A unit pipe pneumatic model to simulate gas kinetics during measurements of embolism in excised angiosperm xylem.

The pneumatic method has been introduced to quantify embolism resistance in plant xylem of various organs by applying a partial vacuum to cut-open xylem. Despite the similarity in vulnerability curves between the pneumatic and other methods, a modeling approach is needed to investigate if changes in xylem embolism during dehydration can be accurately quantified based on gas diffusion kinetics. Therefore, a unit pipe pneumatic (UPPn) model was developed to estimate gas extraction from intact conduits, which were axially interconnected by inter-conduit pit membranes to cut-open conduits. The physical laws used included Fick's law for diffusion, Henry's law for gas concentration partitioning between liquid and gas phases at equilibrium and the ideal gas law. The UPPn model showed that 91% of the extracted gas came from the first five series of embolized, intact conduits and only 9% from the aqueous phase after 15 s of simulation. Considering alternative gas sources, embolism resistance measured with a pneumatron device was systematically overestimated by 2-17%, which corresponded to a typical measuring error of 0.11 MPa for P50 (the water potential equivalent to 50% of the maximum amount of gas extracted). It is concluded that pneumatic vulnerability curves directly measure embolism of intact conduits due to the fast movement of gas across interconduit pit membranes, while gas extraction from sap and diffusion across hydrated cell walls is about 100 times slower. We expect that the UPPn model will also contribute to the understanding of embolism propagation based on temporal gas dynamics.

The theory behind vessel length determination using gas flow rates and comparison between two pneumatic methods based on seven woody species.

In plants, xylem vessel length is important for long-distance water transport; however, the currently used methods for vessel length measurement are inconvenient and time-consuming. The recently developed semi-automated Pneumatron is a device based on the pneumatic theory that is similar to the air-injection method, and can rapidly estimate vessel length. Mean vessel length was compared between the Pneumatron and the air-injection method in seven woody species with a wide range of vessel lengths (2.3-78.7 cm). The results were consistent between the two methods, regardless of whether the same or different samples were used. The theory underlying the gas flow in vessels was improved and expanded, and compared to that underlying the water flow in order to better understand the pneumatic processes within a stem sample. Moreover, a new and simple equation for gas flow in vessels was derived based on the molar gas flow (mol s-1) rather than volume flow, because the former remains constant with distance throughout the stem axis. We strongly recommend using the Pneumatron in future studies owing to its low cost, convenience, rapidity, and simple operation. However, a number of potential issues need to be considered to avoid artifacts during measurements.

How trees thrive in a dry climate: diurnal and seasonal hydrology and water relations in a riparian cottonwood grove.

In semi-arid ecoregions, trees are restricted to river valley floodplains where river water supplements the limited precipitation. To characterize the associated diurnal and seasonal dynamics in hydrology and water relations, we studied narrowleaf cottonwoods (Populus angustifolia) along a prairie river in Canada. From June through August, the shallow soil moisture was depleted but moisture remained higher above the alluvial groundwater table, which dropped to 2.3 m along with river recession. Throughout the summer, with the daily rise in temperature and insolation, foliar stomatal conductance (gs) and transpiration (E) increased to midday and then fell, thus maintaining the midday leaf water potential (Ψmd) above ~-1.7 MPa. This Ψmd approximated the water potential associated with 12% loss of xylem conductivity due to cavitation for branches (P12); the Ψmd and P12 varied independently across eight trees, providing differences in relative hydraulic risk. Sap flux density (Fd) was measured with thermal dissipation probes, and revealed diurnal patterns similar to foliar E. In contrast to our expectation, the daily Fd maxima were consistent through the summer despite the seasonal recession in water supply. Canopy conductances (GS), derived from Fd, sapwood area and canopy area, declined with vapor pressure deficit (D) and fell slightly in late summer along with stomatal sensitivity, which reflects the magnitude of decrease in GS with increasing D. For spatial up-scaling, satellite-derived near-infrared reflectance of vegetation revealed the woodland phenology, with leaf expansion from May through June and gradual decline in photosynthetic productivity through the summer. Thus, the phreatophytic cottonwoods: (i) sustained substantial water use and productivity through the warm and dry summer, by (ii) accessing shallow soil moisture and then deeper alluvial groundwater, and (iii) providing diurnal stomatal regulation, to (iv) avoid xylem cavitation and (v) maintain fairly constant hydraulic conductance.

The hydraulic architecture of an arborescent monocot: ontogeny-related adjustments in vessel size and leaf area compensate for increased resistance.

Bamboos are arborescent monocotyledons that have no secondary growth, but can continually produce conduits with diameters appropriate to the current size of the plant. Here, we studied bamboo hydraulic architecture to address the mechanisms involved in compensating for the increase in hydraulic resistance during ontogeny. We measured the hydraulic weighted vessel diameters (Dh ) at different distances from the apex along the stem of Bambusa textilis. The hydraulic resistance of different components and individuals of different heights were quantified using the high-pressure flowmeter method. The Dh showed tip-to-base widening with a scaling exponent in the range of those reported for trees. Although theoretical hydraulic conductivity decreased from base-to-tip, leaf-specific conductivity did not change. Leaves contributed the most to the whole-shoot hydraulic resistance, followed by the leaf-bearing branches. Roots contributed c. 13% to whole-plant resistance. Interestingly, taller individuals showed lower whole-shoot resistance owing to an increased number of resistances in parallel (side-branches), while leaf-specific resistance was independent of plant size. Tip-to-base vessel widening and height-independent constant leaf-specific conductance could be mechanisms for hydraulic optimization in B. textilis. Similar patterns have also been found in woody plants with secondary growth, but this bamboo exhibits them without secondary growth.

Inferring the role of pit membranes in solute transport from solute exclusion studies in living conifer stems.

The functional role of pits between living and dead cells has been inferred from anatomical studies but amassing physiological evidence has been challenging. Centrifugation methods were used to strip water from xylem conduits, permitting a more quantitative gravimetric determination of the water and solid contents of cell walls than is possible by more traditional methods. Quantitative anatomical evidence was used to evaluate the water volume in xylem conduits and the water content of living cells. Quantitative perfusion of stems with polyethylene glycol of different molecular weight was used to determine the solute-free space. We measured the portioning of water and solute-free space among anatomical components in stems and demonstrated that lignin impeded the free movement of solutes with molecular weight >300. Hence, movement of large solutes from living cells to xylem conduits is necessarily confined to pit structures that permit transmembrane solute transport via primary walls without lignin. The functional role of pits was additionally indicated by combining data in this paper with previous studies of unusual osmotic relationships in woody species that lack pits between dead wood fibers and vessels. The absence of pits, combined with the evidence of exclusion of solutes of molecular weight >300, explains the unexpected osmotic properties.

The Pneumatron: An automated pneumatic apparatus for estimating xylem vulnerability to embolism at high temporal resolution.

Xylem vulnerability to embolism represents an important trait to determine species distribution patterns and drought resistance. However, estimating embolism resistance frequently requires time-consuming and ambiguous hydraulic lab measurements. Based on a recently developed pneumatic method, we present and test the "Pneumatron", a device that generates high time-resolution and fully automated vulnerability curves. Embolism resistance is estimated by applying a partial vacuum to extract air from an excised xylem sample, while monitoring the pressure change over time. Although the amount of gas extracted is strongly correlated with the percentage loss of xylem conductivity, validation of the Pneumatron was performed by comparison with the optical method for Eucalyptus camaldulensis leaves. The Pneumatron improved the precision of the pneumatic method considerably, facilitating the detection of small differences in the (percentage of air discharged [PAD] < 0.47%). Hence, the Pneumatron can directly measure the 50% PAD without any fitting of vulnerability curves. PAD and embolism frequency based on the optical method were strongly correlated (r2 = 0.93) for E. camaldulensis. By providing an open source platform, the Pneumatron represents an easy, low-cost, and powerful tool for field measurements, which can significantly improve our understanding of plant-water relations and the mechanisms behind embolism.

An improved centrifuge method for determining water extraction curves and vulnerability curves in the long-vessel species Robinia pseudoacacia.

Significant improvements to the centrifuge water-extraction method of measuring the percentage loss volume of water (PLV) and corresponding vulnerability curves (VCs) are reported. Cochard and Sperry rotors are both incapable of measuring the VCs of species with long vessels because of premature embolism induced by hypothetical nanoparticles that can be drawn into segments during flow measurement. In contrast, water extraction pushes nanoparticles out of the sample. This study focuses on a long-vessel species, Robinia pseudoacacia, for which many VCs have been constructed by different methods, and the daily water relations have been quantified. PLV extraction curves have dual Weibull curves, and this paper focuses on the second Weibull curve because it involves the extraction of water from vessels, as proven by staining methods. We demonstrate an improved water extraction method after evaporation correction that has accuracy to within 0.5%, shows good agreement with two traditional methods that are slower and less accurate, and is immune to nanoparticle artefacts. Using Poiseuille's Law and the geometry of vessels, we argue that the percentage loss of conductivity (PLC) equals 2PLV-PLV2 in a special case where all vessels, regardless of size, have the same vulnerability curve. In this special case, this equation predicts the data reasonably well.

How does water flow from vessel to vessel? Further investigation of the tracheid bridge concept.

Hydraulic safety and efficiency have become the central concept of the interpretation of the structure and function of vessels and their interconnections. Plants form an appropriate xylem network structure to maintain a balance of hydraulic safety vs efficiency. The term 'tracheid bridge' is used to describe a possible pathway of water transport between neighboring vessels via tracheids, and this pathway could also provide increased safety against embolisms. However, the only physiological study of such a structure thus far has been in Hippophae rhamnoides Linn. To test the function of tracheid bridges, this research examined four species that have relatively long and solitary vessels, which are two of the criteria for efficient tracheid bridges. Tracheids contributed less than 2.2% of the total conductance of the vessels in these species, but in theory, tracheids could serve as very efficient transport connector pathways that may or may not make direct vessel-to-vessel contact via pit fields between adjacent vessels. In some species, tracheid bridges may represent the dominant pathway for water flow between vessels, whereas in other species, tracheid bridges may be sub-dominant or virtually nil. Broader searches of woody taxa are needed to reveal the functional importance of tracheid bridges in vascular plants.

Do nano-particles cause recalcitrant vulnerability curves in Robinia? Testing with a four-cuvette Cochard rotor and with water extraction curves.

Cavitation resistance is a key trait for characterizing the drought adaption in plants and is usually presented in terms of vulnerability curves. Three principal techniques have been developed to produce vulnerability curves, but curves generated with centrifugation are reported to suffer from artifacts when applied to long-vesseled species. The main cause of this artifact is the issue of open vessels, resulting in a nano-particle effect that may seed premature embolism. We used two methods to test the potential mechanism behind the nano-particle effect in centrifuge-based vulnerability curves. A four-cuvette rotor system based on a traditional Cochard rotor was designed to inhibit effervescence while injecting water, but the recalcitrant vulnerability curves in Robinia could not be eliminated. There may be multiple sources, besides effervescence, of hypothetical nano-particles: they may arise from cut surfaces or they may be always present in the injected water, leading to the premature embolisms. To prevent the entry of the hypothetical nano-particles, water extraction curves in terms of PLV (percentage loss volume of extracted water from stems) vs tensions were constructed. The PLV curves of Robinia showed s-shaped characteristics after subtracting the first Weibull components from water extraction curves, which were not related to the water loss from vessels according to dye staining experiments. The differences between T50 (xylem tension at which 50% of hydraulic conductivity is lost) in mean PLV curve and T50 in percentage loss of conductivity curves determined by the four-cuvette rotor system and by the bench dehydration method were 3.9 MPa and 0.7 MPa, respectively. Hence, PLV curves may be a valid way to measure the cavitation resistance in long-vesseled species with centrifugation. Keeping bark intact in the process of measurement is recommended, otherwise it would increase evaporation from the entire system.

Seasonality of cavitation and frost fatigue in Acer mono Maxim.

Although cavitation is common in plants, it is unknown whether the cavitation resistance of xylem is seasonally constant or variable. We tested the changes in cavitation resistance of Acer mono before and after a controlled cavitation-refilling and freeze-thaw cycles for a whole year. Cavitation resistance was determined from 'vulnerability curves' showing the percent loss of conductivity versus xylem tension. Cavitation fatigue was defined as a reduction of cavitation resistance following a cavitation-refilling cycle, whereas frost fatigue was caused by a freeze-thaw cycle. A. mono developed seasonal changes in native embolisms; values were relatively high during winter but relatively low and constant throughout the growing season. Cavitation fatigue occurred and changed seasonally during the 12-month cycle; the greatest fatigue response occurred during summer and the weakest during winter, and the transitions occurred during spring and autumn. A. mono was highly resistant to frost damage during the relatively mild winter months; however, a quite different situation occurred during the growing season, as the seasonal trend of frost fatigue was strikingly similar to that of cavitation fatigue. Seasonality changes in cavitation resistance may be caused by seasonal changes in the mechanical properties of the pit membranes.

Experimental evidence for negative turgor pressure in small leaf cells of Robinia pseudoacacia L versus large cells of Metasequoia glyptostroboides Hu et W.C.Cheng. 1. Evidence from pressure-volume curve analysis of dead tissue.

This paper provides a mini-review of evidence for negative turgor pressure in leaf cells starting with experimental evidence in the late 1950s and ending with biomechanical models published in 2014. In the present study, biomechanical models were used to predict how negative turgor pressure might be manifested in dead tissue, and experiments were conducted to test the predictions. The main findings were as follows: (i) Tissues killed by heating to 60 or 80 °C or by freezing in liquid nitrogen all became equally leaky to cell sap solutes and all seemed to pass freely through the cell walls. (ii) Once cell sap solutes could freely pass the cell walls, the shape of pressure-volume curves was dramatically altered between living and dead cells. (iii) Pressure-volume curves of dead tissue seem to measure negative turgor defined as negative when inside minus outside pressure is negative. (iv) Robinia pseudoacacia leaves with small palisade cells had more negative turgor than Metasequoia glyptostroboides with large cells. (v) The absolute difference in negative turgor between R. pseudoacacia and M. glyptostroboides approached as much as 1.0 MPa in some cases. The differences in the manifestation of negative turgor in living versus dead tissue are discussed.

Experimental evidence for negative turgor pressure in small leaf cells of Robinia pseudoacacia L versus large cells of Metasequoia glyptostroboides Hu et W.C. Cheng. 2. Höfler diagrams below the volume of zero turgor and the theoretical implication for pressure-volume curves of living cells.

The physiological advantages of negative turgor pressure, Pt , in leaf cells are water saving and homeostasis of reactants. This paper advances methods for detecting the occurrence of negative Pt in leaves. Biomechanical models of pressure-volume (PV) curves predict that negative Pt does not change the linearity of PV curve plots of inverse balance pressure, PB , versus relative water loss, but it does predict changes in either the y-intercept or the x-intercept of the plots depending on where cell collapse occurs in the PB domain because of negative Pt . PV curve analysis of Robinia leaves revealed a shift in the x-intercept (x-axis is relative water loss) of PV curves, caused by negative Pt of palisade cells. The low x-intercept of the PV curve was explained by the non-collapse of palisade cells in Robinia in the PB domain. Non-collapse means that Pt smoothly falls from positive to negative values with decreasing cell volume without a dramatic change in slope. The magnitude of negative turgor in non-collapsing living cells was as low as -1.3 MPa and the relative volume of the non-collapsing cell equaled 58% of the total leaf cell volume. This study adds to the growing evidence for negative Pt .

The impact of xylem cavitation on water potential isotherms measured by the pressure chamber technique in Metasequoia glyptostroboides Hu & W.C. Cheng.

Pressure-volume (PV) curve analysis is the most common and accurate way of estimating all components of the water relationships in leaves (water potential isotherms) as summarized in the Höfler diagram. PV curve analysis yields values of osmotic pressure, turgor pressure, and elastic modulus of cell walls as a function of relative water content. It allows the computation of symplasmic/apoplastic water content partitioning. For about 20 years, cavitation in xylem has been postulated as a possible source of error when estimating the above parameters, but, to the best of the authors' knowledge, no one has ever previously quantified its influence. Results in this paper provide independent estimates of osmotic pressure by PV curve analysis and by thermocouple psychrometer measurement. An anatomical evaluation was also used for the first time to compare apoplastic water fraction estimates from PV analysis with anatomical values. Conclusions include: (i) PV curve values of osmotic pressure are underestimated prior to correcting osmotic pressure for water loss by cavitation in Metasequoia glyptostroboides; (ii) psychrometer estimates of osmotic pressure obtained in tissues killed by freezing or heating agreed with PV values before correction for apoplastic water dilution; (iii) after correction for dilution effects, a solute concentration enhancement (0.27MPa or 0.11 osmolal) was revealed. The possible sources of solute enhancement were starch hydrolysis and release of ions from the Donnan free space of needle cell walls.

Stem Hydraulic Conductivity depends on the Pressure at Which It Is Measured and How This Dependence Can Be Used to Assess the Tempo of Bubble Pressurization in Recently Cavitated Vessels.

Cavitation of water in xylem vessels followed by embolism formation has been authenticated for more than 40 years. Embolism formation involves the gradual buildup of bubble pressure (air) to atmospheric pressure as demanded by Henry's law of equilibrium between gaseous and liquid phases. However, the tempo of pressure increase has not been quantified. In this report, we show that the rate of pressurization of embolized vessels is controlled by both fast and slow kinetics, where both tempos are controlled by diffusion but over different spatial scales. The fast tempo involves a localized diffusion from endogenous sources: over a distance of about 0.05 mm from water-filled wood to the nearest embolized vessels; this process, in theory, should take <2 min. The slow tempo involves diffusion of air from exogenous sources (outside the stem). The latter diffusion process is slower because of the increased distance of diffusion of up to 4 mm. Radial diffusion models and experimental measurements both confirm that the average time constant is >17 h, with complete equilibrium requiring 1 to 2 d. The implications of these timescales for the standard methods of measuring percentage loss of hydraulic conductivity are discussed in theory and deserve more research in future.

A comparison of two methods for measuring vessel length in woody plants.

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.

Studies on the tempo of bubble formation in recently cavitated vessels: a model to predict the pressure of air bubbles.

A cavitation event in a vessel replaces water with a mixture of water vapor and air. A quantitative theory is presented to argue that the tempo of filling of vessels with air has two phases: a fast process that extracts air from stem tissue adjacent to the cavitated vessels (less than 10 s) and a slow phase that extracts air from the atmosphere outside the stem (more than 10 h). A model was designed to estimate how water tension (T) near recently cavitated vessels causes bubbles in embolized vessels to expand or contract as T increases or decreases, respectively. The model also predicts that the hydraulic conductivity of a stem will increase as bubbles collapse. The pressure of air bubbles trapped in vessels of a stem can be predicted from the model based on fitting curves of hydraulic conductivity versus T. The model was validated using data from six stem segments each of Acer mono and the clonal hybrid Populus 84 K (Populus alba × Populus glandulosa). The model was fitted to results with root mean square error less than 3%. The model provided new insight into the study of embolism formation in stem tissue and helped quantify the bubble pressure immediately after the fast process referred to above.

Investigations concerning cavitation and frost fatigue in clonal 84K poplar using high-resolution cavitron measurements.

Both drought and freezing-thawing of stems induce a loss of hydraulic conductivity (percentage loss of conductivity [PLC]) in woody plants. Drought-induced PLC is often accompanied by physical damage to pit membranes, causing a shift in vulnerability curves (cavitation fatigue). Hence, if cavitated stems are flushed to remove embolisms, the next vulnerability curve is different (shifted to lower tensions). The 84K poplar (Populus alba × Populus glandulosa) clone has small vessels that should be immune from frost-induced PLC, but results demonstrated that freezing-thawing in combination with tension synergistically increased PLC. Frost fatigue has already been defined, which is similar to cavitation fatigue but induced by freezing. Frost fatigue caused a transition from a single to a dual Weibull curve, but drought-fatigued stems had single Weibull curves shifted to lower tensions. Studying the combined impact of tension plus freezing on fatigue provided evidence that the mechanism of frost fatigue may be the extra water tension induced by freezing or thawing while spinning stems in a centrifuge rather than direct ice damage. A hypothesis is advanced that tension is enhanced as ice crystals grow or melt during the freeze or thaw event, respectively, causing a nearly identical fatigue event to that induced by drought.

An improved sensor for precision detection of in situ stem water content using a frequency domain fringing capacitor.

One role of stems is that of water storage. The water content of stems increases and decreases as xylem water potential increases and decreases, respectively. Hence, a nondestructive method to measure stem water content (StWC) = (volume of water) : (volume of stem), could be useful in monitoring the drought stress status of plants. We introduce a frequency domain inner fringing capacitor-sensor for measuring StWC which operates at 100 MHz frequency. The capacitor-sensor consists of two wave guides (5-mm-wide braided metal) that snugly fit around the surface of a stem with a spacing of 4-5 mm between guides. Laboratory measurements on analog stems reveals that the DC signal output responds linearly to the relative dielectric constant of the analog stem, is most sensitive to water content between the waveguides to a depth of c. 3 mm from the stem surface, and calibrations based on the gravimetric water loss of excised stems of plants revealed a resolution in StWC of < ± 0.001 v/ v. The sensor performed very well on whole plants with a 100-fold increased resolution compared with previous frequency domain and time domain reflectometry methods and, hence, may be very useful for future research requiring nondestructive measurements of whole plants.

Pressure-volume curves: revisiting the impact of negative turgor during cell collapse by literature review and simulations of cell micromechanics.

The Scholander-Hammel pressure chamber has been used in thousands of papers to measure osmotic pressure, πc , turgor pressure, Pt , and bulk modulus of elasticity, ε, of leaf cells by pressure-volume (PV) curve analysis. PV analysis has been questioned in the past. In this paper we use micromechanical analysis of leaf cells to examine the impact on PV curve analysis of negative turgor in living cells (Pt ). Models predict negative Pt (-0.1 to -1.8 MPa) depending on leaf cell size and shape in agreement with experimental values reported by J. J. Oertli. Modeled PV curves have linear regions even when Pt is quite negative, contrary to the arguments of M.T. Tyree. Negative Pt is totally missed by PV curve analysis and results in large errors in derived πc and Pt but smaller errors in ε. A survey of leaf cell sizes vs habitat (arid, temperate, and rainforest), suggests that the majority of published PV curves result in errors of 0.1-1.8 MPa in derived πc and Pt , whereby the error increases with decreasing cell size. We propose that small cell size in leaves is an ecological adaptation that permits plants to endure negative values of water potential with relatively little water loss.