Modification of stomatal aperture
Randomly selected leaves were treated to modify stomatal aperture with
fusicoccin (FC; ‘open’ stomata), abscisic acid (ABA; ‘closed’ stomata),
and water (control) (Jones and Mansfield 1970; Turner and Graniti 1969).
We followed the procedure by Eichert et al. (1998) with some
modifications. Leaves were collected from branches previously
equilibrated at the target Ψ (i.e. at approximately -1.7 and -2.0 MPa
for P. dulcis and P. communis , respectively) and weighed.
Leaf petioles were immersed in tubes filled with aqueous solutions of 10
µM FC (Biomol, Hamburg, Germany), 10 µM ABA (Sigma-Aldrich, Munich,
Germany), or with deionized water for five to six hours. During the
first two hours, leaves were kept at room conditions in a relatively dry
atmosphere, to allow transpiration and enough uptake of the solutions.
Leaves were then fully rehydrated in a dark and humid environment to
restrict transpiration by placing them in individual chambers containing
damp paper towels. At the end of the treatment period, Ψ was confirmed
to be higher than -0.05 MPa. Treatment effectiveness was determined by
stomatal conductance measurements in dehydrating leaves and stomatal
pore aperture in surface micrographs. Treated leaves were then used for
surface rehydration kinetics experiments.