2.2 Bleeding xylem sap sampling and characterization
Bleeding xylem sap (BXS) was collected at bud-break phenological phase (Baggiolini, 1979) from the 15 selected vines. The end of each spur was surface-treated with a diluted solution of sodium hypochlorite (3% active chlorine), then with 95% ethanol and finally rinsed twice with sterile distilled water. The sap exuded for the first 15 min was discarded. A sterile plastic bottle covered with aluminium foil was secured to the end of each bleeding spur to collect the liquid over the following four days. After harvesting, the sap collected from each vine was kept in an ice bag and transported to the laboratory, where it was filtered on 0.45 µm Millipore membranes (Millipore, Bedford, MA, USA).
Dynamic viscosity (ηx) of each BXS was calculated as ηx=[(ρx×tx)/(ρw×tw)]×ηwwhere ρx = sap density, ρw = water density, ηw = water dynamic viscosity (0.8937×10-3 Poiseuille), tx = flow time of sap, tw = water flow time. Measurements were carried at 25±0.1 °C using an Ostwald glass capillary viscometer (Cannon-Fenske Instruments, State College, PA, USA). For each BXS sample, 10 measurements were recorded.
About 2 ml of freshly collected BXS were lyophilized and the resulting powder was treated with 5% metaphosphoric acid (6 ml). After centrifugation (20,000×g , 15 min, 4°C), total ascorbate (T-ASC) and total glutathione (T-GSH) concentrations were measured as described by Zhang & Kirkham (1996).
The auxin and kinetin content of xylem sap was evaluated with the filter paper disk method (Zhao et al, 1992) using the excised cucumber (Cucumis sativus L.) cotyledon root formation (auxin) and the cucumber cotyledon expansion (kinetin) bioassays. Indole-3-acetic acid and 6-furfurylaminopurine were dissolved in 95% ethanol and tested in the range 0.3-50.0 μg ml-1; 95% ethanol was performed as a control.