Ultrastructure of the stomata and the cuticle – TEM microscopy
Cross-section analyses revealed the fine structure of the stomata and the cuticle of the two species (Figure 4). In both species, stomata have prominent outer cuticular ledges, as well as inner cuticular ledges which seem to be more developed in P. communis (Figure 4a, b). With varying but mostly reduced thickness, the abaxial cuticle extends from the ordinary epidermal cells along the stomatal pore, lining the substomatal chamber. This substomatal cuticle is even present in some parenchymatic cells, especially in P. communis (Figure 4c, d). The adaxial cuticle of both species shows an amorphous outer region that appears as a more electron-lucent band than the cuticle underneath, and an inner reticulate region at the interface with the more electron-dense, pectin-rich cell wall region (Figure 4e, f), as previously described for P. communis when prepared following the conventional Spurr’s resin embedding protocol (Guzmán et al. 2014a). The epicuticular wax layer persisting after sample preparation procedures is more conspicuous in P. communis than P. dulcis . For both species, the appearance of the abaxial cuticle is similar, but structural details could not be observed when the cuticle was very thin. Cuticle thickness varied among cells and cell regions in both species(P <0.0001, Table 5 Supporting Information). The outer periclinal cuticle of ordinary epidermal cells was of similar thickness in both leaf sides in P. dulcis (~0.88 µm), and thinner in the adaxial than in the abaxial side in P. communis (~1.65 vs ~1.98 µm). The outer periclinal cuticle of guard cells was thinner than that of ordinary and subsidiary cells, with average values of 0.33 µm in P. dulcis and 0.55 µm in P. communis . The cuticle was thinnest from the central pore region inwards in P. dulcis (~0.05 µm), and at the anticlinal region of guard cells (i.e. along the stomatal pore) and inner periclinal region of ordinary epidermal cells in P. communis (~0.22 µm), i.e. there is an increase in thickness of the internal cuticle at the proximity of the stomatal pore in P. communis . Stomatal pore depth as measured in TEM micrographs (Figure 4a, b) was 6.73 ± 0.02 µm in P. dulcis and 10.02 ± 0.02 µm in P. communis .