Li 1600

Leaf transpiration (E) and stomatal conductance (gs) of fifth fully expanded leaf from the base of stressed and unstressed plants were determined using a portable steady-state porometer LI-1600 (Li-Cor Inc., Lincoln, NE, USA) under the following conditions: temperature 22–25 °C; 16 h/8 h (light/dark) photoperiod; relative humidity 80%; and 250 mol m⁻² s⁻¹ intensity light. The porometer consists of a cuvette with a broadleaf aperture (2 cm²), which permits the precise measurements of water loss by transpiration (µg cm⁻² s⁻¹) and stomatal resistance (s cm⁻¹). Measurements were carried out by attaching the cuvette to the leaf surfaces and simultaneously registered humidity (%) conditions and temperature (°C). Three biological replicates were conducted at each condition.

Leaf water potential (Ψ), transpiration rate (Ε), stomatal conductance (g_s) and leaf temperature (T_l) were measured on the newest fully developed mature leaves of six different individuals from sun-exposed terminal branches. Using the pressure-bomb technique (PMS, Albany, OR, USA), leaf water potential was measured twice during the day, i.e., at predawn (Ψ_p) and midday (Ψ_m). Transpiration rate, g_s and T_l were measured using steady state porometer (Li1600, LI-COR Lincoln, NE, USA). Seasonal measurements of Ψ_p were obtained before sunrise, while the measurements of Ψ_m, E, and g_s were obtained on clear sunny days at around solar noon (12:00–14:30 h), approximately 10–15 days intervals. The presented values, for each of the parameters, are means of six replications per studied shrub.
Leaf hydraulic conductance (K_Leaf) was calculating according to Ohm’s law following the formula: $${\mathrm{K}}_{\mathrm{Leaf}}=\frac{\mathrm{E}}{\left({\mathsf{\Psi }}_{\mathrm{soil}}-{\mathsf{\Psi }}_{\mathrm{Leaf}}\right)}=\frac{\mathrm{E}}{\left({\mathsf{\Psi }}_{\mathrm{p}}-{\mathsf{\Psi }}_{\mathrm{m}}\right)}$$
It has been assumed that Ψ_soil is in equilibrium with Ψ_p and the lowest diurnal Ψ_leaf is equal to Ψ_m [66 ,67 (link),99 (link)]. However, sometimes the first assumption may lead to overestimation of K_plant [100 (link)]. Additionally, values of K_Leaf reflect the capacity of evergreen and deciduous plants, grown under ambient conditions, for water exploitation during a period of soil drying.

Stomatal conductance was measured on 19 April (1 d after the first spray treatment application) in 2012 and on 21 and 28 March in 2013 (3 and 10 days after the first treatment) in 2 fully expanded leaves located on opposite sides of each plant (fifth to seventh basipetally located leaves). Measurements were made between 1:00 and 3:00 p.m. with a steady-state porometer (LI-1600, LI-COR Biotechnology, Lincoln, NE).