Leaf Cell Ultrastructure Analysis

The ultrastructure of the leaf cells was measured according to the methods described by Liu et al. [100 (link)], with minor modification. Leaf samples were cut into small pieces (about 1 cm × 1 cm) and fixed in 2.5% glutaraldehyde solution at 4 °C. The samples were washed five times with 0.1 mol L⁻¹ phosphate buffer and fixed overnight in 1% osmic acid at 4 °C. The materials were dehydrated using a series of acetone solutions (50%, 70% and 90%, v/v), for 15 min at each concentration, and then dehydrated with absolute acetone three times, for 15–20 min each time. The materials were then infiltrated with acetone and resin (EPon812) at proportions of 2:1 (v/v) for 0.5 h at room temperature and at proportions of 1:2 (v/v) for 1.5 h at 37 °C, followed by infiltration with 100% resin for 3 h at 37 °C. After separate polymerization at 37, 45, and 60 °C for 24 h in turn, thin sections were created using an ultramicrotome (Reicher-Jung ULTRACUT, Austria) and the sections were double-stained with uranyl acetate–lead citrate. We examined the samples using a model JEM1200 transmission electron microscope (JEOL, Tokyo, Japan).