Organ xylem vulnerability to cavitation.
The optical vulnerability method (OV) was used to quantify xylem
vulnerability (Brodribb et al 2015). This technique is minimally
invasive and allows simultaneous monitoring of xylem cavitation
throughout the plant. Data produced using the OV technique has been
shown to correspond very closely with vulnerability data collected using
hydraulic and x-ray methods in Tomato (Skelton, Brodribb, & Choat,
2017) and many other species (Gauthey et al., 2020). A combination of
custom-made “cavicams” (opensourceov.org) and a microscope were used
simultaneously on each plant to monitor cavitation during dehydration.
Leaves : a cavicam was secured to a single fully expanded leaf per
plant to view a region 5x5mm that included secondary and tertiary veins.
Tissue was illuminated from underneath by LEDs (white-light emitting
diodes). Images were acquired every 2 min for 8 days. Cameras were
refocussed as necessary.
Petioles : a region of one petiole on an adjacent leaf per plant
was selected close to the main stem. Using a sharp razor blade, a window
of around 15 mm x 8 mm was carefully cut parallel to the surface to
remove the epidermis and cuticle on one side of the petiole, exposing
the xylem. The area was coated in hydrogel (Tensive Gel; Parker
Laboratories Inc., Fairfield, NJ, USA) to improve light transmission and
reduce evaporation from the surface. The section of petiole was then
secured in a cavicam which acquired images as described above using
reflected light.
Peduncles: a region of one peduncle supporting several open
flowers, a variable number of buds and no fruit was selected per plant.
A region of around 15 mm was carefully scraped with a fingernail to
remove the epidermis and cuticle, exposing the xylem. No razor blade was
used as the epidermal tissue was softer than the petiole equivalent and
razor blades easily cut too deep into the vascular tissue. The area was
coated in hydrogel then secured in a cavicam which acquired images using
reflected light.
Petals: Images of one petal per plant from an intact apical
inflorescence were captured with a Leica DFC450 digital camera, mounted
on a Leica M205A stereomicroscope (Leica Microsystems, Wetzlar,
Germany). The petals of a newly opened flower were taped to a glass
plate to prevent movement/shrinkage during image capture. Light was
transmitted through the petal illuminating the midrib xylem bundle and
images captured as described above.