Xylem vulnerability to cavitation can be accurately characterised in species with long vessels using a centrifuge method

Drought-induced cavitation resistance varies considerably between tree species and forest ecosystems (Maherali et al., 2004; Delzon et al., 2010) and is closely linked to survival under severe drought in both conifers (Brodribb & Cochard, 2009; Brodribb et al., 2010) and angiosperms (Kursar et al., 2009; Anderegg et al., 2012; Barigah et al., 2013; Urli et al., 2013). Choat et al. (2012) recently reported that most trees operate very close to their threshold of cavitation, leaving them potentially vulnerable to drought-induced mortality in a warmer/drier world (Engelbrecht, 2012). Indeed, species growing in dry environments are more resistant to droughtinduced cavitation (more negative water potential at 50% cavitation, P50) but experience a more negative minimumwater potential (Pmin) than those growing in wet environments. The so-called hydraulic safety margin, the difference between the level of water stress experienced by a species in the field (Pmin) and the level of water stress leading to hydraulic failure, is, therefore, remarkably narrow, whatever the forest species and biome considered (Choat et al., 2012). This pattern provides clues to the global droughtinduced mortality currently observed, even in very wet environments, such as tropical forests (Allen et al., 2010). Klein et al. (2014) play down the functional significance of the hydraulic safety margin in the vulnerability of forests to drought, pointing out the important role played by additional mechanisms, such as the ability of trees to repair embolism. While it is obvious that drought-induced forest dieback is a complex process involving a number of biotic and abiotic factors, we would like to draw the attention of scientists to the state of evidence for embolism repair, thereby guiding research on tree drought resistance into the most relevant and fruitful directions

A vulnerability curve (VC) describes the extent of xylem cavitation resistance. Centrifuges have been used to generate VCs for decades via static- and flow-centrifuge methods. Recently, the validity of the centrifuge techniques has been questioned. Researchers have hypothesized that the centrifuge techniques might yield unreliable VCs due to the open-vessel artifact. However, other researchers reject this hypothesis. The focus of the dispute is centered on whether exponential VCs are more reliable when the static-centrifuge method is used rather than the flow-centrifuge method. To further test the reliability of the centrifuge technique, two centrifuges were manufactured to simulate the static- and flow-centrifuge methods. VCs of three species with open vessels of known lengths were constructed using the two centrifuges. The results showed that both centrifuge techniques produced invalid VCs for Robinia because the water flow through stems under mild tension in centrifuges led to an increasing loss of water conductivity. In addition, the injection of water in the flow-centrifuge exacerbated the loss of water conductivity. However, both centrifuge techniques yielded reliable VCs for Prunus, regardless of the presence of open vessels in the tested samples. We conclude that centrifuge techniques can be used in species with open vessels only when the centrifuge produces a VC that matches the bench-dehydration VC.

Three methods are in widespread use to build vulnerability curves (VCs) to cavitation. The bench drying (BD) method is considered as a reference because embolism and xylem pressure are measured on large branches dehydrating in the air, in conditions similar to what happens in nature. Two other methods of embolism induction have been increasingly used. While the Cavitron (CA) uses centrifugal force to induce embolism, in the air injection (AI) method embolism is induced by forcing pressurized air to enter a stem segment. Recent studies have suggested that the AI and CA methods are inappropriate in long-vesselled species because they produce a very high-threshold xylem pressure for embolism (e.g., P50) compared with what is expected from (i) their ecophysiology in the field (native embolism, water potential and stomatal response to xylem pressure) and (ii) the P50 obtained with the BD method. However, other authors have argued that the CA and AI methods may be valid because they produce VCs similar to the BD method. In order to clarify this issue, we assessed VCs with the three above-mentioned methods on the long-vesselled Quercus ilex L. We showed that the BD VC yielded threshold xylem pressure for embolism consistent with in situ measurements of native embolism, minimal water potential and stomatal conductance. We therefore concluded that the BD method provides a reliable estimate of the VC for this species. The CA method produced a very high P50 (i.e., less negative) compared with the BD method, which is consistent with an artifact related to the vessel length. The VCs obtained with the AI method were highly variable, producing P50 ranging from -2 to -8.2 MPa. This wide variability was more related to differences in base diameter among samples than to differences in the length of samples. We concluded that this method is probably subject to an artifact linked to the distribution of vessel lengths within the sample. Overall, our results indicate that the CA and the AI should be used with extreme caution on long-vesselled species. Our results also highlight that several criteria may be helpful to assess the validity of a VC.

Xylem hydraulic conductivity (K) is typically defined as K = F/(P/L), where F is the flow rate through a xylem segment associated with an applied pressure gradient (P/L) along the segment. This definition assumes a linear flow-pressure relationship with a flow intercept (F(0)) of zero. While linearity is typically the case, there is often a non-zero F(0) that persists in the absence of leaks or evaporation and is caused by passive uptake of water by the sample. In this study, we determined the consequences of failing to account for non-zero F(0) for both K measurements and the use of K to estimate the vulnerability to xylem cavitation. We generated vulnerability curves for olive root samples (Olea europaea) by the centrifuge technique, measuring a maximally accurate reference K(ref) as the slope of a four-point F vs P/L relationship. The K(ref) was compared with three more rapid ways of estimating K. When F(0) was assumed to be zero, K was significantly under-estimated (average of -81.4 ± 4.7%), especially when K(ref) was low. Vulnerability curves derived from these under-estimated K values overestimated the vulnerability to cavitation. When non-zero F(0) was taken into account, whether it was measured or estimated, more accurate K values (relative to K(ref)) were obtained, and vulnerability curves indicated greater resistance to cavitation. We recommend accounting for non-zero F(0) for obtaining accurate estimates of K and cavitation resistance in hydraulic studies.

Canny's compensating pressure theory for water transport (American Journal of Botany 85: 897-909) has evolved from the premise that cavitation pressures are only a few tenths of a megapascal negative (approximately -0.3 MPa). In contradiction, "vulnerability curves" indicate that xylem pressures can drop below -3 MPa in some species without causing a loss of hydraulic conductivity. Canny claims these curves do not measure the limits to negative pressure by cavitation, but rather the limits to the compensating tissue pressure that otherwise quickly refills cavitated conduits. Compensating pressure is derived from the turgor pressure of the living cells in the tissue. To test this claim, we compared vulnerability curves of Betula nigra stems given three treatments: (1) living control, (2) killed in a microwave oven, and (3) perfused with a -1.5 MPa (10% w/w) mannitol solution. According to Canny's theory, the microwaved and mannitol curves should show cavitation and loss of conductance beginning at approximately -0.3 MPa because in both cases, the turgor pressure would be eliminated or substantially reduced compared to controls. We also tested the refilling capability of nonstressed stems where compensating pressure would be in full operation and compared this with dead stems with no compensating pressure. According to Canny's interpretation of vulnerability curves, the living stems should refill within 5 min. Results failed to support the compensating tissue theory because (a) all vulnerability curves were identical, reaching a -1.5 MPa threshold before substantial loss of conductance occurred, and (b) killed or living stems had equally slow refilling rates showing no significant increase in conductivity after 30 min. In consequence, the cohesion theory remains the most parsimonious explanation of xylem sap ascent in plants.

Xylem cavitation resistance has profound implications for plant physiology and ecology. This process is characterized by a 'vulnerability curve' (VC) showing the variation of the percentage of cavitation as a function of xylem pressure potential. The shape of this VC varies from 'sigmoidal' to 'exponential'. This review provides a panorama of the techniques that have been used to generate such a curve. The techniques differ by (i) the way cavitation is induced (e.g. bench dehydration, centrifugation, or air injection), and (ii) the way cavitation is measured (e.g. percentage loss of conductivity (PLC) or acoustic emission), and a nomenclature is proposed based on these two methods. A survey of the literature of more than 1200 VCs was used to draw statistics on the usage of these methods and on their reliability and validity. Four methods accounted for more than 96% of all curves produced so far: bench dehydration-PLC, centrifugation-PLC, pressure sleeve-PLC, and Cavitron. How the shape of VCs varies across techniques and species xylem anatomy was also analysed. Strikingly, it was found that the vast majority of curves obtained with the reference bench dehydration-PLC method are 'sigmoidal'. 'Exponential' curves were more typical of the three other methods and were remarkably frequent for species having large xylem conduits (ring-porous), leading to a substantial overestimation of the vulnerability of cavitation for this functional group. We suspect that 'exponential' curves may reflect an open-vessel artefact and call for more precautions with the usage of the pressure sleeve and centrifugation techniques.

Progressive diebacks of outer canopy branchlets of Ceanothus crassifolius were repeatedly observed after rainless periods up to 9 mo in duration in the Santa Monica Mountains of southern California. Mean xylem pressures of branchlets near the end of drought were as low as -11.2 MPa (N = 22) with a mean of about 60 dead branchlets per shrub. Inoculation (N = 15) with three species of fungi previously isolated from the same population of C. crassifolius did not promote dieback, suggesting that the observed decline was not fungal induced, as had been proposed. Further, at least 50% of healthy-appearing twigs, without symptoms of dieback, contained isolatible endophytic fungi. We used a centrifugal force method to determine the range of xylem pressure causing cavitation (vulnerability curves) for branchlets (N = 12) and roots (N = 16). We combined vulnerability curves with soil texture data (N = 6) into a water transport model that estimated the critical values (P(Lcrit)) of leaf xylem pressure associated with the loss of water from soil to foliage. Maximum P(Lcrit) was between -10 and -11 MPa and within the range of minimum measured xylem pressures of branchlets during drought and dieback. Branchlet dieback correlated with seasonal declines in xylem pressure in concert with declining safety margins from hydraulic failure. Symptoms of dieback were duplicated in the field by partially severing stem xylem that normally supplied branchlets with water. Taken together, these results indicate that loss of hydraulic conductance to foliage was the probable cause of the observed dieback in C. crassifolius. Partial dieback of peripheral branchlets, and its attendant reduction in evaporative surface area, may be a last-resort mechanism for whole-plant water conservation and drought survival in this species.

In the coldest part of winter, water uptake is blocked by the frozen soil and frozen stems known as 'frost drought' causing severe embolisms in woody plants. Frost drought in stems was simulated in a centrifuge by a synergy between freeze-thaw cycles and the different tensions induced by changing the rotation speed. Frost fatigue was defined as a reduction of embolism resistance after a freeze-thaw cycle and determined from 'vulnerability curves', which showed percent losses of conductivity vs tension (positive value) or xylem pressure (negative value). Different tensions combined with a controlled freeze-thaw cycle were induced to investigate the effects on frost resistance over the course of year. During the growing season, Acer mono Maxim. developed significant frost fatigue, and a significant positive correlation was found between frost fatigue response and exogenous tension. During the dormant season, A. mono showed very high embolism resistance to frost drought, even under a tension of 2MPa. When the exogenous tension was increased to 3MPa while the stem was frozen, significant frost fatigue occurred. Longer freezing times had more serious effects on frost fatigue in A. mono. A hypothesis was raised that at the same low temperature, the severer the drought (higher tension) when stems were frozen, the higher frost fatigue response would be induced.

A centrifugal method is used to measure 'vulnerability curves' which show the loss of hydraulic conductivity in xylem by cavitation. Until recently, conductivity was measured between bouts of centrifugation using a gravity-induced head. Now, conductivity can be measured during centrifugation. This 'spin' method is faster than the 'gravity' technique, but correspondence between the two has not been evaluated. The two methods were compared on the same stem segments for two conifer, four diffuse-porous, and four ring-porous species. Only 17 of 60 conductivity measurements differed, with differences in the order of 10%. When different, the spin method gave higher conductivities at the beginning of the curve and lower at the end. Pressure at 50% loss of conductivity, and mean cavitation pressure, were the same in 14 of 20 comparisons. When different, the spin method averaged 0.32 MPa less negative. Ring-porous species showed a precipitous initial drop in conductivity by both techniques. This striking pattern was confirmed by the air-injection method and native embolism measurements. Close correspondence inspires confidence in both methods, each of which has unique advantages. The observation that ring-porous species operate at only a fraction of their potential conductivity at midday demands further study.

Xylem hydraulic properties are of great significance for plant growth and performance under drought. The ability of plants to avoid drought-induced cavitation and loss of hydraulic conductivity (K) can be characterized with vulnerability curves (VC). A VC describes the sigmoidal relationship between percentage loss of K (PLC) and xylem water potential (ψxyl). The ψ at 50% loss of conductance indicates a commonly used threshold for detrimental embolism (P50). The slope (b) represents cavitation resistance. The standard hydraulic method to determine VC's requires the measurement of water flow rate (WFR) per pressure gradient through stem segments, either by measuring outflow from the stem gravimetrically or inflow using a flow meter. In a comparative study using both measurements of inflow and outflow in asparagus stems, we found considerable disparities in the resulting shapes of VCs (P50 and b). We hypothesized that water uptake of stem tissue occurs during the pressure-driven water transit, particularly at low water potential and that differences in the initial K might result from measurements of inflow or outflow. To determine whether water uptake of stem tissue occurs during K measurements of asparagus plants, we tested for effects of ψxyl on the initial inflow and outflow K at different pressure gradients and investigated if passive water uptake can be estimated by extrapolation from the linear regression between WFR and pressure gradient based on K at two pressures. Initial K differed significantly between inlet and outlet measurements at low ψxyl, whereas maximum K did not, providing evidence of water uptake during transit through droughted stems. The resulting parameters P50 and b, and thus the shape of VCs, differed as well. The extrapolation resulted in the first estimate of passive water uptake, leading to a convergence of the VC at inlet and at outlet. We conclude that differences between in- and outflow may play a major role in K measurements.

Xylem vulnerability to embolism represents an essential trait for the evaluation of the impact of hydraulics in plant function and ecology. The standard centrifuge technique is widely used for the construction of vulnerability curves, although its accuracy when applied to species with long vessels remains under debate. We developed a simple diagnostic test to determine whether the open-vessel artefact influences centrifuge estimates of embolism resistance. Xylem samples from three species with differing vessel lengths were exposed to less negative xylem pressures via centrifugation than the minimum pressure the sample had previously experienced. Additional calibration was obtained from non-invasive measurement of embolism on intact olive plants by X-ray microtomography. Results showed artefactual decreases in hydraulic conductance (k) for samples with open vessels when exposed to a less negative xylem pressure than the minimum pressure they had previously experienced. X-Ray microtomography indicated that most of the embolism formation in olive occurs at xylem pressures below -4.0 MPa, reaching 50% loss of hydraulic conductivity at -5.3 MPa. The artefactual reductions in k induced by centrifugation underestimate embolism resistance data of species with long vessels. A simple test is suggested to avoid this open vessel artefact and to ensure the reliability of this technique in future studies.

Plant resistance to xylem cavitation is a major drought adaptation trait and is essential to characterizing vulnerability to climate change. Cavitation resistance can be determined with vulnerability curves. In the past decade, new techniques have increased the ease and speed at which vulnerability curves are produced. However, these new techniques are also subject to new artefacts, especially as related to long-vesselled species. We tested the reliability of the 'flow rotor' centrifuge technique, the so-called Cavitron, and investigated one potential mechanism behind the open vessel artefact in centrifuge-based vulnerability curves: the microbubble effect. The microbubble effect hypothesizes that microbubbles introduced to open vessels, either through sample flushing or injection of solution, travel by buoyancy or mass flow towards the axis of rotation where they artefactually nucleate cavitation. To test the microbubble effect, we constructed vulnerability curves using three different rotor sizes for five species with varying maximum vessel length, as well as water extraction curves that are constructed without injection of solution into the rotor. We found that the Cavitron technique is robust to measure resistance to cavitation in tracheid-bearing and short-vesselled species, but not for long-vesselled ones. Moreover, our results support the microbubble effect hypothesis as the major cause for the open vessel artefact in long-vesselled species.

During periods of dehydration, water transport through xylem conduits can become blocked by embolism formation. Xylem embolism compromises water supply to leaves and may lead to losses in productivity or plant death. Vulnerability curves (VCs) characterize plant losses in conductivity as xylem pressures decrease. VCs are widely used to characterize and predict plant water use at different levels of water availability. Several methodologies for constructing VCs exist and sometimes produce different results for the same plant material. We directly compared four VC construction methods on stems of black cottonwood (Populus trichocarpa), a model tree species: dehydration, centrifuge, X-ray-computed microtomography (microCT), and optical. MicroCT VC was the most resistant, dehydration and centrifuge VCs were intermediate, and optical VC was the most vulnerable. Differences among VCs were not associated with how cavitation was induced but were related to how losses in conductivity were evaluated: measured hydraulically (dehydration and centrifuge) versus evaluated from visual information (microCT and optical). Understanding how and why methods differ in estimating vulnerability to xylem embolism is important for advancing knowledge in plant ecophysiology, interpreting literature data, and using accurate VCs in water flux models for predicting plant responses to drought.

We studied 15 riparian and upland Sonoran desert species to evaluate how the limitation of xylem pressure (Ψ(x)) by cavitation corresponded with plant distribution along a moisture gradient. Riparian species were obligate riparian trees (Fraxinus velutina, Populus fremontii, and Salix gooddingii), native shrubs (Baccharis spp.), and an exotic shrub (Tamarix ramosissima). Upland species were evergreen (Juniperus monosperma, Larrea tridentata), drought-deciduous (Ambrosia dumosa, Encelia farinosa, Fouquieria splendens, Cercidium microphyllum), and winter-deciduous (Acacia spp., Prosopis velutina) trees and shrubs. For each species, we measured the "vulnerability curve" of stem xylem, which shows the decrease in hydraulic conductance from cavitation as a function of Ψ(x) and the Ψ(crit) representing the pressure at complete loss of transport. We also measured minimum in situ Ψ(x)(Ψ(xmin)) during the summer drought. Species in desert upland sites were uniformly less vulnerable to cavitation and exhibited lower Ψ(xmin) than riparian species. Values of Ψ(crit) were correlated with minimum Ψ(x). Safety margins (Ψ(xmin)-Ψ(crit)) tended to increase with decreasing Ψ(xmin) and were small enough that the relatively vulnerable riparian species could not have conducted water at the Ψ(x) experienced in upland habitats (-4 to -10 MPa). Maintenance of positive safety margins in riparian and upland habitats was associated with minimal to no increase in stem cavitation during the summer drought. The absence of less vulnerable species from the riparian zone may have resulted in part from a weak but significant trade-off between decreasing vulnerability to cavitation and conducting efficiency. These data suggest that cavitation vulnerability limits plant distribution by defining maximum drought tolerance across habitats and influencing competitive ability of drought tolerant species in mesic habitats.

A new technique for generating xylem cavitation and vulnerability curves was evaluated. The centrifugal force was used to lower the negative pressure in a xylem segment and to induce a positive pressure difference between sample's ends. This enabled the determination of sample hydraulic conductance during centrifugation and, hence, its variation with decreasing xylem pressures. The centrifuge technique was compared with standard methods on a large number of species including conifers, diffuse‐porous and ring‐porous woody angiosperms. A very good agreement was found for coniferous and diffuse‐porous species. However, the technique was not appropriate for ring‐porous species, probably because many vessels were cut open in the centrifuged xylem segments. The main advantage of this technique is its rapidity, the vulnerability curve of a xylem segment being constructed typically in less than half an hour. This will greatly facilitate the study of xylem cavitation in ecological or genetic researches.