A comparison of two centrifuge techniques for constructing vulnerability curves: insight into the 'open-vessel' artifact.

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.

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.

Vulnerability to cavitation curves are used to estimate xylem cavitation resistance and can be constructed using multiple techniques. It was recently suggested that a technique that relies on centrifugal force to generate negative xylem pressures may be susceptible to an open vessel artifact in long-vesselled species. Here, we used custom centrifuge rotors to measure different sample lengths of 1-yr-old stems of grapevine to examine the influence of open vessels on vulnerability curves, thus testing the hypothesized open vessel artifact. These curves were compared with a dehydration-based vulnerability curve. Although samples differed significantly in the number of open vessels, there was no difference in the vulnerability to cavitation measured on 0.14- and 0.271-m-long samples of Vitis vinifera. Dehydration and centrifuge-based curves showed a similar pattern of declining xylem-specific hydraulic conductivity (K(s)) with declining water potential. The percentage loss in hydraulic conductivity (PLC) differed between dehydration and centrifuge curves and it was determined that grapevine is susceptible to errors in estimating maximum K(s) during dehydration because of the development of vessel blockages. Our results from a long-vesselled liana do not support the open vessel artifact hypothesis.

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.

Since 1988, researchers have exposed stems to positive pressures to displace water in vessels and measure the impact of applied pressure on hydraulic conductivity. The pressure-sleeve technique has been used in more than 60 publications to measure vulnerability curves (VCs), which are a measure of how water stress impacts the ability of plants to transport water because water stress induces embolism in vessels that blocks water flow. It is thought that the positive pressure in a sleeve required to induce 50% loss of conductivity (PLC), P50 , is the same magnitude as the tension that causes 50% PLC, T50 , where the tension can be induced by either bench-top dehydration or by a centrifuge technique. The unifying concept that P50 =T50 and that the entire VC is the same regardless of method is referred to as the air-seeding hypothesis. In the current study, we performed experiments to further test the air-seeding hypothesis in pressure sleeves and concluded that an "effervescence" mechanism caused embolism formation under positive pressure. This mechanism explains why VCs measured using positive pressure do not always match VCs obtained by other methods that induce water tension.

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.

Vulnerability to cavitation curves describe the decrease in xylem hydraulic conductivity as xylem pressure declines. Several techniques for constructing vulnerability curves use centrifugal force to induce negative xylem pressure in stem or root segments. Centrifuge vulnerability curves constructed for long-vesselled species have been hypothesised to overestimate xylem vulnerability to cavitation due to increased vulnerability of vessels cut open at stem ends that extend to the middle or entirely through segments. We tested two key predictions of this hypothesis: (i) centrifugation induces greater embolism than dehydration in long-vesselled species, and (ii) the proportion of open vessels changes centrifuge vulnerability curves. Centrifuge and dehydration vulnerability curves were compared for a long- and short-vesselled species. The effect of open vessels was tested in four species by comparing centrifuge vulnerability curves for stems of two lengths. Centrifuge and dehydration vulnerability curves agreed well for the long- and short-vesselled species. Centrifuge vulnerability curves constructed using two stem lengths were similar. Also, the distribution of embolism along the length of centrifuged stems matched the theoretical pressure profile induced by centrifugation. We conclude that vulnerability to cavitation can be accurately characterised with vulnerability curves constructed using a centrifuge technique, even 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.

The standard centrifuge method has been frequently used to measure vulnerability to xylem cavitation. This method has recently been questioned. It was hypothesized that open vessels lead to exponential vulnerability curves, which were thought to be indicative of measurement artifact. We tested this hypothesis in stems of olive (Olea europea) because its long vessels were recently claimed to produce a centrifuge artifact. We evaluated three predictions that followed from the open vessel artifact hypothesis: shorter stems, with more open vessels, would be more vulnerable than longer stems; standard centrifuge-based curves would be more vulnerable than dehydration-based curves; and open vessels would cause an exponential shape of centrifuge-based curves. Experimental evidence did not support these predictions. Centrifuge curves did not vary when the proportion of open vessels was altered. Centrifuge and dehydration curves were similar. At highly negative xylem pressure, centrifuge-based curves slightly overestimated vulnerability compared to the dehydration curve. This divergence was eliminated by centrifuging each stem only once. The standard centrifuge method produced accurate curves of samples containing open vessels, supporting the validity of this technique and confirming its utility in understanding plant hydraulics. Seven recommendations for avoiding artefacts and standardizing vulnerability curve methodology are provided.

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.

Loss of hydraulic conductivity occurs in stems when the water in xylem conduits is subjected to sufficiently negative pressure. According to the air-seeding hypothesis, this loss of conductivity occurs when air bubbles are sucked into water-filled conduits through micropores adjacent to air spaces in the stem. Results in this study showed that loss of hydraulic conductivity occurred in stem segments pressurized in a pressure chamber while the xylem water was under positive pressure. Vulnerability curves can be defined as a plot of percentage loss of hydraulic conductivity versus the pressure difference between xylem water and the outside air inducing the loss of conductivity. Vulnerability curves were similar whether loss of conductivity was induced by lowering the xylem water pressure or by raising the external air pressure. These results are consistent with the air-seeding hypothesis of how embolisms are nucleated, but not with the nucleation of embolisms at hydrophobic cracks because the latter requires negative xylem water pressure. The results also call into question some basic underlying assumptions used in the determination of components of tissue water potential using "pressure-volume" analysis.

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.

Centrifuge-based techniques to assess xylem vulnerability to embolism are increasingly being used, although we are yet to reach a consensus on the nature and extent of artefactual embolism observed in some angiosperm species. In particular, there is disagreement over whether these artefacts influence both the spin (Cavitron) and static versions of the centrifuge technique equally. We tested two methods for inducing embolism: bench dehydration and centrifugation. We used three methods to measure the resulting loss of conductivity: gravimetric flow measured in bench-dehydrated and centrifuged samples (static centrifuge), in situ flow measured under tension during spinning in the centrifuge (Cavitron) and direct imaging using X-ray computed microtomography (microCT) observations in stems of two species of Hakea that differ in vessel length. Both centrifuge techniques were prone to artefactual embolism in samples with maximum vessel length longer than, or similar to, the centrifuge rotor diameter. Observations with microCT indicated that this artefactual embolism occurred in the outermost portions of samples. The artefact was largely eliminated if flow was measured in an excised central part of the segment in the static centrifuge or starting measurements with the Cavitron at pressures lower than the threshold of embolism formation in open vessels. The simulations of loss of conductivity in centrifuged samples with a new model, CAVITOPEN, confirmed that the impact of open vessels on the vulnerability to embolism curve was higher when vessels were long, samples short and when embolism is formed in open vessels at less negative pressures. This model also offers a robust and quantitative tool to test and correct for artefactual embolism at low xylem tensions.

Centrifuges provide a fast approach to quantify the embolism resistance of xylem in vulnerability curves (VCs). Since embolism formation is assumingly driven by pressure only, spintime is not standardized for flowcentrifuge experiments. Here, we explore to what extent embolism resistance could be spin-time dependent and hypothesize that changes in hydraulic conductivity (Kh) would shift VCs towards higher water potential (Ψ) values over time. We quantified time-based shifts in flow-centrifuge VCs and their parameter estimations for six angiosperm species by measuring Kh over 15min of spinning at a particular speed before a higher speed was applied to the same sample. We compared various VCs per sample based on cumulative spintime and modelled the relationship between Kh, Ψ and spin-time. Time-based changes of Kh showed considerable increases and decreases at low and high centrifuge speeds, respectively, which generally shifted VCs towards more positive Ψ values. Values corresponding to 50% loss of hydraulic conductivity (P50) became less negative by up to 0.72MPa in Acer pseudoplatanus L., and on average by 8.5% for all six species compared with VCs that did not consider spin-time. By employing an asymptotic exponential model, we estimated time-stable Kh, which improved the statistical significance of VCs in five of the six species studied. This model also revealed the instability of VCs at short spin times with embolism formation in flowcentrifuges following a saturating exponential growth curve. Although pressure remains the major determinant of embolism formation, spin-time should be considered in flow-centrifuge VCs because not considering the time-dependent stability of Kh overestimates embolism resistance. This spin-time artefact is species-specific and likely based on relatively slow gas diffusion that is associated with embolism propagation. The accuracy of VCs is improved by determining time-stable Kh values for each centrifuge speed without considerably extending the experimental time to construct VCs.

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.