
Length of major axis in A-type and B-type starch granules.
Figure 5.
X-ray diffraction patterns of native starch extracts from barley wild type cv. Morex (black) and mutants 1090-GBSSI (grey) and 1517-SSIIa (light grey).
The characteristic peaks of the A-type and V-type polymorphs are indicated.
Table 4.
Percentage crystallinity and relative intensity of diffraction peaks at 15.1° and 19.7°.
Figure 6.
Localization of point mutations in the 3D structures of barley GBSSI, SSI and LDA1.
A) Barley GBSSI was modelled by Swissmodel using the catalytic domain of wild-type rice GBSSI complexed with ADP as a template (pdb 3VUF). The two mature proteins are 84% identical in sequence. The main chain of glycine-493 is represented by blue spheres. In mutant 1090-GBSSI, glycine-493 is substituted by a glutamate (G493E). Co-crystallized ADP of the rice GBSSI structure (3VUF) is superimposed to highlight the adenine nucleotide binding site. B) Crystal structure of barley SSI, co-crystallized with a molecule of maltopentaose (red spheres) (pdb 4HLN). Main chain atoms of mutated residues are represented by coloured spheres: blue (G576D in mutant 5850-SSI), green (T522I in mutant 1132-SSI) and yellow (G509E in mutant 1284-SSI). C) Crystal structure of barley LDA1 (pdb 2X4B). The carbohydrate binding module CBM48 is coloured yellow. Residue no. 270 (blue spheres corresponding to main chain atoms) is part of the CBM48 domain. Mutant 905-LDA1 carries a V270I mutation. A molecule of betacyclodextrine (red spheres) co-crystallized with the protein highlights the putative active site.