Dx 500 system

The side chain distribution of modified starch was determined by HPAEC. The enzyme reaction mixtures were stopped by boiling for 5 min and centrifuged at 13,000 ×g and 4°C for 1 min. The supernatants were filtered through 0.45 μm membranes and then injected into the HPAEC system (DX-500 system, Dionex, USA) with a pulsed electrochemical detector (ED40, Dionex). The ingredients of samples were separated in a CarboPac PA1 column (250 × 4 mm, Dionex) at a flow rate of 1 ml/min with a 10-64% (v/v) gradient of 600 mM sodium acetate in 150 mM NaOH.

High-performance anion-exchange chromatography coupled to a pulsed amperometric detector (HPAEC-PAD) was used for monosaccharide composition analysis^{20 (link),38 (link),39 (link)}. Samples (1 mg/mL) were hydrolyzed with 2 M trifluoroacetic acid at 120 °C for 90 min. After the samples were cooled down to room temperature, t-butyl alcohol was added, and the mixture was evaporated under N₂ flow. The dried samples were solubilized in water, filtered (0.45 µm) and analyzed in a DX 500 system (Dionex, Sunnyvalle, CA, USA) equipped with a CarboPac PA10 column (250 × 4 mm)^{20 (link)}. Neutral sugars analysis was performed in water (1 mL/min; 40 min). Followed by a cleaning sequence with 300 mM NaOH for 10 min with another 10 min of re-equilibration. Uronic acids analysis was performed in 150 mM NaOH (1 mL/min; 30 min) with a 0–220 mM sodium acetate gradient, followed by a cleaning step with 500 mM sodium acetate for 10 min. A post-running adjustment of 10 min with 220 mM and 10 min with 150 mM NaOH followed. Neutral sugars (arabinose (Ara), fucose, galactose (Gal), glucose, mannose, rhamnose (Rha) and xylose), and uronic acids (galacturonic acid (GalA) and glucuronic acid), were used as standards^{40 (link)}.

Starch bound C6 phosphate content was analyzed on the starch purified from the same five biological replicates, each of WT, HP and AO at 10 DAP and MDG. The degree of starch C6 phosphorylation was determined as the content of Glucose 6-P (G6P) after starch hydrolysis, using high- performance anion-exchange chromatography (Dionex, DX 500 system equipped with an S-3500 auto sampler, GP40 pump, ED40 PAD system fitted with a CarboPac PA-1 column) as previously described [67 ]. Standard curves for phosphate content analysis were based on characterized potato starches (potato, cv Dianella, [68 ].