5.4 Xylem embolism vulnerability curves
Vulnerability to hydraulic failure was estimated with cavitation-induced
embolism curves. The relationship between the loss of stem hydraulic
function and xylem water potential (Ψx) (MPa) was
measured on stem tissues (n = 3–5) from 2–3 trees per species
at each stand, resulting in 6–12 curves per species per stand, or 165
total curves. Vulnerability curves were generated using the
air-injection technique (Sperry & Saliendra, 1994; Johnson et
al ., 2016). Branches were harvested from the upper third of the canopy,
and stem samples ~20 cm in length were collected from
the terminal bud of felled branches. Samples were stored at 5 °C
submerged in deionized water that was replenished daily and were
measured within two weeks of collection.
We used a pressure flow meter (XYL’EM embolism meter, Bronkhorst,
Montigny les Cormeilles, France) to measure stem hydraulic conductivity
(Kstem ) (kg m-1s-1 MPa-1), and a pressure sleeve
(Scholander Pressure Chamber model 1505D, PMS Instruments, Corvallis,
OR, USA) to facilitate air-injection. Samples were rehydrated by
flushing native embolism in submerged deionized water under vacuum for
24+ hours. Following rehydration, stem samples were exposed to positive
air pressure in 0.5 to 1.0 MPa increments until >85%
reduction of maximum Kstem (Kmax ) (kg m-1s-1 MPa-1) was reached or the
applied pressure approached instrument limitation. We then correctedKstem to 20 °C to account for changing viscosity
of water with temperature (K20 ) (kg
m-1 s-1 MPa-1).
The percent loss of conductivity (PLC) (%) at a given applied pressure
was calculated as:
\(PLC=100\ \times(1-\frac{K_{20}}{K_{\text{max\ }}}\ )\) (1)
The relationship between PLC and Ψx was then fitted to
the sigmoid function provided by Maherali et al ., (2006):
\(PLC=\ \frac{100}{\left[1+exp(\alpha\left(\Psi_{x}-\ b\right))\ \right]}\)(2)
where α and b are empirical coefficients determined using
nonlinear curve fitting (MATLAB, The Mathworks Inc., Natick, MA, USA; v.
R2018a). The fitted relationship was then used to calculate the
Ψx at which 12% PLC (P12) (MPa) and 50% PLC (denoted
as b = P50) (MPa) occurred. The pressures at 12% PLC (P12 =
2/α + b ) were determined as described by Domec & Gartner
(2001). The value P12, termed the air entry point, is an estimate of the
xylem tension at which the resistance to air entry of pit membranes
within the conducting xylem is overcome and cavitation and embolism
begin.
While the air-injection method is commonly used to assess vulnerability
to embolism (Sperry & Saliendra, 1994; Johnson et al ., 2016),
measurement artifacts from destructive sampling, such as the presence of
open vessels, may over-estimate in-situ vulnerability
(Martin-StPaul et al ., 2014). This bias may be particularly
important for species like Q. alba , which has long xylem vessels
that can reach up to several meters in length (Cochard & Tyree, 1990).
We therefore took multiple steps to minimize bias from open vessel
artifacts. First, we sampled young distal tissues from branch apices,
which have relatively short vessels (Cochard & Tyree, 1990). Second,
before curve generation, the presence of open vessels was checked using
a modified infiltration technique following Cochard et al .
(2010), and samples with suspected open vessels were subsequently not
used for embolism curve measurements.
As a third step, we considered the shape of the vulnerability curve. It
has been suggested that embolism curves that are conspicuously “r”
shaped are likely affected by open vessel artifacts, and that “s”
shaped curves more accurately represent in-situ vulnerability
(Torres-Ruiz et al ., 2014; Skelton et al., 2018). We
defined an “s” shape curve as one that lost less than 7.5% of itsKmax as Ψx declined from 0 to
-0.5 MPa and screened our dataset to use only these curves for the
subsequent analyses. We performed the analysis at alternative cutoff
thresholds of 3%, 5%, and 10% loss of Kmax ,
but there were no noticeable effect on the results. Comparing the “s”
shaped data to the “r” shaped data for Q. alba and L.
tulipifera showed that “r” shaped curves tended to have statistically
higher P12 and P50 than “s” shaped curves (Fig. 2). No A.
saccharum sample displayed “r” shape curves by our criteria. For
species with more vulnerable xylem tissues (e.g., Q. alba ),
including both “s” and “r” shaped curves did not markedly change the
estimated P50 (Fig. 2f). Nevertheless, “r” shaped curves for any
species were not included for subsequent analyses, resulting in a total
of 40, 56, 26 suitable “s” shaped curves for L. tulipifera ,Q. alba , and A. saccharum , respectively (or
~74% of the original 165 curves).
5.5 Xylem anatomy
To understand how changes in xylem vulnerability are linked to
variations in xylem anatomy, we measured vessel lumen area and vessel
density on transverse sections (~40 µm diameter)
extracted from stems of Q. alba and L. tulipifera from the
NC_W, IN, and NC_E chronosequence stands that were used for embolism
vulnerability measurements. Stem samples were softened by boiling in
deionized water (Schweingruber, 2007) and sectioned by hand using a
fresh razor blade. Before analysis under the microscope, the transverse
sections were dried in an oven at 150 °C. Slides from the NC_W and IN
chronosequences were imaged with a stereoscope and color camera at
150\(\times\) magnification (Leica M205F, Leica DFC310FX, Leica
Microsystems, Heerbrugge, Switzerland). Vessel lumen area and density
were then calculated using threshold balance manipulation and the
analyze particle function of ImageJ v1.6 software (National Institutes
of Health, USA) (Scholz et al ., 2013). Slides from the NC_E
chronosequence were photographed at 100\(\times\) and
200\(\times\) magnifications and analyzed using the Motic Images
Advanced 3.2 software (Motic Corporation, Zhejiang, China).