Research progress on the methods for measuring xylem embolism vulnerability

Xylem vulnerability to drought-induced embolism did not differ between stems and petioles of four woody species ( Betula pendula , Liriodendron tulipifera , Populus tremula and Olea europaea ). Our results, together with data compiled from published literature, indicate that hydraulic segmentation during drought stress is not consistently driven by difference in vulnerability to embolism between stem and terminal organs. Hydraulic failure and disconnection of distal organs during protracted drought stress is thought to protect large branches or trunks by reducing water loss and restricting the spread of embolism. Hydraulic segmentation and preferential sacrifice of distal organs such as leaves can be driven by two mechanisms: more negative water potentials at the terminal section of the hydraulic pathway and/or by higher vulnerability to xylem embolism of distal organs. Although vulnerability segmentation has been reported in the literature, the generality of this phenomenon is unclear, in part due to the methodological limitations related to direct measurement of xylem vulnerability to embolism in intact plants. The objective of this study was to evaluate vulnerability segmentation between petioles and stems using non-invasive micro computed tomography (microCT). Vulnerability to embolism was measured in leaf petioles and subtending stems of four woody species (Betula pendula R., Liriodendron tulipifera L., Populus tremula L. and Olea europaea L.) with contrasting drought tolerances. In addition, previously published vulnerability data for petioles and stems were compiled from the literature to investigate the commonality of hydraulic segmentation across a wide range of woody species, with the vulnerability curve methodology distinguished. Using non-invasive imaging on intact plants, we found no evidence of hydraulic segmentation between petioles and stems of four angiosperm tree species, regardless of mechanism. Moreover, the literature dataset indicated that little or no difference in vulnerability to embolism is present between petioles and stems when vulnerability curves were constructed using methods specifically measuring the dynamics of xylem tissue during dehydration (e.g. optical visualization, MicroCT). Our results suggest that vulnerability segmentation between stems and distal organs (petioles and leaves) is limited when only xylem tissue is considered. Large differences in vulnerability between stems and leaves are likely to be driven by extra-xylary components, rather than xylem embolism.

Hydraulic failure is one of the main causes of tree mortality in conditions of severe drought. Resistance to cavitation is known to be strongly related to drought tolerance and species survival in conifers, but the threshold of water-stress-induced embolism leading to catastrophic xylem dysfunction in angiosperms has been little studied. We investigated the link between drought tolerance, survival and xylem cavitation resistance in five angiosperm tree species known to have contrasting desiccation resistance thresholds. We exposed seedlings in a greenhouse to severe drought to generate extreme water stress. We monitored leaf water potential, total plant water loss rate, leaf transpiration, stomatal conductance and CO2 assimilation rate during drought exposure and after rewatering (recovery phase). The time required for the recovery of 50% of the maximum value of a given ecophysiological variable after rewatering was used to determine the critical water potential corresponding to the threshold beyond which the plant failed to recover. We also investigated the relationship between this potential and stem xylem cavitation resistance, as assessed from vulnerability curves. This minimum recoverable water potential was consistent between ecophysiological variables and varied considerably between species, from -3.4 to -6.0 MPa. This minimum recoverable water potential was strongly correlated with P50 and P88, the pressures inducing 50 and 88% losses of stem hydraulic conductance, respectively. Moreover, the embolism threshold leading to irreversible drought damage was found to be close to 88%, rather than the 50% previously reported for conifers. Hydraulic failure leading to irreversible drought-induced global dysfunction in angiosperm tree species occurred at a very high level of xylem embolism, possibly reflecting the physiological characteristics of their stem water-transport system.

Water deficit and soil salinization are the primary abiotic stress factors hindering maize growth. To assess the effect of water and salt stress on xylem embolism in maize and investigate the relationship between drought resistance and xylem vulnerability, a greenhouse experiment was designed using two maize cultivars, Zhengdan 958 (drought-resistant) and Denghai 605 (drought-sensitive). Four treatments were included: control (CK), water deficit (WD), salt stress (SS), and combined water and salt stress (WS). Various hydraulic characteristic indicators, such as stem xylem water potential, leaf xylem water potential, the specific hydraulic conductivity (Ks) and percentage loss of conductivity (PLC), were analyzed. Specific hydraulic conductivity curves and vulnerability curves were constructed, and the hydraulic safety margin (HSM) of the xylem was determined based on stomatal conductance (Gs). The results indicated that the hydraulic conductivity and embolism resistance of maize xylem were not correlated. Compared to Denghai 605, Zhengdan 958 had lower maximum specific hydraulic conductivity Ksmax and P50 values (xylem water potential at 50% PLC) in all treatments, indicating lower water transport capacity but stronger resistance to embolism. Under single-cultivar conditions, salt stress had a greater inhibitory effect on Ksmax and HSM in maize xylem compared to water deficit; thus, more severe embolism was found under salt stress. Under different treatment conditions, Zhengdan 958 had a larger HSM than Denghai 605, showing a wider water transport safety range and overall superior water transport security. To summarize, water and salt stress inhibited the water transport efficiency of the xylem in maize stems, and stronger drought-resistant cultivars showed greater resistance to embolism and larger hydraulic safety margins.

The hydraulic properties of Pinus pinea, Pinus halepensis and Tetraclinis articulata were studied in a coastal dune area from Eastern Spain. The measured variables include vulnerability to xylem embolism (vulnerability curves), hydraulic conductivity and carbon isotopic discrimination in leaves. Leaf water potentials were also monitored in the three studied populations during an extremely dry period. Our results showed that roots had always wider vessels and higher hydraulic conductivity than branches. Roots were also more vulnerable to xylem embolism and operated closer to their hydraulic limit (i.e., with narrower safety margins). Although it was not quantified, extensive root mortality was observed in the two pines during the study period, in agreement with the high values of xylem embolism (> 75%) predicted from vulnerability curves and the water potentials measured in the field. T. articulata was much more resistant to embolism than P. pinea and P. halepensis. Since T. articulata experienced also lower water potentials, safety margins from hydraulic failure were only slightly wider in this species than in the pines. Combining species and tissues, high resistance to xylem embolism was associated with low hydraulic conductivity and with high wood density. Both relationships imply a cost of having a resistant xylem. The study outlined very different water-use strategies for T. articulata and the pines. Whereas T. articulata had a conservative strategy that relied on the low vulnerability of its conducting system to drought-induced xylem embolism, the two pines showed regulatory mechanisms at different levels (i.e., embolism, root demography) that constrained the absorption of water when it became scarce.

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.

Understanding intraspecific variation in the vulnerability of the xylem to hydraulic failure during drought is critical in predicting the response of forest tree species to climate change. However, few studies have assessed intraspecific variation in this trait, and a likely limitation is the large number of measurements required to generate the standard 'vulnerability curve' used to assess hydraulic failure. Here we explore an alternative approach that requires fewer measurements, and assess within species variation in leaf xylem vulnerability in Eucalyptus globulus Labill., an ecologically and economically important species with known genetic variation in drought tolerance. Using this approach we demonstrate significant phenotypic differences and evidence of plasticity among two provenances with contrasting drought tolerance.

Plant hydraulic traits related to leaf drought tolerance, like the water potential at turgor loss point (TLP) and the water potential inducing 50% loss of hydraulic conductance (P50), are extremely useful to predict the potential impacts of drought on plants. While novel techniques have allowed the inclusion of TLP in studies targeting a large group of species, fast and reliable protocols to measure leaf P50 are still lacking. Recently, the optical method coupled with the gas injection (GI) technique has been proposed as a possibility to speed up the P50 estimation. Here, we present a comparison of leaf optical vulnerability curves (OVcs) measured in three woody species, namely Acer campestre (Ac), Ostrya carpinifolia (Oc) and Populus nigra (Pn), based on bench dehydration (BD) or GI of detached branches. For Pn, we also compared optical data with direct micro-computed tomography (micro-CT) imaging in both intact saplings and cut shoots subjected to BD. Based on the BD procedure, Ac, Oc and Pn had P50 values of -2.87, -2.47 and-2.11MPa, respectively, while the GI procedure overestimated the leaf vulnerability (-2.68, -2.04 and -1.54MPa for Ac, Oc and Pn, respectively). The overestimation was higher for Oc and Pn than for Ac, likely reflecting the species-specific vessel lengths. According to micro-CT observations performed on Pn, the leaf midrib showed none or very few embolized conduits at -1.2MPa, consistent with the OVcs obtained with the BD procedure but at odds with that derived on the basis of GI. Overall, our data suggest that coupling the optical method with GI might not be a reliable technique to quantify leaf hydraulic vulnerability since it could be affected by the 'open-vessel' artifact. Accurate detection of xylem embolism in the leaf vein network should be based on BD, preferably of intact up-rooted plants.

Summary Determining physiological mechanisms and thresholds for climate‐driven tree die‐off could help improve global predictions of future terrestrial carbon sinks. We directly tested for the lethal threshold in hydraulic failure – an inability to move water due to drought‐induced xylem embolism – in a pine sapling experiment.In a glasshouse experiment, we exposed loblolly pine (Pinus taeda) saplings (n = 83) to drought‐induced water stress ranging from mild to lethal. Before rewatering to relieve drought stress, we measured native hydraulic conductivity and foliar color change. We monitored all measured individuals for survival or mortality.We found a lethal threshold at 80% loss of hydraulic conductivity – a point of hydraulic failure beyond which it is more likely trees will die, than survive, and describe mortality risk across all levels of water stress. Foliar color changes lagged behind hydraulic failure – best predicting when trees had been dead for some time, rather than when they were dying.Our direct measurement of native conductivity, while monitoring the same individuals for survival or mortality, quantifies a continuous probability of mortality risk from hydraulic failure. Predicting tree die‐off events and understanding the mechanism involved requires knowledge not only of when trees are dead, but when they begin dying – having passed the point of no return.

A common assumption of plant hydraulic physiology is that high hydraulic efficiency must come at the cost of hydraulic safety, generating a trade-off that raises doubts about the possibility of selecting both productive and drought-tolerant herbaceous crops. Wetland plants typically display high productivity, which requires high hydraulic efficiency to sustain transpiration rates coupled to CO2 uptake. Previous studies have suggested high vulnerability to xylem embolism of different wetland plants, in line with expected trade-offs. However, some hygrophytes like Cyperus alternifolius L. can also experience prolonged periods of low water levels leading to substantial drought stress. We conducted an in-depth investigation of this species' hydraulic safety and efficiency by combining gas exchange measurements, hydraulic measurements of leaf hydraulic efficiency and safety, optical measurements of xylem vulnerability to embolism, and determination of cell turgor changes under drought. Our data confirm the high hydraulic efficiency of this wetland species, but at the same time, reveal its surprising drought tolerance in terms of turgor loss point and critical water potential values inducing xylem embolism and hydraulic failure, which were well below values inducing turgor loss and full stomatal closure. C. alternifolius emerges as a highly productive plant that is also well-equipped to tolerate drought via a combination of early stomatal closure and delayed onset of hydraulic damage. The species might represent a model plant to develop crops combining two of the most desirable traits in cultivated plants, i.e., high yield and significant drought tolerance.

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.

Water is transported from roots to foliage through the xylem under negative pressure (=tension). Under this metastable status, water is prone to sudden phase change to water vapor (cavitation). In plants, air can also be aspirated into functioning xylem conduits through inter-conduit pit membranes, and the resulting embolism blocks water transport through the conduit and reduces plant hydraulic conductance and productivity. Xylem embolism and hydraulic failure are major factors contributing to tree mortality and forest decline under global-change-type droughts. However, some plants can tolerate even high embolism levels under drought, recovering hydraulic functionality upon partial or total rehydration via embolism repair and xylem refilling. Here, we review current evidence of embolism repair, highlighting possible physiological mechanisms and suggesting some functional and anatomical determinants making embolism reversal a feasible and successful drought resilience mechanism in some plants.

Because the xylem in leaves is thought to be at the greatest risk of cavitation, reliable and efficient methods to characterize leaf xylem vulnerability are of interest. We report a method to generate leaf xylem vulnerability curves (VCs) by gas injection. Using optical light transmission, we visualized embolism propagation in grapevine (Vitis vinifera) and red oak (Quercus rubra) leaves injected with positive gas pressure. This resulted in a rapid, stepwise reduction of transmitted light, identical to that observed during leaf dehydration, confirming that the optical method detects gas bubbles and provides insights into the air-seeding hypothesis. In red oak, xylem VCs generated using gas injection were similar to those generated using bench dehydration, but indicated 50% loss of conductivity at lower tension (∼0.4 MPa) in grapevine. In determining VC, this method eliminates the need to ascertain xylem tension, thus avoiding potential errors in water potential estimations. It is also much faster (1 h per VC). However, severing the petiole and applying high-pressure gas could affect air-seeding and the generated VC. We discuss potential artifacts arising from gas injection and recommend comparison of this method with a more standard procedure before it is assumed to be suitable for a given species.

Perennial woody plants accumulate native xylem embolisms over time. However, whether this makes the water transport system more vulnerable to drought-induced dysfunction as the percentage of gas-filled vessels increases is unclear. We tested whether increasing the proportion of open (air-filled) vessels changes the overall embolism vulnerability in stems of angiosperm species with long maximum vessel lengths but relatively low vessel connectivity. Using optical vulnerability curves, we measured xylem vulnerability of 57 branches ranging in length from ~ 10 to over 300 cm, from two adult trees (Acacia mearnsii De Wild. and Eucalyptus globulus Labill.) known to have long maximum vessel length (>75 cm) but low vessel connectivity. The fraction of open vessels at different branch lengths was estimated by staining open vessels under suction and with X-ray micro-computed tomography (μCT). To relate this to native field conditions, the percentage of pre-existing native embolisms was measured with μCT on a different set of branches. Our results show that even when a large proportion (> 25%) of open (air-filled) vessels are present, the xylem-embolism thresholds (water potential at 12% (P12), 50% (P50) and 88% (P88) embolized xylem area) resemble those of branches with no open vessels. Scanning of native embolism with μCT revealed 10% (E. globulus) and 20% (A. mearnsii) native embolism under natural conditions. We conclude that even when approximately one-quarter of vessels are air-filled, there is no discernable effect on the overall xylem vulnerability of stem segments with long vessels and low vessel connectivity. Xylem vulnerability to embolism among all the branches measured from each of the two trees was relatively homogeneous with a ~10–20% variation. Our findings also suggest that the presence of pre-existing native embolisms, at the percentages observed in the field (<25%), would not increase vulnerability to xylem embolism in these species with largely isolated individual xylem vessels.

Towards an advanced assessment of the hydrological vulnerability of forests to climate change‐induced drought

The formation of air emboli in the xylem during drought is one of the key processes leading to plant mortality due to loss in hydraulic conductivity, and strongly fuels the interest in quantifying vulnerability to cavitation. The acoustic emission (AE) technique can be used to measure hydraulic conductivity losses and construct vulnerability curves. For years, it has been believed that all the AE signals are produced by the formation of gas emboli in the xylem sap under tension. More recent experiments, however, demonstrate that gas emboli formation cannot explain all the signals detected during drought, suggesting that different sources of AE exist. This complicates the use of the AE technique to measure emboli formation in plants. We therefore analysed AE waveforms measured on branches of grapevine (Vitis vinifera L. 'Chardonnay') during bench dehydration with broadband sensors, and applied an automated clustering algorithm in order to find natural clusters of AE signals. We used AE features and AE activity patterns during consecutive dehydration phases to identify the different AE sources. Based on the frequency spectrum of the signals, we distinguished three different types of AE signals, of which the frequency cluster with high 100-200 kHz frequency content was strongly correlated with cavitation. Our results indicate that cavitation-related AE signals can be filtered from other AE sources, which presents a promising avenue into quantifying xylem embolism in plants in laboratory and field conditions.