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A plumber's-eye view of xylem water transport inwoody plantsJordi Martnez-Vilalta a & Josep Piol aa CREAF/Ecology Unit , Universitat Autnoma de Barcelona , Barcelona, SpainPublished online: 13 Dec 2010.

To cite this article: Jordi Martnez-Vilalta & Josep Piol (2004) A plumber's-eye view of xylem water transport in woodyplants, Journal of Biological Education, 38:3, 137-141, DOI: 10.1080/00219266.2004.9655923

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Practical A plumber's-eye view of xylem water transport in woody plants Jordi Martfnez-Vilalta and Josep Pihol CREAF/Ecology Unit, Universitat Autonoma de Barcelona, Barcelona, Spain

We present a practical for university-level students aimed at measuring and comparing xylem hydraulic properties of co-existing plant species. After sampling branches of several woody species in the field, their main hydraulic properties were measured using a simple set-up. Hydraulic conductivity (Kh) was calculated as the ratio between water flow through a plant segment and the pressure gradient driving the flow. The percent reduction in conductivity due to xylem embolism (i.e. air-filled conduits) was estimated by comparing Kh before and after flushing the measure segments to remove all native embolism. Raw hydraulic conductivity was standardised by cross-sectional wood area or supported leaf area to obtain more meaningful measures of conducting capacity. The results showed differences among study species, particularly between conifers and angiosperms. These differences are briefly discussed in terms of wood anatomy and the general biology of the species. Overall the practical provides a good opportunity for students to appreciate the main aspects of xylem water transport and the constraints it imposes on plant water relations. Key words: Ecophysiology, Hydraulic architecture, Hydraulic conductivity, Plant water transport, Xylem embolism.

Introduction Plants transport enormous amounts of water from the soil to the atmosphere. Of the water absorbed by roots only about 5% is used in metabolism and growth, whereas the remaining 95% is lost through leaves in transpiration. As an example, a single isolated tree may transpire 200-400 litres of water per day (Kozlowski and Pallardy, 1997). Transpiration is intimately linked to carbon acquisition, since both the flow of water out of the leaf and the flow of carbon dioxide into the leaf occur through the same pores (i.e. stomata). At the same time, the huge quantity of water transported from the soil to the leaves is likely to assist the nutrient supply of above ground tissues. How can plants pull up these amounts of water to heights up to more than 100 metres? According to the nearly-hegemonic cohesion-tension theory, solar radiation provides the energy source for water evaporation in leaves, which together with the cohesive strength of liquid water makes the transport along the gradient of water potential possible (Steudle, 1995).

Inside plants water is transported mainly through the xylem, a tissue containing a network of conduits (walls of dead cells) interconnected through porous membranes. A corollary of the cohesion-tension theory is that water transport in the xylem should take place at pressures substantially lower than atmospheric (i.e. under tension). However, at ambient temperature (around 20C) liquid water at pressures below +2.3kPa is thermodynamically unstable. As xylem pressures become increasingly negative, a point is eventually reached at which water evaporates (cavitation) and the affected conduit is filled with air from the surrounding tissue (embolism). Inter-conduit

pit membranes act as filters preventing the propagation of embolisms. However, if xylem pressure is low enough to suck air through inter-conduit pores (the required tension depends on pore size) generalised embolism occurs (Zimmermann, 1983). Embolised conduits no longer contribute to water transport unless they are refilled with liquid water.

High levels of xylem embolism reduce the capacity of plants to transport water and when maintained over long periods of time can cause branch or plant dieback. Therefore, generalised embolism is something to be avoided by plants. But what environmental conditions favour the occurrence of xylem embolism? Drought is clearly one of them, because the lower the water content of the soil the lower the water potential needed by plants to extract and transport water and, thus, the higher the amount of embolism in the xylem. As a result, vulnerability to xylem embolism is one of the key components of drought resistance in plants (Hacke and Sperry, 2001). Freezing can also induce embolism: since the solubility of air is lower in ice than in liquid water, freezing forces air out of the xylem sap solution, forming bubbles. On thawing, these bubbles can either re-dissolve or nucleate cavitation, depending on the size of the conduit (larger conduits being more vulnerable). There is evidence supporting the idea that freezing-induced embolism limits the distribution in cold habitats of many plant species (Hacke and Sperry, 2001).

In this paper we present a practical for university students aimed at measuring and comparing the hydraulic properties of several co-existing plant species. Previous studies have stressed the faulty understanding of transpiration or plant water transport of secondary (Barker, 1998) or university-level students

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Q Xylem water transport Martinez-Vilalta and Pinol (Martinez-Vilalta et al). Although it is clear that a minimum knowledge of the mechanism of sap ascent is required to fully understand the experiments reported in this paper, in our opinion they provide a good opportunity for students to deepen dieir knowledge of die cohesion-tension mechanism and appreciate some of its basic implications. Despite the fact that the study of the hydraulic properties of plant xylem as described in this paper is relatively new (it was systematised during the 1980s, when some of the methods we present here were developed) it is already recognised as one of the key aspects of plant water relations in major textbooks on plant ecology and ecophysiology (five pages out of 23 are devoted to this subject in the chapter on plant water relations in Crawley [1997], and 11 out of 50 in Lambersetal [1998]).

Methodology All sampling and measurements were carried out by 12 groups (20-25 people each) of second year students of biology at the Universitat Autonoma de Barcelona (Spain) as a part of a one-week course on practical ecology. The particular practical we present here lasted for one-two days, depending on the duration of the subsequent class discussion.

Field sampling The first step consists of selecting the group of woody species that will be studied. In the particular example we report here all species (see Table 1) were sampled during early autumn in a Quercus ilex forest from NE Spain (at La Castanya Biological Station [Montseny], 41 46' N, 2 21 ' E). All individuals (N = three - 11 individuals per species) were sampled in the same valley, widiin less than 500 metres. The particular species sampled will obviously depend on the region where the practical is conducted, but in principle any community with more than three or four different woody species is suitable. It is advisable to select species with contrasting life strategies, for example by first providing students with information on the general biology of the dominant woody species in the area and then letting them decide the target species in terms of previous information and personal preferences.

Fully exposed, terminal branches at least 1 m long were collected from four-five different individuals per species. The sampled branch that will be used in all hydraulic measurements should contain a central segment about 20 cm long and 0.5-1 cm in diameter, with no or very few side branches. Because xylem pressures are lower than atmospheric, cutting introduces air (embolism) into severed xylem conduits.

Consequently, the length of sampled branches should be enough that none (or very few) of the conduits embolised during collection extend to measured segments. Although 40-50 cm is normally enough, some species (e.g. typically Quercus spp.) have very long vessels and require the sampling of longer branches. It is possible to know if there is any single vessel extending throughout a given wood segment by injecting pressurised air (at around 60 kPa) into one of the ends of the segment and submerging the other end in water: bubbles will appear at the submerged end only if there is at least one individual vessel crossing the entire segment (Ewers and Fisher, 1989). Immediately after cutting, branches were sealed in plastic bags and carried to the laboratory. Travel time was about four hours. Once in the laboratory samples were conserved at 2C until they were measured within 12 hours.

Hydraulic properties Hydraulic conductivity (KJ is defined as the ratio between a water flow (7) and the pressure gradient required to produce it (Vp), and is, thus, a measure of capacity to transport water. We measured xylem hydraulic conductivity following Sperry et al (1988). Segments about 20cm long and with a diameter of 0.5-1 cm were re-cut underwater from the sampled branches. Their proximal ends were connected and clamped to a tubing system filled with filtered (4> = 0.22pm), deionised water, and connected to a water reservoir (see Figure 1). This reservoir was around 60cm above the level of the branches, equivalent to a

height'pressure difference

water reservoir

tubing

Figure 1 Diagram of the set-up we used to measure hydraulic conductivity of branch segments.

Table 1 Main ecological and wood anatomical attributes of the study species1.

Plant species

Abies sp.2

Alnus glutinosa Castanea sativa Celtis australis Cistus salviifolius Hedera helix Quercus ilex

Family

Pinaceae Betulaceae Fagaceae Ulmaceae Cistaceae Araliaceae Fagaceae

Life form

Tree Tree Tree Tree Shrub Liana Tree

Wood type

Conifer Semi-ring-porous Ring-porous Ring-porous Diffuse porous Semi-ring-porous Diffuse porous

Leaf habit

Evergreen Deciduous Deciduous Deciduous Summer dec. Evergreen Evergreen

Macroclimate

Temperate Temperate Temperate Mediterranean Mediterranean Temperate Mediterranean

'Data compiled from Baas and Schweingruber (1987). 2Sampled trees correspond to an exotic Abies species (probably a hybrid between A. alba and A. pinsapo) planted in the area.

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f ) Xylem water transport

pressure difference of approximately 6kPa. The resulting water flow was measured gravimetrically, by weighing previously zeroed, cotton-filled vials that had been attached to the distal ends of the segments for a known period of time. Hydraulic conductivity (Ky in m4 MPa"1 s"1) was calculated as the ratio between the flow through the segment and the pressure gradient (pressure difference divided by the length of the segment]. This initial hydraulic conductivity [Kh.) corresponds to the conditions under which the sampled individuals were operating in the field and, therefore, includes any native embolism they were experiencing.

In order to obtain their maximum hydraulic conductivity {KhmaJ the segments were flushed at high pressure (600 kPa] with water for around 60 min to remove all native embolisms. In our case this was achieved by lifting the water reservoir to the ceiling (about six metres above]. After flushing, the water reservoir was lowered again and hydraulic conductivity was re-measured exactly as before. The percentage loss of hydraulic conductivity (PLC) due to the amount of embolism originally present in the segments can be evaluated by comparing their hydraulic conductivities before and after flushing:

PIC = 100- l j ^ ! Equation 1 \ h, max /

In the few cases in which Khi>Kh (probably because measured flows approached the limits of the resolution of our 0.001-g precision balance] it was assumed that PLC ~ 0. Since hydraulic conductivity is clearly size-dependent it is always advisable to relate it to other branch properties, like cross-sectional wood area or supported leaf area. Specific hydraulic conductivity [K$, in m2 MPa"1 s"1) was calculated as the ratio between Khmax and cross-sectional area of the segment (without bark]; and leaf-specific conductivity [Ku in m2 MPa"1 s"1], as the quotient between Kh max and supported leaf area. An additional variable of interest to characterise hydraulic architecture is the ratio between cross-sectional area and leaf area [A^AJ (Zimmerman, 1983]. In our case, leaf area was measured with a LI-COR 3100 leaf area meter (LI-COR). If similar equipment is not available it is possible to estimate mass per unit leaf area [LMA, in mg cm2] in a reduced amount of leaves and use this value to convert total leaf weight into total leaf area.

Data analysis Hydraulic properties were compared among species by means of one-way AN OVA (plus Tuckey HSD a posteriori test]. Since none of the variables studied was normally distributed they were normalised (logarithmic transformation for Ks, KL and AS:AL; and arcsine transformation for PLC] prior to analysis. Consequently the averages and confidence intervals (CI] provided in the text and figures correspond to those calculated for the transformed variables back-transformed to the original scale.

Results Typical water flows through measured segments were 0.1-1 mg s"1 for angiosperms and 0.01-0.1 mg s"1 for Abies sp. (globally 10 - 1000 mg deposited in a 15-minute measurement; enough to be measured with a 0.001 g-precision balance]. When standardised according to cross-sectional area of wood and leaf area, hydraulic conductivity ranged, respectively, between 1.44 x 10" 5 {Abies sp.] and 9.99 x 10"4 m2 MPa"1 s"1 [Alnus glutinoso) [Ks),

Journal of Biological Education (2004) 38(3)

Martinez-Vilaltaand Pinol

and between 8.91 x 10"9 m2 MPa"1 s"1 [Abies sp.] and 1.03 x 10"6

[Castanea sativd) [KA. In both cases hydraulic conductivity was significantly lower in Abies sp. than in any other species (see Figures 2a and 2b]. High KL of C. sativa (Figure 2b] is related with larger AS:AL in this species (Figure 2d), which was starting to lose leaves at the time of measurement. There was a wide range of inter-specific variation in estimated loss of conductivity due to xylem embolism (PLC), from around 0% in Abies sp. to > 80% in Celtis australis. Loss of conductivity was significantly lower in Abies sp. than in any other species, and larger in C australis and Q. ilex than in A. glutinosa; the rest of species showing intermediate values (see Figure 2c).

Figure 2 Hydraulic properties of branches of the study species, including: specific hydraulic conductivity (K^ (a), leaf-specific hydraulic conductivity (KJ (b), percent loss of conductivity due to xylem embolism under field conditions (c), and wood-to-leaves area ratio (d). Column heights and error bars correspond, respectively, to averages and 95% confidence intervals calculated for normalised variables back-transformed to the original scale (see 'Data analysis' section). Different letters indicate differences between species at the 0.05 significance level. See Table 1 for species' full names.

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f ) Xylem water transport

Discussion This practical offers several opportunities for discussion. Measured water flows illustrate very clearly one essential property of wood: its capacity to transport water longitudinally. Xylem occupies the main part of the cross sectional area of a typical stem or root and has a huge hydraulic conductivity compared to other tissues (e.g. Ks of parenchyma is = 10~9 m2 MPa"1 s"1

[Raven, 1993], four to six orders of magnitude lower than those measured in this study for wood segments). Without this high wood hydraulic conductivity terrestrial plants would never have reached the heights we currently observe in most forests. Measured hydraulic conductivities can be scaled up to the tree level to provide a rough idea of the amount of water that can be transported by a whole individual of the study species. If it is assumed that measured Ks is representative of above-ground parts, the flow of water (7) through the trunk can be obtained from:

J = Ks A s VM* Equation 2

If we take, for example, a 20 cm-diameter Q. ilex tree and consider a reasonable gradient of water potential (Vi); = 0.1 MPa m"1], we obtain that 7=2 .4 x 10~6 m3 s_l = 8.5 1 h"1. This value is reasonably similar to the actual flows estimated for Q. ilex trees of the same size in a similar forest (Martinez-Vilalta et al, unpublished) and gives us an idea of the magnitude of the flow that can be supported by the xylem.

What determines the hydraulic conductivity of the xylem? As a first approximation xylem conduits can be assimilated to capillaries and, consequently, it is possible to describe the water flow across them using the Hagen-Poiseuille equation:

N n v y 11 K, Equation 3 * i 8 p

where N is die number of functional conduits, R. is die radius of die i conduit (in m), and u, is the viscosity of water (1.002 x 10"9

MPa s at 20C). In angiosperms xylem water transport occurs basically through relatively wide and long multicellular conduits, termed vessels (diameter = 10-200 |xm). In conifers, which do not have vessels, water moves dirough smaller conduits (tracheids], typically less than 10 u,m in diameter (see Figure 3). The dependence of hydraulic conductivity on conduit radius raised to the fourth power (Equation 3) explains why Ks and KL were lowest in Abies sp. (see Figure 2]. Although we have not tried it in the practicals, it is easy to cut wood sections from the measured segments using a hand microtome or a razor blade and, with the

Figure 3 Photographs of representative cross-sections of the xylem of an angiosperm (left, Quercus ilexj and a conifer (right, Pinus pineaj. Both photographs are at the same scale (white bar length = 100 fim). Note the difference in the general structure of the xylem and, particularly, the much wider conduits in the angiosperm.

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aid of a microscope, obtain representative values of conduit size and density for each study species (see Ewers and Fisher, 1989 for detailed methods]. These values can be used to estimate a theoretical Kh max. Alternatively, representative values (if they are known] can be provided by the tutor. Theoretical calculations normally overestimate measured conductivities because of the existence of resistances not accounted for in Equation 3 (e.g. inter-conduit membranes].

The fact that initial hydraulic conductivity increased after flushing the segments (i.e. PLC > 0) is a clear illustration of one of the main drawbacks of the mechanism of sap ascent in plants. In most plants xylem hydraulic conductivity under field conditions is substantially lower than Ki because of air-filled, nonfunctional conduits. What explains the presence of high levels of embolism in some of the study species (Figure 2c]? In our case it is probably a combination of residual summer embolism (Q. ilex, for example is known to be highly vulnerable to drought-induced xylem embolism; Martinez-Vilalta et al, 2002] and freezing-induced xylem embolism, particularly in winter deciduous species (see Table 1].

Embolism and the timing of leaf fall are known to be related: high levels of embolism during the autumn are associated with early leaf fall and vice versa (Hacke and Sperry, 2001]. The apparent exception of A. glutinosa, a deciduous species with very low PLC, arises from the fact that this species was sampled at the bottom of the valley while all the other species were sampled in the mid slopes of the same valley. In the area where A. glutinosa was sampled, soil water potentials were probably close to 0 and, therefore, no (or little] embolism could be formed.

Words of caution Before concluding, several words of caution are needed. It has been shown that a considerable insight can be gained from the consideration of plant xylem as a tissue composed of dead cells, acting as passive conduits. However, recent studies suggest that living parenchyma cells within the xylem may have a more important role in water transport than previously thought, for example in the refilling of previously embolised conduits (Holbrook and Zwieniecki, 1999].

Moreover, there is some degree of controversy on the validity of the cohesion-tension theory (Zimmermann et al, 2000], although the vast majority of researchers agree on its main aspects. Finally, in this paper we have adopted a relatively simple view, focusing on xylem water transport, without explicitly considering other aspects, notably soil-root and leaf-atmosphere interfaces, which together with xylem transport determine whole plant water economy.

Acknowledgements We would like to thank the students of second year of biology at the Universitat Autonoma de Barcelona (academic years 2001 - 2002 and 2002 - 2003], who obtained all the results we present here and improved the practical with their suggestions. The comments by two anonymous reviewers substantially improved the manuscript.

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Appendix

Suppliers LI-COR, inc., 4421 Superior St., Lincoln, NE 68504, USA. Tel: 402-467-3576; Fax: 402-467-2819; Email: envsales@licor.com.

Jordi Martinez-Vilalta (corresponding author) is currently Research Fellow at the School of GeoSciences, University of Edinburgh, Edinburgh EH9 3JU, UK. Tel: +44 131 6505427. Fax: +44 131 6620478. Email: jordi.martinez-vilalta@ed.ac.uk Josep Pinol is Professor of Ecology at the CREAF/ Ecology Unit at the Universitat Autbnoma de Barcelona. Email: Josep.Pinol@uab.es.

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