Visualizing Embolism Propagation in Gas-Injected Leaves.

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.

Despite many studies of the percent loss of hydraulic conductivity in excised branches, there is doubt as to whether cutting stems in air introduces unnatural embolism into the xylem at the cut surface. To address this question, hydraulic conductivity was measured in seedlings of northern red oak (Quercus rubra L.) and rooted scions of eastern cottonwood (Populus deltoides Bartr. ex Marsh.) that had been droughted in pots. Results indicate that in situ dehydration produced a very similar vulnerability curve (% loss of conductivity versus water potential) to those previously obtained by bench-top dehydration of excised branches of eastern cottonwood and red oak. In eastern cottonwood cuttings, conductivity loss increased sharply below water potentials of -1.0 MPa, with 100% loss of conductivity occurring by -2.0 MPa, whereas conductivity loss in red oak seedlings was more gradual, i.e., increasing below -1.5 MPa and sustaining 100% loss of conductivity by about -4.0 MPa.

Oak (Quercus spp.) research and management often focus on northern red oak (Quercus rubra) and assume that associated upland oaks have similar growth patterns. To test this premise, we measured the survival and development of four species of acorn-origin oak seedlings growing in four different levels of understory sunlight for 8 years. Northern red oak had better survival than black (Quercus velutina), chestnut (Quercus montana), and white oak (Quercus alba) in 5% sunlight, but none of the species exhibited much growth. In 15 and 40% sunlight, survival was equal among species, but for growth the seedlings formed two groups with chestnut/northern red oak growing more than black/white oak. In 75% sunlight, survival was equal among species, but northern red oak grew faster than the other three species. Assuming that other oaks have growth habits similar to those of northern red oak could lead to a reduction in or the inadvertent loss of an oak species. Management and Policy Implications This study points out key differences in the survival and growth of black, chestnut, northern red, and white oak seedlings at the light levels created by a three-cut shelterwood sequence. Important management recommendations based on those differences include the following: In advance of an acorn crop, decrease dense shade to diffuse or partial shade (15–40% sunlight) by removing the midstory and understory canopies. If premasting shade control is not possible, begin regeneration treatments no later than 1–2 years after black, chestnut, and white oaks mast or no later than 3–5 years after northern red oak produces acorns. Conduct species-specific inventories for oak seedlings rather than grouping them together as oak reproduction. To emphasize chestnut or northern red oak, use the two-cut or three-cut shelterwood sequence because seedlings of these two oak species can grow reasonably well in 15–40% sunlight. To emphasize black or white oak, use a two-cut shelterwood because seedlings of these species need approximately 40% sunlight or more to initiate and sustain vigorous height growth.

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 resistance to cavitation is an important trait that is related to the ecology and survival of plant species. Vessel network characteristics, such as vessel length and connectivity, could affect the spread of emboli from gas-filled vessels to functional ones, triggering their cavitation. We hypothesized that the cavitation resistance of xylem vessels is randomly distributed throughout the vessel network. We predicted that single vessel air injection (SVAI) vulnerability curves (VCs) would thus be affected by sample length. Longer stem samples were predicted to appear more resistant than shorter samples due to the sampled path including greater numbers of vessels. We evaluated the vessel network characteristics of grapevine (Vitis vinifera L.), English oak (Quercus robur L.) and black cottonwood (Populus trichocarpa Torr. & A. Gray), and constructed SVAI VCs for 5- and 20-cm-long segments. We also constructed VCs with a standard centrifuge method and used computer modelling to estimate the curve shift expected for pathways composed of different numbers of vessels. For all three species, the SVAI VCs for 5 cm segments rose exponentially and were more vulnerable than the 20 cm segments. The 5 cm curve shapes were exponential and were consistent with centrifuge VCs. Modelling data supported the observed SVAI VC shifts, which were related to path length and vessel network characteristics. These results suggest that exponential VCs represent the most realistic curve shape for individual vessel resistance distributions for these species. At the network level, the presence of some vessels with a higher resistance to cavitation may help avoid emboli spread during tissue dehydration.

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.

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.

The hydraulic performance of woody species during drought is currently of high interest in the context of climate change. It is known that woody species have the capacity to mitigate water shortage by using internally stored water. Elastic shrinkage of living cells and also water release during cavitation contribute to the so-called 'hydraulic capacitance' (C) of the plant, which adds water to the transpiration stream and buffers fluctuations in water potential. Although sap-conducting conduits may ultimately serve as a water pool, cavitation will hamper the conduction of sap. Both hydraulic conductivity and C are thus inextricably linked and the interaction between both should be studied to better understand hydraulic functioning of woody species during drought. However, measurements of C are scarce and no distinction is usually made between C from elastic storage and C supplied by cavitation. In this paper, we propose a new method to assess both the decrease in hydraulic conductivity and the change in C during bench dehydration of a whole-branch segment using continuous measurements of acoustic emissions, radial diameter shrinkage and gravimetrical water loss. With this method we could establish proper vulnerability curves for grapevine (Vitis vinifera L. 'Johanniter') and quantify C during dehydration. Our results showed that loss in hydraulic conductivity during the cavitation phase was accompanied by 22-92% gain in hydraulic capacitance; therefore, a certain degree of cavitation may be tolerated in grapevine during periods of drought stress.

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.

Two upland sites in Arkansas were studied to test the performance of 1-0 northern red oak (Quercus rubra L.) and white oak (Quercus alba L.) seedlings planted in group selection openings. Both red and white oak seedlings were planted at one location in the Ozark Mountains,and only red oak seedlings were planted at a second site along Crowleys Ridge. Holes were dug with power augers and seedlings were planted by hand. At the time of planting, the mean height of red oak and white oak seedlings at the Ozark site were 3.4 and 1.9 ft, respectively. Red oak seedlingsat Crowleys Ridge averaged 3.0 ft tall when planted. After 4 years at the Ozark site, 77% of red oak and 86% of white oak were alive. After 3 years at Crowleys Ridge, red oak survival was 80%. Seedlings at both sites grew slowly. Mean 4-year height increment at the Ozarksite was 2.1 ft for red oak and 2.5 ft for white oak, and mean 3-year height increment for red oak at Crowleys Ridge was 1.6 ft. Three years after planting in the Ozark Mountains and 2 years after planting at Crowleys Ridge, naturally regenerating competition had suppressed over one-thirdof the red oak and about one-half of the white oak. This necessitated a release treatment around planted seedlings at both sites. Oaks that decreased in total height over a given growing season were common. Most seedlings that decreased in height had been pulled over or crushed by other vegetationor exhibited top dieback. South. J. Appl.For. 30(3):142–146.

The gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), is an introduced defoliator that preferentially feeds on oaks, Quercus spp. (Fagaceae) in the north‐eastern USA. As the gypsy moth expands its geographic range, the extensive oak component in forests and urban environments of the USA assure its successful establishment. Given their economic and ecological importance, and the gypsy moth's potential to cause mortality, we evaluated caterpillar preference and performance on various oaks prevalent in the central hardwoods region. Most of the physical and chemical characteristics we measured, from budbreak phenology to foliar chemistry, varied significantly among the oak species tested. Similarly, insect preference and performance varied significantly, though not always in predictable ways. Caterpillar preference was compared for black, Q. velutina Lamarck, burr, Q. macrocarpa Michaux, cherrybark, Q. pagoda Rafinesque, northern red, Q. rubra L., pin, Q. palustris Muenchhausen, swamp white, Q. bicolor Willdenow, white, Q. alba L., and willow, Q. phellos L., oaks. Gypsy moth preference was greatest for black and burr, and least for northern red, pin, and willow oaks. We assessed foliar characteristics and caterpillar performance on foliage from burr, cherrybark, northern red, pin, and willow oaks. Caterpillar preference did not always correlate with performance. Gypsy moth consumption and growth were highest, and development most rapid, on pin oak, which had high nitrogen and tannin levels, and was among the least preferred. Northern red and willow oaks were also among the least preferred and were the least suitable tested, producing caterpillars with moderate to low consumption and growth rates, as well as the longest development. Northern red oak contained the lowest foliar tannins; willow oak foliage was lowest in carbohydrates and nitrogen. Our results suggest that a combination of foliar characteristics may be responsible for gypsy moth preference and performance, and that an optimal combination of foliar components serves to maximize host suitability. These data will provide information useful for planning and managing urban forests in the presence of expanding gypsy moth populations.

Individual variation in acorn production by five species of southern Appalachian oaks

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.

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.

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.