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.