Pressure–volume traits
Pressure–volume (P-V) measurements were carried out during spring (from
the end of May to the beginning of June) and late summer (at the end of
August) to assess leaf water relations. P-V measurements were generated
for a minimum of four twigs using the bench-drying method in accordance
with Tyree and Hammel (Tyree & Hammel, 1972). Within each site, five
trees were selected, and one terminal woody branch of each tree was
excised (leaf attached) and immediately recut under deionized water at
6:00 AM to remove any air emboli. The newly recut ends of the branches
were rehydrated in deionized water for more than 4 h with entire branch
and assimilating organs covered in black plastic bags to facilitate full
rehydration (Arndt, Irawan, & Sanders, 2015). One leaf of each woody
branch was cut, and its turgid weight measured using a digital balance
(0.0001 g resolution). Then, the corresponding Ψ was measured
using the model 3005 pressure chamber. If this measurement was less than
-0.5 MPa, then the twig was discarded, because it had not fully
rehydrated. The leaves were left on a bench at room temperature
(~ 22°C) to slowly dehydrate. This process was repeated
several times until the correlation between
1/Ψand fresh weight formed a straight line consisting of at least four
measurements with R 2 > 0.98. The
twig was then oven-dried at 70°C for 1 week (until a constant weight was
reached) and then weighed to determine the relative water content (RWC)
as follows:
RWC (%) = 100 × (FW-DW)/(TW-DW), (2)
where FW represents the fresh weight of the twig; DW represents the dry
weight; and TW represents the turgid weight. The π at full turgor
(π 100) and turgor loss point
(ΨTLP) were derived from the curve of 1/Ψ against
RWC using a P-V curve-fitting method available online
(http://landflux.org) based on Schulte and Hinckley (Schulte &
Hinckley, 1985).