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.