James K. Wheeler and N. Michele Holbrook, Department of Organismic and Evolutionary Biology, Harvard University

March, 2007

Xylem cavitation diminishes a plant’s capacity to transport water from the soil to the leaves. This reduction in xylem hydraulic conductivity can impair rates of carbon fixation by inducing stomatal closure to prevent further cavitation and desiccation of leaf tissues. Several mechanisms by which plants can restore xylem transport capacity are mentioned in the textbook. This web essay outlines these processes in more detail and explores the possibility that plants might be able to refill cavitated vessels while tension exists in adjacent conduits— a process referred to here as “local refilling.”

Mechanisms by which plants can restore hydraulic conductivity after cavitation

1. Produce new xylem conduits: Any plant that possesses the capacity for secondary growth can simply produce new xylem to replace the cavitated conduits. This is a common mechanism for maintaining hydraulic conductivity in trees and shrubs, but is not available to plants lacking secondary growth.

2. Refill cavitated conduits: Embolized (gas-filled) conduits can be refilled if the pressure on the gas phase increases to the extent that the gas dissolves into the surrounding liquid phase. This immediately raises several questions: How much pressure is needed to force gas into solution? For how long must this pressure be sustained? These questions are addressed here and the mechanisms that plants can employ to generate positive pressure are evaluated in the following section.

The pressure required for refilling depends upon the composition of the gas contained within the embolized conduit. Immediately following a cavitation event, the embolus will largely consist of water vapor, which has an absolute vapor pressure of 2.3 kPa at 20°C (which is –99 kPa relative to atmospheric pressure). At this point, the pressure need only exceed this value to force these molecules back into solution. However, over time, cavitated conduits become air-filled as dissolved gases in the surrounding liquid phase come out of solution. Forcing an air bubble back into solution requires pressure greater than 100 kPa (1 atm).

How long this pressure has to be sustained also depends on the gas composition. Water vapor dissolves easily as the added molecules have essentially no effect on the concentration of the solvent; refilling of cavitated conduits containing only water vapor should occur almost instantaneously once the vapor pressure of water is exceeded. In contrast, forcing air into solution is more difficult as the dissolving gas molecules locally increase their concentration in the liquid surrounding the embolus. The movement of more gas into solution is held in check by the rate at which the newly dissolved gases diffuse away from the gas–liquid boundary. Thus, the time needed to force an air-embolus into solution depends upon both the magnitude of the applied pressure and the diffusional limitations imposed by the surrounding environment (Yang and Tyree 1992).

In considering the generation of positive pressures needed to effect refilling, it is important to take into account both the hydrostatic pressures in the liquid phase and the forces arising from the surface tension of curved gas-liquid interfaces. While the hydrostatic pressure can be either positive or negative, the surface tension of water always produces a force directed towards the inward (concave) side of the meniscus (see Figures 3.4 to 3.6 in the textbook). This means that the hydrostatic pressures in the xylem needed for refilling depends upon the size of the conduit being refilled, because the curvature of the gas:water interface, and therefore the force produced by surface tension, will be set by the dimensions of the conduit. Thus, a small conduit containing only water-vapor (i.e., recently cavitated) can refill even when the xylem is under tension. For example, Hacke and Sperry (2003) calculate that a water vapor filled bubble with a radius of 8 μm the minimum hydrostatic pressure allowing dissolution of a water-vapor filled bubble with a radius of 8 μm is –118 kPa relative to atmospheric pressure at sea level (or –18 kPa absolute pressure). However, for larger conduits and particularly those that become air-filled, positive pressures in the liquid phase are needed to refill embolized conduits.

Mechanisms for Refilling

Plants may generate the positive pressures necessary for refilling within the roots, throughout the stem, or locally, within the cavitated conduit. The mechanisms by which these processes occur range from fairly well understood in the case of root pressure to poorly understood for the phenomenon of local pressurization.

A. Root pressure: Many species are able to raise the hydrostatic pressure throughout their vascular system by actively loading solutes into their root stele. Osmotically produced “root” pressures, which may reach several hundred kPa, either force emboli into solution or push the air out of hydathodes or open vessel ends (Sperry et al. 1987, Fisher et al. 1997). Most evidence suggests that this process can only occur when the soil is saturated and transpiration is low, but the critical thresholds at which root pressure is inhibited have not been established experimentally. While the phenomenon is well documented in herbs and vines (Tyree et al. 1986, Sperry et al. 1987, Fisher et al. 1997, Stiller et al. 2003), it has only been reported in a small number of trees (e.g., Sperry et al. 1994, Ewers et al. 2001). Diurnal refilling via root pressure has been reported in some crop species (Tyree et al. 1986, Stiller et al. 2003), but it is unclear whether trees employ root pressure for daily recovery of hydraulic conductivity.

B. Stem pressure: Several temperate species draw water into their stems when temperatures drop near or below freezing. Upon thawing, the positive pressure built up by this influx of water refills embolism induced over the winter by both freezing and dehydration (Sperry et al. 1988, Marvin and Greene 1951). It is this process that causes sugar maple trees to bleed in the spring, which is the raw material for maple syrup. The pressures produced by these species are generated throughout the stem, but can co-occur with some degree of root pressure (Ewers et al. 2001, Sperry et al. 1988). While the precise mechanism for this is still not fully understood, it is clear that this process is limited to the spring before leaf flush when the soil is saturated and transpiration is minimal.

C. Local pressurization: Local refilling, where pressurization occurs solely within an embolized conduit or a few adjacent cells, is the most ecologically interesting mechanism, as it could allow plants to recover conductivity during periods of active transpiration and therefore adjust to changing conditions throughout the day. However, it is also the most controversial. The remainder of this Web Essay explores the limitations and possible mechanisms by which plants could generate pressure within individual conduits while the remainder of the xylem remains under tension.

How strong is the evidence for local refilling?

Evidence for local refilling derives from a suite of studies employing a variety of experimental and measurement techniques: artificially inducing embolism by injecting pressurized air into the xylem (Salleo et al. 1996, 2004, Tyree et al. 1999), recording cycles of embolism and refilling by freezing the xylem or perfusing dye through the xylem and then visualizing the number of filled conduits under a microscope (Canny 1997, McCully et al. 1998, Zwieniecki and Holbrook 1999, Canny et al. 2001), recording diurnal variation in conductance in situ (Zwieniecki and Holbrook 1999, Bucci et al. 2003, Domec et al. 2006), and inducing cavitation by drying plants in the laboratory (Hacke and Sperry 2003, Stiller et al. 2005). Because all of these approaches involve destructive sampling and/or could themselves create artifacts (e.g., Cochard et al. 2000, 2001), unequivocal evidence for local refilling that restores the ability of previously cavitated conduits to transport water under tension remains elusive.

The strongest evidence to date for the occurrence of local refilling is provided by studies in which treatments designed to alter physiological parameters such as phloem transport (girdling), starch reserves (shading), or membrane activities (addition of sodium orthovanadate or fusicoccin) reduce the degree of refilling compared with control plants. These “perturbation” studies have led to general agreement on certain points. If local refilling occurs it requires a metabolic source of energy, most likely sugars either stored in the xylem parenchyma as starch or supplied by the phloem. Refilling also requires a source of water, which is generally thought to be the phloem. However, how local refilling actually occurs remains a matter of active discussion.

Proposed mechanisms for local refilling

The basic questions surrounding local refilling center on the development of a sufficient driving force to move water into the embolized vessel as well as a way to prevent water that has entered the cavitated conduit from being pulled away by tensions present in the apoplast. Several mechanisms have been suggested to overcome these problems, but each either lacks experimental evidence or has failed to withstand rigorous theoretical analysis. Below is a brief outline of the mechanisms currently proposed; a more thorough treatment is available in a review by Clearwater and Goldstein (2005).

1. Hacke and Sperry (2003) proposed that refilling occurs osmotically, with xylem parenchyma cells secreting solutes of sufficient size that they cannot pass through the inter-vessel pit membranes. The key element of this hypothesis is that the refilling vessel remains hydraulically connected to adjacent conduits due to the proposed semi-permeable action of the pit membranes. Thus adjacent conduits could serve as a source of water for refilling. While the simplicity of this hypothesis is appealing, the presence of large solutes has not been detected.

2. All of the other proposed mechanisms for local refilling require the embolized conduit to be isolated from the transpiration stream during the refilling process and then reconnected once refilling has occurred. A hypothesis for how such hydraulic compartmentalization might occur was first proposed by Holbrook and Zwieniecki (1999). The authors suggest that the shape of the pit chamber and its hydrophobic walls may lead to the formation of a meniscus in the neck of the pit chamber that traps a small gas barrier preventing contact between the water in the refilling vessel and the transpiration stream. Based on measurements of the pit chambers of six species they conclude the maximum pressure this meniscus could sustain ranges from 70 to 300 kPa (Zwieniecki and Holbrook 2000).

Figure 1   Hydraulic compartmentalization of vessel refilling. Left: Living cells adjacent to the embolized vessel create a driving gradient that draws water into the vessel lumen (blue arrows). Droplets are retained on the wall due to the nonzero contact angle (θ). Low permeability of the secondary wall prevents tension in adjacent vessels from being transmitted. Influx of water into the lumen compresses the gas phase (black arrows), forcing it into solution (yellow arrows). The dissolved gas then diffuses away from the refilling vessel, where it may be carried off by the transpiration stream. Right: Bordered pit geometry (inverted funnel with angle α) prevents water from entering the pit channel before the lumen is entirely filled. The upper conduit is actively refilling and the water is under positive pressure; the lower vessel is under tension. Arrows indicate the effects of hydrostatic pressure (black) and surface tension force (red) on the gas/liquid interface. (Figure reprinted from Holbrook and Zwieniecki 1999.)

Although this mechanism could, at least in theory, isolate a refilling xylem conduit, difficulty arises when the refilled conduit must be reconnected to the transpiration stream. If the pit chambers do not all fill simultaneously, the gas that isolated the vessel from the transpiration stream may induce cavitation and lead to a futile cycle of cavitation and refilling. Mathematical models based on the geometry and surface chemistry of bordered pits have been used to argue how such coordination might be achieved (Konrad and Roth-Nebelsick 2005), but the details of how this might occur in nature remain unexplored.

Two proposals exist to describe the driving force for water movement into hydraulically isolated conduits. The first is that refilling occurs osmotically (Holbrook and Zwieniecki 1999, Tyree et al. 1999), with water is being drawn across the cellular membrane of adjacent xylem parenchyma. In this case much smaller solutes (potentially monatomic ions such as K⁺) could be used as cellular membranes are much less porous than inter-vessel pit membranes. An alternative hypothesis, first postulated by Canny (1995) and later expanded by Bucci et al. (2003) and Domec et al. (2006), suggests that an increase in “tissue pressure” in the cortex induces centripetal water flow that can refill embolized conduits. This hypothesis requires a build-up in cortical turgor pressure due to an accumulation of solutes, followed by the net inward movement of water due to cortical cells being physically constrained by surrounding rigid tissues.

Both of these proposed mechanisms remain, to a large degree, unsubstantiated. To date no one has demonstrated sufficient osmotic concentrations in xylem sap to induce refilling. This may be due to the difficulty in accurately determining the osmotic concentration of nanoliter to picoliter samples extracted from single vessels or dilution of the contents of refilling vessels with water from full vessels when larger volumes are extracted. A variation on osmotic loading that could account for this apparent lack of a sufficient osmotic driving force is described by Pickard (2003). Tissue pressure, as a mechanism that could effect refilling, remains hypothetical, although experiments in which mechanical damage to the outer cortex eliminated recovery of hydraulic conductivity have been proposed as evidence that the build up of cortical turgor pressure plays a role in refilling (Bucci et al. 2003, Domec et al. 2006). However, other authors have argued that it requires water to flow in opposite directions simultaneously and that the mechanism is based upon a transient change in pressure that can not be maintained once water begins to move between tissues (Tyree 1999, Tyree 1999 et al., Comstock 1999).

Ecological consequences of local refilling

The idea that plants might be capable of embolism repair while tension exists in the xylem has substantial ecological consequences. Cavitation appears to occur frequently and to the extent that the loss in transport capacity can limit gas exchange. Until recently it was generally believed that the losses of conductivity from cavitation were persistent. The ability to refill embolized conduits would not alter the fact that cavitation sets an absolute limit on the conditions under which plants can photosynthesize, but it could permit plants to restore some of their photosynthetic capacity under conditions of partially reduced tensions. This could have important ramifications in seasonally arid environments that experience small rainfall events during the dry season. Plants possessing the ability to refill could exploit this moisture even if they had sustained substantial cavitation. The ability to refill would also allow plants to recover conductive ability in the afternoon following midday extremes in vapor pressure deficit (as suggested by the evidence of daily cycles of cavitation and refilling). This would be advantageous because it would allow plants to transpire near their hydraulic limit during midday and then recover from cavitation in the afternoon, thus increasing total daily assimilation.

The fact that plants employ a variety of structural and physiological mechanisms to limit cavitation (Zimmermann 1983) suggests that if a repair mechanism exists, it is frequently more expensive than avoiding cavitation. To understand how refilling might affect inter-specific interactions it is necessary to understand the costs involved. A mechanistic understanding of refilling would provide insight into these costs and under what conditions investment in a refilling strategy might be more cost effective than simply avoiding cavitation.

References

Bucci S J, Scholz F G, Goldstein G, Meinzer F C, and Sternberg L D L. (2003) Dynamic changes in hydraulic conductivity in petioles of two savanna tree species: factors and mechanisms contributing to the refilling of embolized vessels. Plant, Cell and Environment 26: 1633–1645.

Canny M J. (1995) A new theory for the ascent of sap-cohesion supported by tissue pressure. Annals of Botany 75: 343–357.

Canny M J. (1997) Vessel contents during transpiration-embolisms and refilling. American Journal of Botany 84: 1223–1230.

Canny M J, McCully M E, and Huang C X. (2001) Cryo-scanning electron microscopy observations of vessel content during transpiration in walnut Petioles. Facts or Artefacts? Plant Physiology and Biochemistry 39: 555–563.

Clearwater M J and Goldstein G. (2005) Embolism repair and long distance water transport. In Vascular Transport in Plants, Holbrook N M and Zwieneicki M A.,eds. Elsevier Academic Press Burlington, MA: 375–400.

Cochard H, Bodet C, Ameglio T and Cruiziat P. (2000) Cryo-scanning electron microscopy observations of vessel content during transpiration in walnut petioles. Facts or artifacts? Plant Physiology 124: 1191–1202.

Cochard H, Ameglio T and Cruiziat P. (2001) The cohesion theory debate continues. TRENDS in Plant Science 6:456.

Comstock J P. (1999) Why Canny’s theory doesn’t hold water. American Journal of Botany 86: 1077–1081.

Domec J C, Scholz F G, Bucci S J, Meinzer F C, Goldstein G, and Villalobos-Vega R. (2006) Diurnal and seasonal variation in root xylem embolism in neotropical savanna woody species: impact on stomatal control of plant water status. Plant, Cell and Environment 29: 26–35.

Ewers F W, Ameglio T, Cochard H, Beaujard F, Martignac M, Vandame M, Bodet C, and Cruizat P. (2001) Seasonal variation in xylem pressure of walnut trees: root and stem pressures. Tree Physiology 21: 1123–1132.

Fisher J B, Angles G, Ewers F W, Lopez-Portillo J. (1997) Survey of root pressure in tropical vines and woody species. International Journal of Plant Sciences 158: 44–50.

Hacke U G and Sperry J S. (2003) Limits to xylem refilling under negative pressure in Laurus nobilis and Acer negundo. Plant, Cell and Environment 26: 303–311.

Holbrook N M and Zwieniecki M A. (1999) Embolism repair and xylem tension: Do we need a miracle? Plant Physiology 120, 7–10.

Konrad Wand Roth-Nebelsick A. (2005) The significance of pit shape for hydraulic isolation of embolized conduits of vascular plants during novel refilling. Journal of Biological Physics 31, 57–71.

Marvin J W and Greene M T. (1951) Temperature-induced sap flow in excised stems of Acer. Plant Physiology 26: 565–580. McCully M E, Huang C X, and Ling L E C. (1998) Daily embolism and refilling of xylem vessels in the roots of field-grown maize. New Phytologist 138: 327–342.

Pickard W F. (2003) The riddle of root pressure. I. Putting Maxwell’s Demon to rest. Functional Plant Biology 30: 121–134.

Salleo S, Lo Gullo M A, de Paoli D, and Zippo M. (1996) Xylem recovery from cavitation-induced embolism in young plants of Laurus nobilis: a possible mechanism. New Phytologist 132: 47–56.

Salleo S, Lo Gullo M A, Trifilo P, and Nardini A. (2004) New evidence for a role of vessel-associated cells and phloem in the rapid xylem refilling of cavitated stems of Laurus nobilis L. Plant Cell and Environment 27: 1065–1076.

Sperry J S, Holbrook N M, Zimmermann M H, and Tyree M T. (1987) Spring filling of xylem vessels in wild grapevine. Plant Physiology 83: 414–417.

Sperry J S, Donnelly J R, and Tyree M T. (1988) Seasonal occurrence of xylem embolism in sugar maple (Acer saccharum). American Journal of Botany 75: 1212–1218.

Sperry J S, Nicols K L, Sullivan J E M, and Eastlack S E. (1994) Xylem embolism in ring-porous, diffuse-porous, and conifer trees of northern Utah and interior Alaska. Ecology 75: 1736–1752.

Stiller V, LaFitte H R and Sperry J S. (2003) Hydraulic properties of rice and the response of gas exchange to water stress. Plant Physiology 132: 1698–1706.

Stiller V, Sperry J S, and Lafitte R. (2005) Embolized conduits of rice (Oryza sativa, Poaceae) refill despite negative xylem pressure. American Journal of Botany 92: 1970–1974.

Tyree M T, Fiscus E L, Wullschleger S D and Dixon M A (1986) Detection of xylem cavitation in corn under field conditions. Plant Physiology 82: 597–599.

Tyree M T. (1999) The forgotten component of plant water potential: A reply- tissue pressures are not additive in the way M. J. Canny suggests. Plant Biology 1:598–601.

Tyree M T, Salleo S, Nardini A, Lo Gullo M A and Mosca R. (1999) Refilling of embolized vessels in young stems of laurel. Do we need a new paradigm? Plant Physiology 120: 11–21.

Yang S and Tyree M T. (1992) A theoretical model of hydraulic conductivity recovery from embolism with comparison to experimental data on Acer saccharum. Plant, Cell and Environment 15: 633–643.

Zimmermann MH (1983) Xylem Structure and the Ascent of Sap, Springer, Berlin, Heidelberg, New York.

Zwieniecki M A and Holbrook N M. (1999) Diurnal variation in xylem hydraulic conductivity in white ash (Fraxinus americana L.) red maple (Acer rubrum L.) and red spruce (Picea rubens Sarg.). Plant, Cell and Environment 21: 1173–1180.

Zwieniecki M A and Holbrook N M. (2000) Bordered pit structure and vessel wall surface properties. Implications for embolism repair. Plant Physiology 123: 1015–1020.