Further Test of Pneumatic Method in Constructing Vulnerability Curves Using Six Tree Species with Contrasting Xylem Anatomy

Vessels are the chief conduit for long-distance water transport in the majority of flowering plants. Vessel length is a key trait that determines plant hydraulic efficiency and safety, yet relatively little is known about this xylem feature. • We used previously published studies to generate a new global data set of vessel length in woody plants. These data were used to examine how evolutionary history, plant habit, environment, and growth ring porosity influenced vessel length. We also examined the relationship between mean vessel length and mean vessel diameter and maximum vessel length. • Data on mean vessel length were available for stems of 130 species and on maximum vessel length for stems of 91 species. A phylogenetic analysis indicated that vessel length did not exhibit significant phylogenetic signal. Liana species had longer vessel lengths than in tree or shrub species. Vessel diameter was not predictive of mean vessel length, but maximum vessel length strongly predicted mean vessel length. Vessel length did not vary between species that differed in growth ring porosity. • Many traits often assumed to be linked to vessel length, including growth ring porosity and vessel diameter, are not associated with vessel length when compared interspecifically. Sampling for vessel length has been nonrandom, e.g., there are virtually no data available for roots, and sampling for environment has been confounded with sampling for habit. Increased knowledge of vessel length is key to understanding the structure and function of the plant hydraulic pathway.

A comparison of five methods to assess embolism resistance in trees

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.

Vessel length in xylem of woody species is a key functional trait for understanding water transport mechanisms, directly influencing xylem hydraulic efficiency and safety. However, the accurate measurements of vessel length are susceptible to methodological variations. This study employed silicone injection, air injection and pneumatic method on eight subtropical woody branches to compare their applicability, accuracy and limitations for measuring vessel length and its distribution pattern. The results demonstrated that the vessel length distribution curves in three methods all showed an asymmetric unimodal right-skewed distribution. Both mean and mode vessel lengths of eight species obtained from the pneumatic and air injection methods showed no significant differences and were all higher than that from the silicone injection method. Besides, across all vessel diameter ranges in eight species, both mean and mode vessel lengths showed significant positive correlations among the three methods. However, the correlations became weakened in wide vessel species, especially for the silicone injection data. Moreover, vessel lengths were significantly positively related to the vessel diameter in eight species. This study provides empirical evidence for selecting appropriate methods to measure vessel length, which has a crucial role in determining water transport functions in the xylem of plants.

Changes in age of the hydraulic architecture of Ulmus minor and U. minor × U. pumila juvenile wood were studied and related to tolerance to Dutch elm disease (DED). The xylem vessel dimensions and the conductivity to air of 2- to 7-year-old branches were analyzed and quantified. No obvious differences in vessel length distribution and conductivity were found to explain differences in DED tolerance among the U. minor clones, or, at the taxon level, the higher DED tolerance of U. minor × U. pumila. Among the U. minor clones, the more susceptible one had wider vessels and a higher maximum vessel diameter than the more tolerant clone. Relations between vessel lengths, vessel diameters and branch sizes were highly significant, and varied between taxa. The diameter and length of vessels increased with age, and average values stabilized 1–2 years earlier for U. minor than for U. minor × pumila. Mean maximum vessel length was significantly higher in U. minor and increased more with age and maximum vessel diameter than in U. minor × pumila. With each 0.2 m increase in height up the stem, conductivities for U. minor and U. minor × U. pumila decreased by 59 and 50 %, respectively, probably due to shortening of the vessels. The implications of xylem structure for the means of pathogen movement and resistance to DED are discussed.

A crucial phase in plants - it's a gas, gas, gas!

ABSTRACTPlant water relations, xylem anatomy and the hydraulic architecture of 1‐year‐old twigs of Spartium junceum, both healthy and affected by a phytoplasm disease, were studied. The disease causes twigs to be either shortened (witches broom disease, WBD) or flat (fasciate disease, FD). WBD twigs show a sevenfold increase in total leaf area, smaller and shorter xylem conduits, a higher stomatal conductance (gl) and a decline of minimum leaf water potentials (Ψl) below the turgor loss point. FD twigs had nearly twice the leaf area of the healthy controls as well as high gl values and Ψl values below the turgor loss point. Moreover, significant differences between healthy and affected twigs in stem stomatal conductance (gs) and in the total stem area were recorded. Affected twigs die back under drought stress, which is explained by a pronounced loss of hydraulic conductivity of the infected stems (40 and 60%) in FD and WBD as well as by the unfavourable ratio of weighted conduit radius (Σr4) to total surface area (At), so that the efficiency of the stem in supplying the whole transpiring area with water is strongly reduced.

Knowledge of xylem vessel length is important for several reasons, including the accurate calculation and comparison of hydraulic conductivity studies in excised stems. Vessel length data and distributions are also relevant in some anatomical, ecological, evolutionary, pathological and compatible hydraulic studies. However, determining vessel length is tedious, so is often either avoided or undertaken arbitrarily in hydraulic conductivity studies. We examined four injection media (paints and inks) under transmission electron microscopy to ascertain which was most suitable for determining vessel length. Hunt’s Speedball India ink, with evenly distributed, uniform spherical carbon particles of 33 nm, would avoid premature vessel blockage and, therefore, coupled with the fact that it is non-toxic, is the preferred medium to determine vessel length in Acacia amoena Wendl. terminal stems. The longest vessel was 10 cm, which accounted for 0.4% of vessels. Vessel length distributions were then determined and compared using the same dataset and four established methods. All four methods produced distributions which indicated that the most common vessel length class was short (0–2 cm), and no method was significantly different from the other; however, for ease of calculation, the Christman et al. (2009) method is recommended. Whether vessel length or distribution is necessary for hydraulic-conductivity studies will depend on whether or not merely indicative rates of flow are required, but to provide comparative information for global datasets, then they are needed.

Key messageTree species in a temperate floodplain forest had leaf turgor loss point values similar to those of upland forest trees, suggesting physiological drought tolerance in this generally non-water-limited system.Leaf turgor loss point (TLP) is a key plant trait associated with drought tolerance. In the bottomland hardwood (BLH) forests that grow in floodplains of the southeastern USA, drought stress is generally low but may increase with climate change. To address drought tolerance among BLH trees, we measured TLP among 20 species in a BLH forest in Louisiana, USA. We tested whether (1) TLP is higher in BLH tree species than in upland temperate-zone trees; (2) lower TLP is associated with higher drought tolerance among BLH species; (3) TLP drops during the growing season within BLH trees; and (4) within species, TLP is lower in more water limited, non-flooded BLH habitats than in seasonally flooded habitats. Among BLH tree species, TLP was −2.23 ± 0.28 (mean ± SD) and, contrary to our hypothesis, weakly positively correlated with drought tolerance. Within BLH species, TLP was lower in non-flooded habitats than seasonally flooded habitats and TLP decreased between the early and late growing season, more so in the non-flooded habitat. Overall, our results show that TLP among BLH trees is relatively low and plastic for a system that is generally not water limited, which may contribute to drought tolerance in future scenarios.

Vessel lengths are important to plant hydraulic studies, but are not often reported because of the time required to obtain measurements. This paper compares the fast dynamic method (air injection method) with the slower but traditional static method (rubber injection method). Our hypothesis was that the dynamic method should yield a larger mean vessel length than the static method. Vessel length was measured by both methods in current year stems of Acer, Populus, Vitis and Quercus representing short- to long-vessel species. The hypothesis was verified. The reason for the consistently larger values of vessel length is because the dynamic method measures air flow rates in cut open vessels. The Hagen-Poiseuille law predicts that the air flow rate should depend on the product of number of cut open vessels times the fourth power of vessel diameter. An argument is advanced that the dynamic method is more appropriate because it measures the length of the vessels that contribute most to hydraulic flow. If all vessels had the same vessel length distribution regardless of diameter, then both methods should yield the same average length. This supports the hypothesis that large-diameter vessels might be longer than short-diameter vessels in most species.

Embolism, the formation of air bubbles in the plant water transport system, has a major impact on plant water relations. Embolism formation in the water transport system of plants disrupts plant water transport capacity, impairing plant functioning and triggering plant mortality. Measuring embolism with traditional hydraulic methods is both time-consuming and requires large amounts of plant material. While the stem hydraulic methods measure loss of xylem hydraulic conductance due to embolism formation, the pneumatic method directly quantifies the amount of emboli inside the xylem as changes in xylem air content. The pneumatic method is an easy and fast (8+ embolism curves per day) method to measure plant embolism requiring minimal plant material. Here, we provide detailed descriptions and recent technical improvements on the pneumatic method.

Using the Pneumatic method to estimate embolism resistance in species with long vessels: A commentary on the article “A comparison of five methods to assess embolism resistance in trees”

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.

Vessel length is an important but understudied dimension of variation in angiosperm vascular anatomy. Among other traits, vessel length mediates an important tradeoff between hydraulic efficiency and safety that could influence how plants respond to extreme weather with climate change. However, the functional significance of vessel length variation within individual stems is poorly known, in part because existing data analysis methods handle uncertainty in a way that makes vessel length distributions difficult to compare. We provide a solution to this problem through a hierarchical Bayesian framework for estimating vessel lengths and we demonstrate the flexibility of this method by applying it to data from serial cross sections of dye injected stems. Our approach can accelerate data collection and accommodate associated uncertainties by statistically correcting for bias and error that result from subsampling images. We illustrate our analytical framework by estimating and comparing vessel length distributions for 21 woody species characteristic of a North American forest. The best-fit model corrected for both bias due to secondary growth and sampling error within and among species. Vessel length estimates from this model varied by almost an order of magnitude and parameters of these distributions correlated with point estimates derived from a different, commonly used method. Furthermore, we show how key contrasts can be estimated with the Bayesian framework, and in doing so, we show that the shape of the vessel length distribution differed between ring- and diffuse-porous species, suggesting that within-stem vessel length variation corresponds to water stress seasonality and contributes to landscape-level habitat segregation. Our analysis method revealed the importance of within-stem variation in vessel length, and our results complement work on between-species variation in average vessel length, further illuminating how vascular anatomy can influence woody plants’ responses to water stress.

Methods to estimate xylem embolism resistance generally rely on hydraulic measurements, which can be far from straightforward. Recently, a pneumatic method based on air flow measurements of terminal branch ends was proposed to construct vulnerability curves by linking the amount of air extracted from a branch with the degree of embolism. We applied this novel technique for 10 temperate tree species, including six diffuse, two ring-porous and two gymnosperm species, and compared the pneumatic curves with hydraulic ones obtained from either the flow-centrifuge or the hydraulic-bench dehydration method. We found that the pneumatic method provides a good estimate of the degree of xylem embolism for all angiosperm species. The xylem pressure at 50% and 88% loss of hydraulic conductivity (i.e., Ψ50 and Ψ88) based on the methods applied showed a strongly significant correlation for all eight angiosperms. However, the pneumatic method showed significantly reduced Ψ50 values for the two conifers. Our findings suggest that the pneumatic method could provide a fast and accurate approach for angiosperms due to its convenience and feasibility, at least within the range of embolism resistances covered by our samples.