D glucose

GAS cell cultures were grown in either BHP-Glucose or BHP-Galactose with or without the addition of 50 μM zinc (a sub-inhibitory zinc concentration). Samples (2 ml) were collected at start-, middle- and end-exponential and stationary phase for metabolite analysis. These samples were pelleted by centrifugation (5000 g, 5 min). The resulting supernatant was filter sterilised and used to assay metabolite concentrations using the D-glucose, L-lactic acid, acetic acid and ethanol determination kits according following manufacturer’s instructions (Megazyme).

The β-glucan concentration of the supernatant was analyzed by a competitive enzyme-linked immuno-sorbent assay (ELISA) based on Streptococcus (S.) pneumoniae serotype 37 antibodies for the quantification of the bacterial β-glucan [86 (link)]. The assay was performed as previously described [36 (link)]. The D-glucose concentrations of the supernatant samples were determined using a glucose oxidase/peroxidase assay (GOPOD, Megazyme Ltd., Bray, Ireland) according to the manufacturer’s protocol. The assay was adapted to a microtiter plate volume with a 50 μL sample volume and 150 μL of the GOPOD reagent. A standard curve was used for D-glucose (Megazyme Ltd., Bray, Ireland) calculations. All experiments were carried out using four biological replicates.

For frozen samples, all filled grains from one side of the rachis were pooled while kept frozen. Grains were lyophilized at −15�C for 5 d followed by 24 h at room temperature. All grains from matured, dried samples were lyophilized at room temperature (24 h). Dried grains were crushed, cooled in liquid nitrogen and ground to flour in a SPEX SamplePrep2010 Geno/Grinder� (Metuchen, New Jersey, USA).
Determination of total starch was done using the AA/AMG kit from Megazyme (Bray, Ireland). D-Glucose (1 mg/ml) and Regular Maize Starch from Megazyme (Bray, Ireland) served as controls. Amylose content and amylose: amylopectin ratio was determined using the K-AMYL kit (Megazyme, Ireland). β-glucans were quantified using the K-BGLU kit (Megazyme, Ireland). Five milligram aliquots of flour were used, otherwise manufacturer’s recommendations were followed. Grubbs test for outliers, ANOVA and ANCOVA were made using XLSTAT add-on for Microsoft Excel (Addinsoft, New York, NY, USA). ANOVA was applied to mature samples and ANCOVA to distinct stages during development (HT: 240–393�Cd; recovery: 384–601�Cd; desiccation: 464–681�Cd). For samples taken during development n = 3, for samples taken at maturity n = 5 plants were used for measurements.