title: Waxy and non-waxy barley cultivars exhibit differences in the targeting and catalytic activity of GBSS1a.
abstract: Amylose synthesis is strictly associated with activity of granule-bound starch synthase (GBSS) enzymes. Among several crops there are cultivars containing starch types with either little or no amylose known as near-waxy or waxy. This (near) amylose-free phenotype is associated with a single locus (waxy) which has been mapped to GBSS-type genes in different crops. Most waxy varieties are a result of either low or no expression of a GBSS gene. However, there are some waxy cultivars where the GBSS enzymes are expressed normally. For these types, single nucleotide polymorphisms have been hypothesized to represent amino-acid substitutions leading to loss of catalytic activity. We here confirm that the HvGBSSIa enzyme from one such waxy barley variety, CDC_Alamo, has a 90% reduction in catalytic activity. We also engineered plants with expression of transgenic C-terminal green fluorescent protein-tagged HvGBSSIa of both the non-waxy type and of the CDC_Alamo type to monitor their subcellular localization patterns in grain endosperm. HvGBSSIa from non-waxy cultivars was found to localize in discrete concentric spheres strictly within starch granules. In contrast, HvGBSSIa from waxy CDC_Alamo showed deficient starch targeting mostly into unknown subcellular bodies of 0.5-3 µm in size, indicating that the waxy phenotype of CDC_Alamo is associated with deficient targeting of HvGBSSIa into starch granules.
authors.affiliation: Department of Molecular Biology and Genetics, Section of Crop Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark.
authors.forename: Kim H
authors.initials: KH
authors.lastname: Hebelstrup
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 København V, Denmark.
authors.forename: Morten Munch
authors.initials: MM
authors.lastname: Nielsen
authors.affiliation: Department of Molecular Biology and Genetics, Section of Crop Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark.|Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 København V, Denmark.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Carciofi
authors.affiliation: Department of Molecular Biology and Genetics, Section of Crop Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark.
authors.forename: Olga
authors.initials: O
authors.lastname: Andrzejczak
authors.affiliation: Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Shahnoor Sultana
authors.initials: SS
authors.lastname: Shaik
authors.affiliation: Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Andreas
authors.initials: A
authors.lastname: Blennow
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 København V, Denmark.
authors.forename: Monica M
authors.initials: MM
authors.lastname: Palcic
mesh_terms: D019943:Amino Acid Substitution; D000688:Amylose / Q000378:metabolism*; D002384:Catalysis; D001467:Hordeum / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D017423:Sequence Analysis, RNA; D013214:Starch Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D000688:Amylose; C434607:granule-bound starch synthase I; D013214:Starch Synthase
keywords: Amylose; GBSS; starch biosynthesis; starch functionality; subcellular targeting; waxy.