Microscopy (ESEM)
Leaves treated with FC and ABA and control leaves were analyzed with an
environmental scanning electron microscope (ESEM, FEI Quanta 200, North
America NanoPort, Portland, OR, USA; acceleration potential 10 kV;
working distance 10 mm; Gaseous Secondary Electron Detector 6.2 Torr).
This technique enables the observation of samples in their native state,
i.e. without involving any previous sample preparation, as well as the
study of condensation processes at high resolution (Burkhardt and
Hunsche 2013; Cheng et al. 2005). For each species, three randomly
selected leaves per treatment were used. Sections of approximately 20
mm2 were obtained from the mid-region of the leaves,
avoiding venation as much as possible, and placed on the microscope
stage. A Peltier chip attached to the stage allowed temperature and thus
RH control. Temperature was kept between 7 and 5 ºC to achieve RH values
between 50 and 100% during examination. Stomatal pore aperture (width)
and density were measured in the micrographs using ImageJ.
Pore
aperture was measured in stomata that allowed clear pore limit
identification, with 54 (FC), 65 (ABA) and 45 (control) stomata being
analyzed in P. dulcis , and 44 (FC), 40 (ABA) and 36 (control) inP. communis (with a minimum of 15 (P. dulcis ) and 12
(P. communis ) stomata per leaf). Stomatal density was determined
in six leaves per species. Pore width was analyzed using a linear
mixed-effect model with treatment as fixed factor and leaf and
micrograph as random factors (micrograph nested within the leaf), and
Tukey tests in R.