Microscopy (TEM)
Freshly collected leaves were analyzed by transmission electron
microscopy (TEM). Samples were prepared as described by Guzmán et al.
(2014a) with slight modifications. Leaf segments
(4-mm2) were cut from the mid-region of the blade.
Segments were then fixed in ice-cold 2.5% glutaraldehyde and 2%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, and kept at 4 ºC
prior to further preparation steps. Samples were then rinsed with cold
phosphate buffer (x4) and post-fixed in a 2% osmium tetroxide and 3%
potassium ferrocyanide aqueous solution for 1.5 h. Tissues were washed
with distilled water (x3), dehydrated in a graded series of 30, 50, 70,
80, 90, 95 and 100% acetone (x2, 15 min per concentration) and embedded
in acetone-Spurr’s resin (Spurr’s low viscosity embedding media,
Electron Microscopy Sciences, Hatfield, PA, USA) solutions (3:1, 2h;
1:1, 2h; 1:3; 3h (v:v)) and in pure resin (overnight) at room
temperature (20 ºC). Tissues were finally embedded in blocks which were
incubated for 3 days at 70 ºC. Ultrathin sections were cut, and
post-stained with uranyl acetate and lead citrate. Sections were
subsequently observed with a FEI Talos 120C TEM (Tokyo, Japan) operated
at 80 kV and equipped with a FEI Ceta CMOS camera. Stomatal pore depth
and cuticle thickness were measured in the micrographs using ImageJ.
Pore depth was measured in six open stomata per species. Cuticle
thickness (excluding the epicuticular wax layer) was measured in
ordinary epidermal cells, subsidiary cells, and guard cells, both in the
external and internal (substomatal) periclinal regions, as well as in
guard cell anticlinal regions (along the pore). Measurements were
performed in a minimum of six cells of each type with about 10
repetitions, and results were compared using ANOVA and Tukey tests with
R.