Waxy and non-waxy barley cultivars exhibit differences in the targeting and catalytic activity of GBSS1a.

Kim H Hebelstrup,Andreas Blennow,Morten Munch Nielsen,Olga Andrzejczak,Massimiliano Carciofi,Shahnoor Sultana Shaik,Monica M Palcic

doi:10.1093/jxb/erw503

Kim H Hebelstrup, Andreas Blennow + Show 5 more

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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.

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Starch is the main polysaccharide in which carbon and energy are stored in higher plants and is the principal dietary source of energy for humans (Zeeman et al, 2010; Tetlow, 2011)
The highly branched amylopectin is the major component of starch and has an estimated molecular mass of Abbreviations: DIC, differential interference contrast; eGFP, enhanced green fluorescent protein; GBSS, granule-bound starch synthase; GP, Golden Promise; SNP, single nucleotide polymorphism; SS, starch synthase
We mapped Asp219, M490, and I491 to this structure (Fig. 1B). None of these amino acids is located in the presumed catalytic site or in the transit peptide. To test if they are important for the efficiency of GBSSIa starch synthesis, we cloned and successfully expressed six different versions of HvGBSSIa in Escherichia coli: (i) a wild-type consensus (HvGBSSIaWT) cloned from the nonwaxy barley line Vogelsanger; (ii) The CDC Alamo variant (HvGBSSIaCDC_Alamo) containing all three amino acid substitutions D219V/M490V/I491V; and four different combinations of these, namely (iii) HvGBSSIaM490V, (iv) HvGBSSIa I491V, (v) HvGBSSIa M490V/I491V, and (vi) HvGBSSIa D219V

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Starch is the main polysaccharide in which carbon and energy are stored in higher plants and is the principal dietary source of energy for humans (Zeeman et al, 2010; Tetlow, 2011). The ratio between amylose and amylopectin in plant starches varies depending on the botanical source, but typically 15–30% of the starch granule is composed of amylose (Tetlow, 2011). This ratio seems to have been optimized in nature for optimal starch granule robustness, digestibility and carbohydrate remobilization as demonstrated for barley seedling establishment (Shaik et al, 2014). The cloning and characterization of starch modifying enzymes is increasingly important for industrial and agro-biotechnological modification of starches and other carbohydrates (Hebelstrup et al, 2015)

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