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 .