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).