Iodine staining and starch content determination in leaves

Flag leaves were harvested at the end of the day and the end of the night, decolorized in 80% (v/v) ethanol, and stained in Lugol solution (Sigma) for 30 min. After staining, excess Lugol was removed by soaking the samples in ddH₂O for optimal visualization of the amylose–iodine complex in starch. To quantify starch levels, leaves were ground in liquid nitrogen and dried in an oven at 80°C to constant weight. The total starch and amylose contents in the leaf samples were determined with a starch determination kit, Megazyme K-TSTA (Wicklow, Ireland), and an amylose determination kit, Megazyme K-AMYL.