Root xylem embolisms and refilling. Relation To water potentials of soil, roots, and leaves, and osmotic potentials of root xylem Sap

Embolism and refilling of vessels was monitored directly by cryomicroscopy of field-grown corn (Zea mays L.) roots. To test the reliability of an earlier study showing embolism refilling in roots at negative leaf water potentials, embolisms were counted, and root water potentials (Psiroot) and osmotic potentials of exuded xylem sap from the same roots were measured by isopiestic psychrometry. All vessels were full at dawn (Psiroot -0.1 MPa). Embolisms were first seen in late metaxylem vessels at 8 AM. Embolized late metaxylem vessels peaked at 50% at 10 AM (Psiroot -0.1 MPa), fell to 44% by 12 PM (Psiroot -0.23 MPa), then dropped steadily to zero by early evening (Psiroot -0.28 MPa). Transpiration was highest (8.5 μg cm-2 s-1) between 12 and 2 PM when the percentage of vessels embolized was falling. Embolized vessels were refilled by liquid moving through their lateral walls. Xylem sap was very low in solutes. The mechanism of vessel refilling, when Psiroot is negative, requires further investigation. Daily embolism and refilling in roots of well-watered plants is a normal occurrence and may be a component of an important hydraulic signaling mechanism between roots and shoots.     

In vivo observation of cavitation and embolism repair using magnetic resonance imaging

Magnetic resonance imaging (MRI) was used to noninvasively monitor the status of individual xylem vessels in the stem of an intact, transpiring grape (Vitis vinifera) plant over a period of approximately 40 h. Proton density-weighted MRI was used to visualize the distribution of mobile water in the stem and individual xylem vessels were scored as either water or gas filled (i.e. embolized). The number of water-filled vessels decreased during the first 24 h of the experiment, indicating that approximately 10 vessels had cavitated during this time. Leaf water potentials decreased from -1.25 to -2.1 MPa during the same period. Watering increased leaf water potentials to -0.25 MPa and prevented any further cavitation. Refilling of xylem vessels occurred as soon as the lights were switched off, with the majority of vessels becoming refilled with water during the first 2 to 3 h in darkness. These measurements demonstrate that MRI can be used to monitor the functional status of individual xylem vessels, providing the first method to study the process of cavitation and embolism repair in intact plants.     

Some properties of the walls of metaxylem vessels of maize roots,including tests of the wettability of their lumenal wall surfaces

Background and Aims

Since the proposal of the cohesion theory there has been a paradox that the lumenal surface of vessels is rich in hydrophobic lignin, while tension in the rising sap requires adhesion to a hydrophilic surface. This study sought to characterize the strength of that adhesion in maize (Zea mays), the wettability of the vessel surface, and to reconcile this with its histochemical and physical nature.

Methods

Wettability was assessed by emptying the maize root vessels of sap, perfusing them with either water or oil, and examining the adhesion (as revealed by contact angles) of the two liquids to vessel walls by cryo-scanning electron microscopy. The phobicity of the lumenal surface was also assessed histochemically with hydrophilic and hydrophobic probes.

Key Results

Pit borders in the lumen-facing vessel wall surface were wetted by both sap/water and oil. The attraction for oil was weaker: water could replace oil but not vice versa. Pit apertures repelled oil and were strongly stained by hydrophilic probes. Pit chambers were probably hydrophilic. Oil never entered the pits. When vessels were emptied and cryo-fixed immediately, pit chambers facing away from the vessels were always sap-filled. Pit chambers facing vessel lumens were either sap- or gas-filled. Sap from adjoining tracheary elements entering empty vessels accumulated on the lumenal surface in hemispherical drops, which spread out with decreasing contact angles to fill the lumen.

Conclusions

The vessel lumenal surface has a dual nature, namely a mosaic of hydrophilic and hydrophobic patches at the micrometre scale, with hydrophilic predominating. A key role is shown, for the first time, of overarching borders of pits in determining the dual nature of the surface. In gas-filled (embolized) vessels they are hydrophobic. When wetted by sap (vessels refilling or full) they are hydrophilic. A hypothesis is proposed to explain the switch between the two states.     

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.     

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.     

Capacitive effect of cavitation in xylem conduits: results from a dynamic model

Embolisms decrease plant hydraulic conductance and therefore reduce the ability of the xylem to transport water to leaves provided that embolized conduits are not refilled. However, as a xylem conduit is filled with gas during cavitation, water is freed to the transpiration stream and this transiently increases xylem water potential. This capacitive effect of embolism formation on plant function has not been explicitly quantified in the past. A dynamic model is presented that models xylem water potential, xylem sap flow and cavitation, taking into account both the decreasing hydraulic conductance and the water release effect of xylem embolism. The significance of the capacitive effect increases in relation to the decreasing hydraulic conductance effect when transpiration rate is low in relation to the total amount of water in xylem conduits. This ratio is typically large in large trees and during drought.     

Vulnerability to cavitation differs between current‐year and older xylem: non‐destructive observation with a compact magnetic resonance imaging system of two deciduous diffuse‐porous species

Development of xylem embolism during water stress in two diffuse‐porous hardwoods, Katsura (Cercidiphyllum japonicum) and Japanese white birch (Betula platyphylla var. japonica), was observed non‐destructively under a compact magnetic resonance imaging (MRI) system in addition to conventional quantitation of hydraulic vulnerability to cavitation from excised stem segments. Distribution of white and dark areas in MR images corresponded well to the distribution of water‐filled/embolized vessels observed by cryo‐scanning electron microscopy in both species. Water‐filled vessels were observed in MR images as white areas in Katsura and as white dots in Japanese white birch, respectively, and embolisms could be detected as a change to dark areas. The increase in the relative embolized area (REA: %) in the cross‐sectional area of total xylem during water stress, which was estimated from the binarized MR images, was consistent with the hydraulic vulnerability curves of these species. From the non‐destructive MRI observations, cavitation induced by water stress was shown to develop earlier in 1‐ or 2‐year‐old xylem than in the current‐year xylem in both species; that is, the vulnerability to cavitation differs between vessels in the current‐year xylem and those in older annual rings.     

Sap flow measurements in grapevines (Vitis vinifera L.) 1. Stem morphology and use of the heat balance method

The heat balance method was evaluated in detail for its use in older, mature grapevines with stems of 35 – 45 mm in diameter. Dye colouring of the xylem vessels revealed that even 21 year old grapevines did not show any development of heartwood and that xylem vessels of that age still have the capacity to transport water. A comparison of weight loss of potted vines on a balance and sap flow measurements demonstrated that the heat balance system reflected rapid changes in flow rate without any time delay. However, since even 20 year old xylem vessels of grapevines have the capacity to conduct water, the heater band was not able to heat the sap in all year rings evenly. Apparently, at low flow rates this effect was small and sap flow was calculated correctly. With increasing flow rates large thermal heterogeneities developed upsetting the calculation of the heat balance and mass flow. Consequently, actual sap flow was overestimated by 50 to 100% at high flow rates. This could be attributed to thermal gradients in these relatively thick stems excluding the use of this technique for measurements of long term as well as short term water use patterns in older grapevines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ion-mediated enhancement of xylem hydraulic conductivity is not always suppressed by the presence of Ca2+ in the sap

The physiological significance of ion-mediated enhancement of xylem hydraulic conductivity (K(h)) in planta has recently been questioned. The phenomenon has been suggested to be an artefact caused by the use of deionized water as a reference fluid during measurements of the impact of different ions on K(h). In the present study, ion-mediated changes in K(h) were measured in twigs of five woody species during perfusion with 25 mM KCl compared with different reference fluids like deionized water, a commercial mineral water containing different ions (including 0.5 mM Ca(2+)), and a 1 mM CaCl(2) solution. Both fully hydrated twigs and twigs with about 50% loss of hydraulic conductivity due to cavitation-induced embolism were tested. Adding 25 mM KCl to the three reference fluids caused K(h) to increase by about 20%. The KCl-mediated increase of K(h) was even larger (up to 100%) in embolized twigs. The presence of Ca(2+) in the reference solution decreased, but not suppressed, the KCl-mediated enhancement of K(h) in fully hydrated twigs of three species, but not in the other two species tested. Ca(2+) did not affect the K(h) response to KCl in embolized twigs. These data suggest that the recently reported suppression of the 'ionic effect' by the presence of calcium in the xylem sap is not a general phenomenon and that ion-mediated changes of K(h) may play a role in planta partially to compensate for cavitation-induced loss of xylem hydraulic conductivity.     

Xylem dysfunction caused by water stress and freezing in two species of co-occurring chaparral shrubs

Water transport from the roots to leaves in chaparral shrubs of California occurs through xylem vessels and tracheids. The formation of gas bubbles in xylem can block water transport (gas embolism), leading to shoot dieback. Two environmental factors that cause gas embolism formation in xylem conduits are drought and freezing air temperatures. We compared the differential vulnerabilities of Rhus laurina and Ceanothus megacarpus, co-dominant shrub species in the coastal regions of the Santa Monica Mountains of southern California, to both water stress-induced and freezing-induced embolism of their xylem. Rhus laurina has relatively large xylem vessel diameters, a deep root system, and a large basal burl from which it vigorously resprouts after wildfire or freezing injury. In contrast, Ceanothus megacarpus has small-diameter vessels, a shallow root system, no basal burl and is a non-sprouter after shoot removal by wildfire. We found that R. laurina became 50% embolized at a water stress of –3 MPa and 100% embolized by a freeze–thaw cycle at all hydration levels. In contrast, C. megacarpus became 50% embolized at a water stress of –9 MPa and 100% embolized by freeze–thaw events only at water potentials lower than –3 MPa. Reducing thaw rates from 0·8 °C min^?1 to 0·08 °C min^?1 (the normal thaw rate measured in situ) had no effect on embolism formation in R. laurina but significantly reduced embolism occurrence in well-hydrated C. megacarpus (embolism reduced from 74 to 35%). These results were consistent with the theory of gas bubble formation and dissolution in xylem sap. They also agree with field observations of differential shoot dieback in these two species after a natural freeze–thaw event in the Santa Monica Mountains.     

Limits to xylem refilling under negative pressure in Laurus nobilis and Acer negundo

The ability of juvenile Laurus nobilis and Acer negundo plants to refill embolized xylem vessels was tested under conditions of soil drought when xylem sap pressure was substantially negative, thus violating the expected condition that pressure must rise to near atmospheric for refilling. Intact potted plants were dried to a stem water potential (Σ_W) corresponding with approximately 80% loss of hydraulic conductivity (PLC) in shoots. Then plants were re‐watered and kept at a less negative target Ψ_W for 1–48 h. The Ψ_W was measured continuously with stem psychrometers. Rewatered L. nobilis held at the target Ψ_W for 1 h showed no evidence for refilling unless Ψ_W was within a few tenths of a MPa of zero. In contrast, re‐watered L. nobilis held for 24 and 48 h at water potentials well below zero showed a significant reduction in PLC. The recovery was highly variable, being complete in some stem segments, and scarcely evident in others. Embolism repair was accompanied by a significant but moderate decrease in the osmotic potential (Ψ) of the bulk xylem sap (Ψ = ?67 kPa in recovering plants versus ?31 kPa in controls). In contrast, embolized and re‐watered A. negundo plants held for 24 h at target Ψ_W of ?0·9 and ?0·3 MPa showed no embolism reversal. The mechanism allowing L. nobilis plants to refill under negative pressure is unknown, but does not appear to operate in A. negundo, and is slower to act for drought‐induced embolism than when embolism was artificially induced by air injection as previously shown for L. nobilis.     

Blood supply of the bed nucleus of the stria terminalis in rat

Summary The topographical distribution of the blood vessels in the bed nucleus of the stria terminalis (NIST) has been mapped in rats. Arteries and veins were visualized in red and blue by using a double-ink perfusion technique. Arteries supplying the NIST arise from the anterior cerebral artery directly or through the anterior communicating and interhemispheric arteries. Only a few, dorsal branches derive from the medial cerebral artery through thalamostriatal arteries. According to their terminal branches, NIST arteries can be divided into five groups: medial, ventral, lateral, septal and dorsal, which have only a relatively small overlap in their territories. About 90% of veins from the NIST drain into the major basal veins. Medial branches run into the perioptic and interhemispheric veins, while the ventral branches and the large lateral vein drain directly into the anterior cerebral vein. A small proportion of NIST veins run dorsalward into the vena cerebri magna via thalamostriatal veins.     

Significance of source and size in the mechanical response of human cerebral blood vessels

Cerebral blood vessels are frequently damaged in traumatic brain injury. Mechanical properties of fresh human cerebral vessels obtained through surgeries have been reported. Because surgical sources of human specimens are rare and produce a limited amount of material, we sought to compare the properties of more readily available cerebral arteries and veins obtained from cadavers to fresh vessel data. Additionally, because the previous study was limited to small vessels available in surgery, it was unknown how generally applicable the results were to larger cerebral arteries and veins. In the current study, large and small cerebral vessels from autopsy were stretched axially. Data from these and similar tests on fresh vessels were combined to determine the significance of source and size on mechanical properties. Structural comparisons of histological samples were additionally utilized to characterize differences. Results indicate that specimens from autopsy and surgery behave similarly except that vessels from autopsy tend to be less extensible. While tests on large vessels were limited, small arteries obtained from autopsy tended to be slightly stiffer than large arteries. In contrast, bridging veins from cadavers were typically stiffer and stretched less before structural failure than cortical veins from the same source. These effects are, however, secondary to differences identified between arteries and veins in the previous study.     

Water relations in silver birch during springtime: How is sap pressurised?

• Positive sap pressures are produced in the xylem of birch trees in boreal conditions during the time between the thawing of the soil and bud break. During this period, xylem embolisms accumulated during wintertime are refilled with water. The mechanism for xylem sap pressurization and its environmental drivers are not well known.
• We measured xylem sap flow, xylem sap pressure, xylem sap osmotic concentration, xylem and whole stem diameter changes, and stem and root non‐structural carbohydrate concentrations, along with meteorological conditions at two sites in Finland during and after the sap pressurisation period.
• The diurnal dynamics of xylem sap pressure and sap flow during the sap pressurisation period varied, but were more often opposite to the diurnal pattern after bud burst, i.e. sap pressure increased and sap flow rate mostly decreased when temperature increased. Net conversion of soluble sugars to starch in the stem and roots occurred during the sap pressurisation period. Xylem sap osmotic pressure was small in comparison to total sap pressure, and it did not follow changes in environmental conditions or tree water relations.
• Based on these findings, we suggest that xylem sap pressurisation and embolism refilling occur gradually over a few weeks through water transfer from parenchyma cells to xylem vessels during daytime, and then the parenchyma are refilled mostly during nighttime by water uptake from soil. Possible drivers for water transfer from parenchyma cells to vessels are discussed. Also the functioning of thermal dissipation probes in conditions of changing stem water content is discussed.

     

Spring filling of xylem vessels in wild grapevine

Xylem vessels in grapevines Vitis labrusca L. and Vitis riparia Michx. growing in New England contained air over winter and yet filled with xylem sap and recovered their maximum hydraulic conductance during the month before leaf expansion in late May. During this period root pressures between 10 and 100 kilopascals were measured. Although some air in vessels apparently dissolved in ascending xylem sap, results indicated that some is pushed out of vessels and then out of the vine. Air in the vessel network distal to advancing xylem sap was compressed at about 3 kilopascals; independent measurements indicated this was sufficient to push air across vessel ends, and from vessels to the exterior through dead vine tips, inflorescence scars, and points on the bark. Once wetted, vessel ends previously air-permeable at 3 kilopascals remained sealed against air at pressures up to 2 and 3 megapascals. Permeability at 3 kilopascals was restored by dehydrating vines below −2.4 megapascals. We suggest that the decrease in permeability with hydration is due to formation of water films across pores in intervascular pit membranes; this water seal can maintain a pressure difference of roughly 2 megapascals, and prevents cavitation by aspirated air at xylem pressures less negative than −2.4 megapascals.     

Vessel contents during transpiration - embolisms and refilling

A test was made of the previous unexpected observation that embolized vessels were refilled during active transpiration. The contents of individual vessels in petioles of sunflower plants were examined, after snap-freezing at 2-h intervals during a day's transpiration, in the cryo-scanning electron microscope, and assessed for the presence of liquid or gas (embolism) contents. Concurrent measurements were made of irradiance, leaf temperature, transpiration rate, and leaf water potential (by pressure chamber). Up to 40% of the vessels were already embolized by 0900 (transpiration rate ~5 _g_cm-2_s-1, water potential about -300 J/kg), and the proportion declined to a minimum (as low as 4%) at 1500. This was the time of highest transpiration rate (~25 _g_cm-2_s-1) and most negative water potential (-600 to -700 J/kg). Images of vessels with mixed gas and liquid contents showed water being extruded through pits in the walls of the vessels to refill them. The data indicate that: (1) the water columns are weak and break under quite small tensions; (2) embolisms are repaired by refilling the vessels with water on a short time scale (minutes) throughout the day; (3) the vigor of this refilling process is adjusted by the plant on a longer time scale (hours) to the intensity of the water stress; (4) the pressure chamber balance pressure (P) does not measure tension in the vessels; (5) P is also not a measure of water stress (as measured by vessel embolization); and (6) P is a measure of the plant's response to water stress, i.e., a measure of the vigor of the refilling process. The test confirms the previous observations and negates all the assumptions and evidences of the Cohesion Theory. The data are fully consistent with the Compensating Pressure Theory, which predicted the relations demonstrated in this experiment. Using the assumptions of that theory it is easy to outline a simple mechanism by which the refilling of vessels might be achieved by reverse osmosis, and the adjustment in (3) might be achieved by osmoregulation in the starch sheath.     

Lymphatic vessels of the upper extremity and their relation to nodes in the axillary area in healthy subjects and lymph flow blockade

Large and small lymphatic vessels have been studied roentgenologically on the medial, lateral, posterior and anterior surfaces of the upper extremity in 113 patients at the age of 19-63 years at blockade of the lymphatic stream. On the medial and lateral surfaces the lymphatic vessels are filled with the contrast substance via anatomical approaches from the palmar and dorsal sides of the forearm. With isolated contrasting of various large lymphatic vessels, zones in the skin and in the subcutaneous fatty layers drained by them are revealed, as well as distribution of small vessels in the forearm and shoulder in each region. Variants of large lymphatic vessels and their tributaries are defined; an essential variability of their inflow into the axillary lymph nodes from various anatomical areas of the upper extremity is found. Into every 1-4 groups of the lymph nodes of the axillary area, 1-3 large vessels inflow, through them the contrast substance switches from the same anatomical zone repeatedly.     

Effect of stem water content on sap flow from dormant maple and butternut stems: induction of sap flow in butternut

Sap flow from excised maple stems collected over the winter (1986/87) was correlated with stem water content. Stem water content was high in the fall (>0.80) and decreased rapidly during 2 weeks of continuous freezing temperatures in late winter (<0.60). Exudation of sap from stem segments subjected to freeze/thaw cycles was small (<10 mL/kg) in the fall, but substantial exudation (45-50 mL/kg) occurred following the decline in water content. These observations are consistent with Milburn's and O'Malley's models (J.A. Milburn, P.E.R. O'Malley [1984] Can J Bot 62: 2101-2106; P.E.R. O'Malley, J.A. Milburn [1983] Can J Bot 61:3100-3106) of sap absorption into gas-filled fibers during freezing. Exudation volume was increased 200 to 300% in maple stems originally at high water content (>0.80) after perfusion with sucrose and dehydration at −12°C. Sap flow was also induced in butternut stem segments after the same treatment. Thus, sap flow may not be unique to maples. Sap flow could not be increased in stem segments dehydrated at 4°C. Migration of water molecules from small ice crystals in fibers to larger crystals in vessels while stems were frozen may account for increase exudation after dehydration at −12°C. This would result in preferential dehydration of fibers and a distribution of gas and sap favorable for stem-based sap flow.     

Is Xylem Embolism and Refilling Involved in the Rapid Wilting and Recovery of Plants Following Root Cooling and Rewarming?

Abstract: Rapid wilting and subsequent rapid recovery of the shoots of plants whose roots are cooled and rewarmed (first described by Sachs, 1860^[23]), has been investigated by cryo-scanning electron microscopy. Squash plants began to wilt within 5 min and were completely wilted 1 h after their roots were placed in nutrient solution at 4C. Recovery began in 5 min and was complete by 45 min when the roots were returned to solution at 22C. Some stomata on the abaxial leaf surface remained fully or partially open in the wilted plants and transpiration continued at a low level. Both control and wilted plants had the same proportion (60%) of large root vessels partially or totally gas-filled, showing that the supply of water was not limited by the reduction of axial hydraulic conductance due to vessel embolism. However, only 10% of these embolized vessels in the wilted plants contained any liquid, compared to ≅ 80% of similar vessels in control and recovered plants. This is visual evidence of reduced radial hydraulic conductance into the vessels in the cold roots, and that this reduced conductance, together with still open-stomata, produces wilting. These effects were reversed by rewarming.