2.4 Scanning electron microscopy
To evaluate the size of pores that make up the sieve plates, samples of
all leaf veins and stem axial areas (see above description) were cut,
frozen in liquid nitrogen, and transferred to super-chilled ethanol,
prior to being sectioned in different angles with respect to the main
axis with the goal of obtaining sections aligned with the highly angled
sieve plates. These sections were incubated within a mixture of 0.1%
w/v proteinase K dissolved in 50 mM Tris-HCl buffer, 1.5 mM
Ca2+ acetate and 8% Triton X-100, pH 8.0 (Mullendore
et al., 2010), in a water bath at 60°C for 2 weeks. After rinsing with
ethanol once, and with distilled water thrice, the sections were
incubated with a 1% w/v aqueous solution of α-amylase for 2 days at
60°C, then rinsed thrice in water and finally freeze-dried for 24 h with
a Freeze Dyer (Labconco Freeze Dry System). Samples were then mounted on
SEM studs and sputter coated with gold-palladium using a Denton Vacuum
Desk II Sputter Coater for 180 s at 20 V and 6.67 Pa. Samples were
imaged with a JEOL-6010LV scanning electron microscopy (SEM) (JEOL,
Peabody, MA, USA), using high vacuum and an accelerating voltage of
10–15 kV. The size of sieve pores, of sieve areas, and pore density
were measured. Although sieve pore radius was measured for each vein
order, due to the small size of the sieve plates within higher order
veins, the preservation of intact sieve plates was poor. As a result, we
were only able to determine sieve plate size and pore density for the
midrib (n=5). The total number of pores measured was large (n=105 for
petiole; n=262 for primary vein; n= 84 for minor vein orders). In the
thinnest stems, the stiffness of the external fiber layer, along with
the fragility of the sieve tubes, limited preservation –and thus
visualization- of the pores, relative to the base of the stem, where
preservation was much better and resulted in n=600 pores measured.
2.5 Phloem sap velocity in Austrobaileya
scandens
Phloem transport velocity was measured by tracking the movement of the
fluorescent dye esculin (reviewed by Knoblauch et al., 2015) in the
secondary veins of a five-year-old vine at 11:00 h for several days. A
young plant (five years approximately) with fully formed leaves, was
watered to saturation, and then one of the long branches immobilized on
the microscope stage, with the abaxial surface exposed upward. A 50 μL
droplet of 5mM aqueous esculin mixture, combined with 0.1% of the
SilEnergy surfactant (RedRiver Specialties, Shreveport, LA, USA), was
applied with a micropipette to the junction of the second with minor
order veins (adapted from Jensen et al., 2011, Savage et al., 2013). To
allow better permeabilization of the dye, the tip of the pipette was
used to slightly abrade the thick cuticle, and a small window was
opened, and was covered with the liquid during the experiment to prevent
desiccation. To track the movement of the dye, we used a portable Stereo
Microscope Fluorescence Adapter with 510–540 nm excitation wavelength,
and a long pass 405 UV nm filter band (NIGHTSEA, Lexington, MA, USA).
Time-lapse images were obtained every 10 s for 30min with a Zeiss v12
dissecting microscope using the 0.63× PlanApo objective and an AxioCam
512 Color camera connected to the AxioVision software. Despite multiple
attempts, the movement of the dye could only be observed in two leaves.
In both cases, fluorescence increased gradually downstream the vein a
few minutes after the dye was applied. Velocity was measured by
thresholding the images (Hue 119-188; Saturation: 97-255; Brightness:
0-255nm), cleaning the dark outliers within 2 pixels’ radius, and then
calculating the time elapsed by pixel accumulation two centimeters away
from the area where the dye was applied (n=2).