Refilling of a hydraulically isolated embolized xylem vessel: model calculations

When they are hydraulically isolated, embolized xylem vessels can be refilled, while adjacent vessels remain under tension. This implies that the pressure of water in the refilling vessel must be equal to the bubble gas pressure, which sets physical constraints for recovery. A model of water exudation into the cylindrical vessel and of bubble dissolution based on the assumption of hydraulic isolation is developed. Refilling is made possible by the turgor of the living cells adjacent to the refilling vessel, and by a reflection coefficient below 1 for the exchange of solutes across the interface between the vessel and the adjacent cells. No active transport of solutes is assumed. Living cells are also capable of importing water from the water-conducting vessels. The most limiting factors were found to be the osmotic potential of living cells and the ratio of the volume of the adjacent living cells to that of the embolized vessel. With values for these of 1.5 MPa and 1, respectively, refilling times were in the order of hours for a broad range of possible values of water conductivity coefficients and effective diffusion distances for dissolved air, when the xylem water tension was below 0.6 MPa and constant. Inclusion of the daily pattern for xylem tension improved the simulations. The simulated gas pressure within the refilling vessel was in accordance with recent experimental results. The study shows that the refilling process is physically possible under hydraulic isolation, while water in surrounding vessels is under negative pressure. However, the osmotic potentials in the refilling vessel tend to be large (in the order of 1 MPa). Only if the xylem water tension is, at most, twice atmospheric pressure, the reflection coefficient remains close to 1 (0.95) and the ratio of the volume of the adjacent living cells to that of the embolized vessel is about 2, does the osmotic potential stay below 0.4 MPa.     

The dynamics of gas bubbles in conduits of vascular plants and implications for embolism repair

Pressure-induced tensions in the xylem, the water conducting tissue of vascular plants, can lead to embolism in the water-conducting cells. The details and mechanisms of embolism repair in vascular plants are still not well understood. In particular, experimental results which indicate that embolism repair may occur during xylem tension cause great problems with respect to current paradigms of plant water transport. The present paper deals with a theoretical analysis of interfacial effects at the pits (pores in the conduit walls), because it was suggested that gas-water interfaces at the pit pores may be involved in the repair process by hydraulically isolating the embolized conduit. The temporal behaviour of bubbles at the pit pores was especially studied since the question of whether these pit bubbles are able to persist is of crucial importance for the suggested mechanism to work. The results indicate that (1) the physical preconditions which are necessary for the suggested mechanism appear to be satisfied, (2) pit bubbles can achieve temporal stability and therefore persist and (3) dissolving of bubbles in the conduit lumen may lead to the final breakdown of the hydraulic isolation. The whole process is, however, complex and strongly dependent on the detailed anatomy of the pit and the contact angle.     

The Significance of Pit Shape for Hydraulic Isolation of Embolized Conduits of Vascular Plants During Novel Refilling

During plant water transport, the water in the conducting tissue (xylem) is under tension. The system is then in a metastable state and prone to bubble development and subsequent embolism blocking further water transport. It has recently been demonstrated, that embolism can be repaired under tension (= novel refilling). A model (Pit Valve Mechanism = PVM) has also been suggested which is based on the development of a special meniscus in the pores (pits) between adjacent conduits. This meniscus is expected to be able to isolate embolized conduits from neighbouring conduits during embolism repair. In this contribution the stability of this isolating meniscus against perturbations is considered which inevitably occur in natural environments. It can be shown that pit shape affects the stability of PVM fundamentally in the case of perturbation. The results show that a concave pit shape significantly supports the stability of PVM. Concave pit shape should thus be of selective value for species practicing novel refilling.     

A model of bubble growth leading to xylem conduit embolism

The dynamics of a gas bubble inside a water conduit after a cavitation event was modeled. A distinction was made between a typical angiosperm conduit with a homogeneous pit membrane and a typical gymnosperm conduit with a torus-margo pit membrane structure. For conduits with torus-margo type pits pit membrane deflection was also modeled and pit aspiration, the displacement of the pit membrane to the low pressure side of the pit chamber, was found to be possible while the emboli was still small. Concurrent with pit aspiration, the high resistance to water flow out of the conduit through the cell walls or aspirated pits will make the embolism process slow. In case of no pit aspiration and always for conduits with homogeneous pit membranes, embolism growth is more rapid but still much slower than bubble growth in bulk water under similar water tension. The time needed for the embolism to fill a whole conduit was found to be dependent on pit and cell wall conductance, conduit radius, xylem water tension, pressure rise in adjacent conduits due to water freed from the embolising conduit, and the rigidity and structure of the pits in the case of margo-torus type pit membrane. The water pressure in the conduit hosting the bubble was found to occur almost immediately after bubble induction inside a conduit, creating a sudden tension release in the conduit, which can be detected by acoustic and ultra-acoustic monitoring of xylem cavitation.     

Mechanism of water stress-induced xylem embolism

We investigated the hypothesis that water stress-induced xylem embolism is caused by air aspirated into functional vessels from neighboring embolized ones (e.g. embolized by physical damage) via pores in intervessel pit membranes. The following experiments with sugar maple (Acer saccharum Marsh.) support the hypothesis. (a) Most vessels in dehydrating stem segments embolized at xylem pressures < −3 megapascals; at this point the pressure difference across intervessel pits between air-filled vessels at the segment's ends and internal water-filled vessels was >3 megapascals. This same pressure difference was found to be sufficient to force air across intervessel pits from air injection experiments of hydrated stem segments. This suggests air entry at pits is causing embolism in dehydrating stems. (b) Treatments that increased the permeability of intervessel pits to air injection also caused xylem to embolize at less negative xylem pressures. Permeability was increased either by perfusing stems with solutions of surface tension below that of water or by perfusion with a solution of oxalic acid and calcium. The mechanism of oxalic-calcium action on permeability is unknown, but may relate to the ability of oxalate to chelate calcium from the pectate fraction of the pit membrane. (c) Diameter of pores in pit membranes measured with the scanning electron microscope were within the range predicted by hypothesis (≤0.4 micrometer).     

Analysis of circular bordered pit function II. Gymnosperm tracheids with torus-margo pit membranes

A model of xylem conduit function was applied to gymnosperm tracheids with torus-margo pit membranes for comparison with angiosperm vessels. Tracheids from 17 gymnosperm tree species with circular bordered pits and air-seed pressures from 0.8 to 11.8 MPa were analyzed. Tracheids were more reinforced against implosion than vessels, consistent with their double function in transport and support. Tracheid pits were 3.3 to 44 times higher in hydraulic conductivity than vessel pits because of greater membrane conductivity of the torus-margo configuration. Tight scaling between torus and pit size maximized pit conductivity. Higher pit conductivity allowed tracheids to be 1.7-3.4 times shorter than vessels and still achieve 95% of their lumen-limited maximum conductivity. Predicted tracheid lengths were consistent with measured lengths. The torus-margo structure is important for maximizing the conductivity of the inherently length-limited tracheid: replacing the torus-margo membrane with a vessel membrane caused stem tracheid conductivity to drop by 41%. Tracheids were no less hydraulically efficient than vessels if they were long enough to reach their lumen-limiting conductivity. However, this may only be possible for lumen diameters below approximately 60-70 μm.     

Chorioallantoic placenta formation in the rat: II. Angiogenesis and maternal blood circulation in the mesometrial region of the implantation chamber prior to placenta formation.

Rat gestation sites were examined on days 7 through 9 of pregnancy by light microscopy and transmission and scanning electron microscopy to determine the extent of vascular modifications in the vicinity of the mesometrial part of the implantation chamber (mesometrial chamber). At a later time, the mesometrial chamber is, in conjunction with the uterine lumen, the site of chorioallantoic placenta formation. On day 7, in the vicinity of the mesometrial chamber, vessels derived from a subepithelial capillary plexus and venules draining the plexus were dilating. By early day 8, this network of thin-walled dilated vessels (sinusoids) was further enlarged and consisted primarily of hypertrophied endothelial cells with indistinct basal laminas. Sinusoids were frequently close to the mesometrial chamber's luminal surface which was devoid of epithelial cells but was lined by decidual cell processes and extracellular matrix. By late day 8, cytoplasmic projections of endothelial cells extended between healthy-appearing decidual cells and out onto the mesometrial chamber's luminal surface, and endothelial cells were sometimes found on the luminal surface indicating that endothelial cells were migrating. The presence of maternal blood cells in the mesometrial chamber lumen suggested that there was continuity between the chamber and blood-vessel lumens. On day 9, the mesometrial chamber was completely lined with hypertrophied endothelial cells, and sinusoid lumens were clearly continuous with the lumen of the mesometrial chamber. Mesometrial sinusoids and possibly the mesometrial chamber lumen were continuous with vessels in vicinity of the uterine lumen that were fed by mesometrial arterial vessels. Clearing of the mesometrial chamber lumen during perfusion fixation via the maternal vasculature indicated the patency of this luminal space and its confluence with mesometrial arterial vessels and sinusoids. The conceptus occupied an antimesometrial position in the implantation chamber on days 7 through 9, and it was not in direct contact with uterine tissues in the vicinity of the mesometrial chamber. These observations suggest that angiogenesis, not trophoblast invasion or decidual cell death, plays a major role in the opening of maternal vessels into the mesometrial chamber lumen before the formation of the chorioallantoic placenta.     

Possible causes for embolism repair in xylem

Wood sections of eight species of angiosperm and gymnosperm were made and observed under microscope. When a dehydrated section was rewet, the air inside its conduits contracted under the force of surface tension for several seconds to form elongated or spherical bubbles. The elongated bubbles in smaller conduits shortened till vanished. In addition, we also discorved that bubbles in larger conduits extended at first, then collapsed and disappeared; the bubbles outside conduits appeared gradualy or popped up in the field of view one after another; for some samples, they originated mainly from the cross sections of the wood rays. The smaller ones also collapsed and the larger ones grew up gradually. We suspected that air might transfer from the bubbles with short radii to those with large radii, both inside and outside conduits. The calculation of the amount of gas in all bubbles in a field of view supported our hypothesis. There are two possible mechanisms to explain the phenomena. First, based on the capillay equation, air can move from a smaller bubble to a larger one. Another reason is that the dissolving air from smaller bubbles can enter into the adjacent bubbles with larger curvature radii. Gas movement should obey the same rules in living plants. Therefore, we suggest that after cavitation events, instead of air moving from xylem into ambient atmosphere, two mechanisms could induce air to transfer from smaller conduits into larger conduits or the regions with lower pressures, leading the embolized conduits in the smaller conduits to repair. Furthermore, the differnce of values of contact angles in conduits might promote the refilling of embolism at lower xylem pressure.     

Vessel contents of leaves after excision - a test of Scholander's assumption

A test was attempted of the assumption that, when a leaf is cut, the xylem still contains water under tension beyond the first vessel cross walls. This assumption enabled Scholander to argue that the balance pressure in his pressure chamber measured the tension in water columns in the vessels before cutting. The numbers of embolized vessels were counted, after rapid freezing of petiole and midrib samples of sunflower leaves, in the cryo-scanning electron microscope. Counts were made on leaves still attached to the plant and at intervals after cutting from the plant (up to 16 min) during a short spring day's transpiration. The lengths of vessels in the leaves, measured by latex particle perfusion, showed that 8% of vessels in the mid-petioles and 0% in the midribs should be opened by cutting. The changing percentages of embolized vessels (E) with time showed that: (1) in intact plants E was close to zero until midday when it rose to ~40%, and then fell progressively to near zero by 1600; (2) in excised leaves there was no detectable change in E immediately after cutting, and, in all but two time courses, no change as large as the 8% of opened vessels within 16 min; (3) but briefly, when E was high (midday), it rose further after cutting to a plateau (_E = 30-40%) in 4 min. From this rate of emptying, the estimated maximum pressure difference between vessels and parenchyma was of the order of 0.05-0.2 MPa (0.5 to 2 bar) at this time. (4) All these changes occurred in the petioles 1 h before they were found in the midribs. The test failed because the expected large pressure difference between vessels and parenchyma was not present. Further, the embolized vessels were refilled at the time of peak transpiration, which would be impossible with any substantial tension in the vessels. Because these results contradict the whole basis of the Cohesion Theory, a second experiment was carried out to test them, and is reported in a companion paper.     

Penetration of stomata by liquids: dependence on surface tension, wettability, and stomatal morphology

Wettability of the leaf surface, surface tension of the liquid, and stomatal morphology control penetration of stomata by liquids. The critical surface tension of the lower leaf surface of Zebrina purpusii Brückn. was estimated to be 25 to 30 dyne cm⁻¹. Liquids having a surface tension less than 30 dyne cm⁻¹ gave zero contact angle on the leaf surface and infiltrated stomata spontaneously while liquids having a surface tension greater than 30 dyne cm⁻¹ did not wet the leaf surface and failed to infiltrate stomata. Considering stomata as conical capillaries, we were able to show that with liquids giving a finite contact angle, infiltration depended solely on the relationship between the magnitude of the contact angle and the wall angle of the aperture. Generally, spontaneous infiltration of stomata will take place when the contact angle is smaller than the wall angle of the aperture wall. The degree of stomatal opening (4, 6, 8, or 10 μm) was of little importance. Cuticular ledges present at the entrance to the outer vestibule and between the inner vestibule and substomatal chamber resulted in very small if not zero wall angles, and thus played a major role in excluding water from the intercellular space of leaves. We show why the degree of stomatal opening cannot be assessed by observing spontaneous infiltration of stomata by organic liquids of low surface tension.     

Do Woody Plants Operate Near the Point of Catastrophic Xylem Dysfunction Caused by Dynamic Water Stress? : Answers from a Model

We discuss the relationship between the dynamically changing tension gradients required to move water rapidly through the xylem conduits of plants and the proportion of conduits lost through embolism as a result of water tension. We consider the implications of this relationship to the water relations of trees. We have compiled quantitative data on the water relations, hydraulic architecture and vulnerability of embolism of four widely different species: Rhizophora mangle, Cassipourea elliptica, Acer saccharum, and Thuja occidentalis. Using these data, we modeled the dynamics of water flow and xylem blockage for these species. The model is specifically focused on the conditions required to generate `runaway embolism,' whereby the blockage of xylem conduits through embolism leads to reduced hydraulic conductance causing increased tension in the remaining vessels and generating more tension in a vicious circle. The model predicted that all species operate near the point of catastrophic xylem failure due to dynamic water stress. The model supports Zimmermann's plant segmentation hypothesis. Zimmermann suggested that plants are designed hydraulically to sacrifice highly vulnerable minor branches and thus improve the water balance of remaining parts. The model results are discussed in terms of the morphology, hydraulic architecture, eco-physiology, and evolution of woody plants.     

Architecture of Branch-root Junctions in Maize: Structure of the Connecting Xylem and the Porosity of Pit Membranes

The architecture of the connecting xylem network in the vascularplexus linking branch and main root vessels has been examinedusing cryoSEM, and the limiting porosity of the network determinedwith tracers (dye, and particles of known size: latex, polystyreneand gold sols). Dye and water move freely throughout the xylemnetwork, while particles are constrained to follow tortuousvessel-like conduits of irregularly-shaped elements linked bylarge-diameter perforations. These conduits end at special pitmembranes (boundary pit membranes) at the periphery of mainroot vessels. Particles accumulate on the outer side of thesefilters, often filling the terminal elements of these conduitsadjacent to the main root vessels. Some vessel elements withinthe plexus are isolated from the convoluted conduits by normalpit membranes, and often also from each other, by pit membranesand still-intact end walls in otherwise mature elements. Theseextra-conduit elements may be an auxiliary filtering system.The boundary pit membranes filtered out particles with meandiameters as small as 4.9 ± 0.7 nm, indicating a poresize one or two orders of magnitude smaller than most previousmeasurements for pit membranes, but close to pore sizes determinedfor hydrated primary cell walls. It is concluded that boundarypit membranes at branch-root junctions are efficient filtersfor microbes and particulates entering damaged branch roots.They would also restrict entry of air/water interfaces whenmain root xylem tension was less than approx. 100 MPa. Copyright2000 Annals of Botany Company Zea mays, air-seeding, branch-root junction, cryoSEM, embolisms, maize, pit membrane, pore size, xylem, water transport     

The Dynamics of Embolism Repair in Xylem: In Vivo Visualizations Using High-Resolution Computed Tomography

Water moves through plants under tension and in a thermodynamically metastable state, leaving the nonliving vessels that transport this water vulnerable to blockage by gas embolisms. Failure to reestablish flow in embolized vessels can lead to systemic loss of hydraulic conductivity and ultimately death. Most plants have developed a mechanism to restore vessel functionality by refilling embolized vessels, but the details of this process in vessel networks under tension have remained unclear for decades. Here we present, to our knowledge, the first in vivo visualization and quantification of the refilling process for any species using high-resolution x-ray computed tomography. Successful vessel refilling in grapevine (Vitis vinifera) was dependent on water influx from surrounding living tissue at a rate of 6 × 10⁻⁴ μm s⁻¹, with individual droplets expanding over time, filling vessels, and forcing the dissolution of entrapped gas. Both filling and draining processes could be observed in the same vessel, indicating that successful refilling requires hydraulic isolation from tensions that would otherwise prevent embolism repair. Our study demonstrates that despite the presence of tensions in the bulk xylem, plants are able to restore hydraulic conductivity in the xylem.Vascular plants have evolved a simple but elegant system for long-distance transport of water and minerals through a network of nonliving, pipe-like cells. Whereas long-distance transport in animals is actively driven by positive pressure, most water transport in plants is passively driven by tension as explained by the Cohesion-Tension (C-T) theory (Dixon and Joly, 1894; Tyree, 2003). Water under tension is metastable however (Hayward, 1971), making the transport system inherently vulnerable to cavitation and blockage by gas embolisms (Tyree and Sperry, 1989). Direct measurements of negative pressures (tensions) in xylem (Wei et al., 1999) have confirmed the fundamental basis for the C-T theory of water transport in plants (e.g. Tyree, 2003), but many details regarding the susceptibility of the xylem network to cavitation and blockage by embolisms, and a thermodynamically plausible mechanism for the repair of these embolisms, remain unclear (Clearwater and Goldstein, 2005).Plants have apparently evolved mechanisms, including root pressure, to remove embolisms and restore water transport in vessels (Sperry et al., 1987; Tibbetts and Ewers, 2000; Isnard and Silk, 2009). Refilling of embolized vessels far from roots (Holbrook et al., 2001) and under a state of tension (Salleo and Gullo, 1986) is not well understood, but most hypotheses involve localized solute export into embolized vessels from adjacent living xylem parenchyma, osmotic movement of water into these vessels, and isolation of the refilling vessel from the tension in its local water environment (Tyree et al., 1999; Hacke and Sperry, 2003; Clearwater and Goldstein, 2005; Salleo et al., 2006). Embolism repair is complicated by the fact that xylem conduits (tracheids and vessels) form an interconnected network. While such a network will provide a low-resistance pathway for the bulk flow of water when the conduits are filled, if a cavitation event and subsequent embolism (gas bubble) either spontaneously occurs within a conduit, or spreads to it from another conduit, the presence of tension in this network should also quickly drain a conduit of its water and prevent its refilling. The spread of embolisms is limited by the small effective pore size of the connections between conduits (known as pit membranes), but under conditions of low plant water availability, embolisms do occur and spread (Tyree and Zimmermann, 2002; Choat et al., 2008), and evidence for the repair of embolized vessels, despite the presumed presence of a tension throughout the plant xylem, has been obtained in many species (Salleo et al., 1996; McCully et al., 1998; Zwieniecki and Holbrook, 1998; Kaufmann et al., 2009).A major limitation to the testing of these hypotheses and to our understanding of embolism repair has been the lack of in vivo observations at a sufficient resolution and an appropriate temporal scale to document how the refilling occurs. Here we present a new method for imaging the functional status of vessels using high-resolution x-ray computed tomography (HRCT), providing, to our knowledge, the first in vivo visualization of the refilling process for any species. Previous in vivo measurements of vessel refilling have been performed using NMR imaging, but the resolution was insufficient to determine the source of the refilling water (Holbrook et al., 2001; Scheenen et al., 2007). In vivo imaging at this scale allows for nondestructive visualization and measurement of the change in both air and water volume within the vessel lumen, giving unprecedented access to the mechanisms of embolism repair.     

Changes of zero-stress state of rat pulmonary arteries in hypoxic hypertension

Zero-stress state of the main pulmonary arteries, from the main trunk to a vessel with a lumen diameter approximately 60 microns, was determined in 25 normal control and 38 hypoxic pulmonary hypertensive rats. Pulmonary hypertension was induced by placing the rats in a hypoxic chamber with 10% O2-90% N2 at atmospheric pressure. The zero-stress state of each vessel was obtained by first cutting the vessel transversely into a series of rings and then cutting each ring radially, whereupon the ring opened into a sector, which is characterized by an opening angle defined as the angle subtended between two lines originating from the midpoint of the inner wall (endothelium) to the tips of the inner wall. Whereas the pulmonary blood pressure increased monotonically during the development of pulmonary hypertension, the opening angle followed a different course; e.g., the values (means +/- SD) of the opening angle at the pulmonary trunk at times 0 (control) and 2, 12, 28, 96, 144, 240, 480, and 720 h after exposure to hypoxia are, respectively, 294 +/- 30 degrees, 378 +/- 24 degrees, 385 +/- 12 degrees, 374 +/- 11 degrees, 246 +/- 63 degrees, 267 +/- 49 degrees, 193 +/- 19 degrees, 195 +/- 83 degrees, and 239 +/- 38 degrees. Trends at other places on the artery are similar, but the magnitudes differ. In this period of time, intimal edema and thickening were found. The intima media thickened rapidly from 48 to 240 h and then more slowly from 240 to 720 h. Adventitia thickened later; its thickness exceeded that of the intima media at approximately 96 h. Thus the changes of zero-stress state of the pulmonary arteries are seen to be related to the nonuniform remodeling of the vessel wall as revealed by the edema, blebs, and thickening of different layers.     

Induction of air embolism in xylem conduits of pre-defined diameter.

A new method is presented that enables the induction of embolisms in a fraction of all xylem vessels, based on diameter, at one cut end of a stem segment. The method is based on the different capillary characteristic of xylem vessels of different cross-sectional size. To verify the method, air embolisms were induced in cut xylem vessels of chrysanthemum (Dendranthemaxgrandiflorum Tzvelev cv. Cassa) stem segments at different xylem tensions and compared with the distribution of gas-filled vessels as visualized by cryo-scanning electron microscopy (Cryo-SEM). At -6 kPa xylem pressure, air-entrance was only induced in large diameter vessels (>30 microm), while at -24 kPa embolisms were induced in almost all xylem vessels (>10 microm). Although the principle of the embolization method worked well, smaller diameter vessels were observed to be embolized than was expected according to the calculations. The role of cross-sectional shape and contact angle between xylem sap and vessel wall at the menisci are discussed. After correction for the observed (diameter independent) deviation from circularity of the cross-sectional vessel shape the contact angle was calculated to be approximately 55 degrees. Hydraulic resistance (Rh) measurements before and after embolization showed that the effect of embolizing only large diameter cut xylem vessels had only a small influence on overall Rh of a stem segment. Embolizing all cut xylem vessels at one cut end almost trebled overall Rh. The difference was discussed in the light of the networking capacity of the xylem system.     

Direct measurements of intervessel pit membrane hydraulic resistance in two angiosperm tree species

The hydraulic resistance of pit membranes was measured directly in earlywood vessels of Fraxinus americana and Ulmus americana. The area-specific resistance of pit membranes (r(mem)) was higher than modeled or measured values obtained previously for hardwood species, with r(mem) of 5.24 × 10(3) MPa·s·m(-1) for Fraxinus and 2.56 × 10(3) MPa·s·m(-1) for Ulmus. The calculated resistance of pit canals was three orders of magnitude below total pit resistance indicating that pit membranes contributed the majority of resistance. Scanning electron microscopy indicated that pit membranes of Ulmus were thinner and more porous than those of Fraxinus, consistent with the difference in r(mem) between the species. Measurements of average vessel diameter and length and area of wall overlap with neighboring vessels were used to partition the vascular resistance between vessel lumen and pit membrane components. Pit membrane resistance accounted for 80% of the total resistance in Fraxinus and 87% in Ulmus in 2-yr-old branch sections. However, measurements of vessel dimensions in the trunk suggest that the division of resistance between pit membrane and lumen components would be closer to co-limiting in older regions of the tree. Thus, pit membrane resistance may be of greater relative importance in small branches than in older regions of mature trees.     

Easy Come,Easy Go: Capillary Forces Enable Rapid Refilling of Embolized Primary Xylem Vessels

Protoxylem plays an important role in the hydraulic function of vascular systems of both herbaceous and woody plants, but relatively little is known about the processes underlying the maintenance of protoxylem function in long-lived tissues. In this study, embolism repair was investigated in relation to xylem structure in two cushion plant species, Azorella macquariensis and Colobanthus muscoides, in which vascular water transport depends on protoxylem. Their protoxylem vessels consisted of a primary wall with helical thickenings that effectively formed a pit channel, with the primary wall being the pit channel membrane. Stem protoxylem was organized such that the pit channel membranes connected vessels with paratracheal parenchyma or other protoxylem vessels and were not exposed directly to air spaces. Embolism was experimentally induced in excised vascular tissue and detached shoots by exposing them briefly to air. When water was resupplied, embolized vessels refilled within tens of seconds (excised tissue) to a few minutes (detached shoots) with water sourced from either adjacent parenchyma or water-filled vessels. Refilling occurred in two phases: (1) water refilled xylem pit channels, simplifying bubble shape to a rod with two menisci; and (2) the bubble contracted as the resorption front advanced, dissolving air along the way. Physical properties of the protoxylem vessels (namely pit channel membrane porosity, hydrophilic walls, vessel dimensions, and helical thickenings) promoted rapid refilling of embolized conduits independent of root pressure. These results have implications for the maintenance of vascular function in both herbaceous and woody species, because protoxylem plays a major role in the hydraulic systems of leaves, elongating stems, and roots.There is a pressing need to understand how plants manage the maintenance of water transport from roots through leaves under changing environmental conditions (Allen et al., 2010; Choat et al., 2012). The problem arises because water is transported through the xylem under tension (i.e. under negative absolute pressure). As tension increases, conduits become increasingly vulnerable to cavitation, which causes the conduits to lose their ability to transport water. Conduits can become embolized during normal diurnal function as a result of tensions induced by transpiration and in response to environmental conditions such as drought or freezing stress (Zimmermann and Tyree, 2002). Vulnerability to cavitation and embolism formation suggests that plants have mechanisms to regain lost hydraulic capacity, either through the formation of new conduits or by refilling embolized ones.The vulnerability of conduits to embolisms and the capacity for repair are related to the structural diversity of xylem tissue (Zwieniecki and Holbrook, 2009; Lens et al., 2011; Cai et al., 2014). In vascular plants, the classification of xylem tissues depends on the meristem that produced them (Evert and Eichhorn, 2006). Primary xylem is produced by apical meristems and includes both protoxylem and metaxylem conduits, which are distinguished by their wall structure and the timing of their development. Protoxylem matures during organ elongation, which results in loss of function due to stretching in some tissues and species, while in many others, functionality is maintained throughout the life of the organ. In contrast, metaxylem matures in elongated tissue. In herbaceous plants, primary xylem is the major hydraulic system of the roots, stems, and leaves. In woody plants, the primary xylem remains the main hydraulic system of the leaves, while the radial growth of stems occurs through the activity of a vascular cambium, which produces secondary xylem with only metaxylem conduits. As a woody plant grows, the secondary xylem (and hence the metaxylem) thus becomes of increasing importance to stem hydraulic function. However, protoxylem remains an integral component of the plant hydraulic system due to its function in leaves and elongating stems and roots.As discussed in a recent review (Brodersen and McElrone, 2013), refilling of embolized vessels has been shown to depend on the generation of positive pressure by roots in many monocots, herbaceous plants, and a few woody species. However, many species lack root pressure; thus, attention has focused on so-called novel refilling, which involves adjacent living cells in the repair of embolized metaxylem or secondary xylem in stems of mature plants. Novel refilling has been studied with a variety of methods to visualize temporal variation in the presence and subsequent absence of embolized vessels, including cryo-scanning electron microscopy (Cryo-SEM; Canny, 1997; McCully et al., 2014), double staining (Zwieniecki and Holbrook, 1998; Zwieniecki et al., 2000), NMR imaging (Holbrook et al., 2001; Zwieniecki et al., 2013), and high-resolution x-ray computed tomography (Lee and Kim, 2008; Brodersen et al., 2010; Kim and Lee, 2010; Lee et al., 2013; Suuronen et al., 2013). These observations, in combination with other measurements, led to a working hypothesis of an osmotically driven repair mechanism in which sugars pumped into embolized vessels by adjacent paratracheal parenchyma provide the osmotic pressure difference that refills the vessel (Nardini et al., 2011).Little is known about embolism and its repair in protoxylem, which has structural features that make it potentially more vulnerable to embolism than metaxylem in the same plant or tissue (Choat et al., 2005). These include a greater exposed area of the primary cell wall with annular or helical thickenings instead of secondary walls. This could enhance stretching of the primary wall when large pressure differences develop between functional and embolized vessels, thereby decreasing the pressure required for air seeding of bubbles (Choat et al., 2004). Choat et al. (2005) suggested that greater vulnerability of protoxylem to embolism might underpin the roles of petioles, leaves, and small stems in the hydraulic segmentation hypothesis of Zimmermann (1983), in which sacrifice of the most easily replaceable tissues protects the function of the main structure of a plant during water stress. If ease of protoxylem embolism were to contribute to the function of hydraulic fuses during mild water stress, then ease of refilling would be required to rapidly reset the system.This study focuses on embolism repair in two distantly related, vascular species, Azorella macquariensis (Apiaceae) and Colobanthus muscoides (Caryophyllaceae), that depend exclusively on protoxylem for vascular water transport. Both species form cushions, with the former being an endemic, keystone species in the alpine zone of subantarctic Macquarie Island and the latter being a regional endemic that plays a major role in rocky coastal areas often within the supralittoral zone (Selkirk et al., 1990; Orchard, 1993). Both species are of ecological interest, because the subantarctic region is under increasing threat from climate change (Adams, 2009). Specifically, the climate on Macquarie Island is progressively changing from one that is perpetually wet and misty to one with increased exposure to periodic drying (Bergstrom et al., 2015). Dieback of alpine vegetation was first observed in 2008, and by 2010, extensive and unprecedented decline of A. macquariensis led to its listing as critically endangered (Bricher et al., 2013).In this study, protoxylem structure was studied in relation to embolism repair. Refilling of gas-filled vessels was compared between excised tissue and that in intact, detached shoots. The results showed that the physical properties of the protoxylem facilitated refilling by capillary forces and that rapid refilling in detached shoots supplied with water occurred without root pressure.     

The spatial pattern of air seeding thresholds in mature sugar maple trees

Air seeding threshold (P_a) of xylem vessels from current year growth rings were measured along the vertical axis of mature sugar maple trees (Acer saccharum Marsh.), with sampling points in primary leaf veins, petioles, 1-, 3-, and 7-year-old branches, large branches, the trunk and roots. The air seeding threshold was taken as the pressure required to force nitrogen gas through intervessel pit membranes. Although all measurements were made on wood produced in the same year, P_a varied between different regions of A. saccharum, with distal organs such as leaves and petioles having lower P_a than basal regions. Mean (SE) P_a ranged from 1.0 (± 0.1) MPa in primary leaf veins to 4.8 (± 0.1) MPa in the main trunk. Roots exhibited a P_a of 2.8 (± 0.2) MPa, lower than all other regions of the tree except leaf veins and petioles. Mean xylem vessel diameter increased basipetally, with the widest vessels occurring in the trunk and roots. Within the shoot, wider vessels had greater air seeding thresholds, contrasting with trends previously reported. However, further experimentation revealed that differences in P_a between regions of the stem were driven by the presence of primary xylem conduits, rather than differences in vessel diameter. In 1-year-old branches, P_a was significantly lower in primary xylem vessels than in adjacent secondary xylem vessels. This explained the lower values of P_a measured in petioles and leaf veins, which possessed a greater ratio of primary xylem to secondary xylem than other regions. The difference in P_a between primary and secondary xylem was attributed to the greater area of primary cell wall (pit membrane) exposed in primary xylem conduits with helical or annular thickening.     

Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers.

On days 7 and 8 of pregnancy, mesometrial regions of rat gestation sites were examined by light microscopy and transmission electron microscopy to determine what changes occur before the chorioallantoic placenta forms in that region. By day 7, gestation sites contained a uterine lumen mesometrially and an antimesometrial extension of the uterine lumen, the implantation chamber. The implantation chamber consisted of a mesometrial chamber between the uterine lumen and the conceptus, an antimesometrial chamber that contained the conceptus, and a decidual crypt antimesometrial to the conceptus. Stromal cells that formed the walls of the implantation chamber were closely packed decidual cells, while those that surrounded the uterine lumen were loosely arranged. Late on day 7, a portion of the epithelium lining the mesometrial chamber was degenerating, but this area of initial degeneration was never adjacent to the antimesometrial chamber. By early day 8, most of the epithelial cells lining the mesometrial chamber were degenerating and were being sloughed into the chamber lumen. Although degeneration of these epithelial cells morphologically resembled necrosis, it was precisely controlled, since adjacent epithelial cells lining the uterine lumen remained healthy. The space that separated the denuded luminal surface of the mesometrial chamber from underlying decidual cells became wider and was occupied by an extracellular matrix rich in cross-banded collagen fibrils. Decidual cell processes, that earlier had penetrated the basal lamina beneath healthy epithelial cells, protruded into this matrix and penetrated the basal lamina at the luminal surface. By late day 8, large areas of denuded chamber wall were covered with decidual cell processes, little remained of the basal lamina, and cross-banded collagen fibrils were scarce in the area occupied by decidual cell processes. During the times studied, uterine tissues that formed the walls of the mesometrial chamber were not in direct contact with the conceptus. This study indicates that trophoblast does not play a direct role in epithelial degeneration, basal lamina penetration, or extracellular matrix modifications in the mesometrial region of implantation chambers where part of the chorioallantoic placenta forms, although trophoblast may be required to trigger or modulate some of the changes.     

From the sap's perspective: The nature of vessel surfaces in angiosperm xylem

Premise of the Study

Xylem sap in angiosperms moves under negative pressure in conduits and cell wall pores that are nanometers to micrometers in diameter, so sap is always very close to surfaces. Surfaces matter for water transport because hydrophobic ones favor nucleation of bubbles, and surface chemistry can have strong effects on flow. Vessel walls contain cellulose, hemicellulose, lignin, pectins, proteins, and possibly lipids, but what is the nature of the inner, lumen‐facing surface that is in contact with sap?

Methods

Vessel lumen surfaces of five angiosperms from different lineages were examined via transmission electron microscopy and confocal and fluorescence microscopy, using fluorophores and autofluorescence to detect cell wall components. Elemental composition was studied by energy‐dispersive X‐ray spectroscopy, and treatments with phospholipase C (PLC) were used to test for phospholipids.

Key Results

Vessel surfaces consisted mainly of lignin, with strong cellulose signals confined to pit membranes. Proteins were found mainly in inter‐vessel pits and pectins only on outer rims of pit membranes and in vessel‐parenchyma pits. Continuous layers of lipids were detected on most vessel surfaces and on most pit membranes and were shown by PLC treatment to consist at least partly of phospholipids.

Conclusions

Vessel surfaces appear to be wettable because lignin is not strongly hydrophobic and a coating with amphiphilic lipids would render any surface hydrophilic. New questions arise about these lipids and their possible origins from living xylem cells, especially about their effects on surface tension, surface bubble nucleation, and pit membrane function.