Pneumatic measurements
An automatic Pneumatron apparatus was applied to measure gas diffusion
kinetics of desiccating leaves (Pereira et al. , 2020a, Jansenet al. , In press). The principle of this apparatus was similar to
the manual approach of Pereira et al. (2016) and Zhang et
al. (2018), but with a much higher accuracy and temporal resolution.
When samples for pneumatic measurements were prepared, the cut-open
conduits embolised intentionally, which happened quickly by shaving the
sample ends with a fresh razor blade in air.
Pneumatic measurements were taken every 15 min. For this a 40 kPa of
absolute pressure was created with a vacuum pump, which extracted gas
from a petiole end via a solenoid valve. The amount of gas extracted in
a discharge tube with known volume could be measured with a pressure
sensor. The vacuum pump reached 40 kPa (i.e., the initial pressure
Pi) within less than a second. Pressure data were
recorded in a SD card at a time interval of 500ms. The final pressure
(Pf) was taken after 30s. According to the ideal gas
law, the moles of air extracted from vessels (Δn) could be calculated
following the equation below:
Δn = nf-ni =\(\frac{(Pf-Pi)\times V}{\text{RT}}\) (Eqn 1)
where ni and nf represented the moles of
air molecules at the initial and final pressure, V was the fixed volume
of the discharge tube (1.1 mL), R was the gas constant (8.134 kPa L
mol-1 K-1), and T was the room
temperature in the lab (around 25°C). Since a small V is needed to
increase the measurement precision when a tiny amount of air is sucked
from plant tissue, which is the case for detached leaves (Pereiraet al. , 2020a, Jansen et al. , In press), V was estimated
as the maximum gas volume that could be extracted when leaves were fully
dehydrated (ADmax, see below) divided by 510.2 (Pereiraet al. , 2020a). The volume of air discharged (AD, µL) from
vessels could then be calculated based on the ideal gas law, with
Patm being the atmospheric pressure: AD =
106×Δn R T/ Patm (Eqn 2),
Finally, the Percentage of Air Discharged (PAD, %) was calculated:
PAD =
100×(AD-ADmin)/(ADmax-ADmin)
(Eqn 3)
where ADmin was the minimum volume of air discharged
when the leaf was well hydrated, and ADmax was the
maximum volume of air discharged when the leaf was strongly dehydrated.
Vulnerability curves were generated by plotting PAD or PEP against the
corresponding leaf water potential (Ψ), with a fitting by the following
equation (Pammenter and Vander Willigen, 1998):
PAD or PEP =100/ (1+exp (S /25) (Ψ-P50)) (Eqn 4)
S represented the slope of the fitted curve, and
P50 represented the water potential at 50% of air
discharged, or 50% of the total embolised pixels of the leaf area
scanned. Values of P12 (water potential at 12% of air
discharged or embolised pixels) and P88 (water potential
at 88% of air discharged or embolised pixels) were calculated following
the equations by Domec and Gartner (2001):
P12 = 2/(S /25) +P50 (Eqn 5)
P88 = -2/(S /25) +P50 (Eqn 6)