Nitric Acid Digestion and Metal Analysis

Nitric acid digestion and metal concentration analysis were carried out, as described in Kobayashi et al. (2013 (link)), with slight modifications as below. For each replicate, 6–10 brown seeds were dehusked and dried for 2 d at 70 °C, weighed, and wet-ashed with 1.0–1.5 mL of 13.4 M nitric acid and 1.0–1.5 mL of 8.8 M hydrogen peroxide for 20 min at 220 °C using a MarsXpress oven (CEM, Matthews, NC, USA). Straw segments were cut into 3–4 cm pieces and dried for 2–3 d at 70 °C, and portions weighing 100–200 mg were wet-ashed with 2.0 mL of 13.4 M nitric acid and 2.0 mL of 8.8 M hydrogen peroxide for 20 min at 230 °C. Inductively coupled plasma optical emission spectrometry (5800 ICP-OES, Agilent, Japan) was used to measure Fe, Zn, manganese (Mn) and copper (Cu) concentrations.