American Journal of Botany 93(7): 993–1000. 2006.

DIRECT MEASUREMENTS OF INTERVESSEL PIT MEMBRANE HYDRAULIC RESISTANCE IN TWO ANGIOSPERM SPECIES1

BRENDAN CHOAT,2,4 TYLER W. BRODIE,2 ALEXANDER R. COBB,2 MACIEJ A. ZWIENIECKI,3 AND N. MICHELE HOLBROOK2

2Department of Organismic and Evolutionary Biology, Harvard University. Cambridge, Massachusetts 02138 USA; and 3Arnold Arboretum, Harvard University, Cambridge, Massachusetts 02138 USA

The hydraulic resistance of pit membranes was measured directly in earlywood vessels of americana and Ulmus americana. The area-specific resistance of pit membranes (rmem) was higher than modeled or measured values obtained 3 1 3 1 previously for hardwood species, with rmem of 5.24 3 10 MPasm for Fraxinus and 2.56 3 10 MPasm 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 rmem 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 .

Key words: Fraxinus; resistance partitioning; Ulmus; xylem vessel.

The transport of water to substantial heights against the force pit membranes is more difficult to measure or model because of gravity at rates sufficient to allow the net uptake of CO2 is little is known about the fine structure of pit membranes. a topic that has fascinated botanists for centuries. achieve Indeed, the contribution of pits to vascular resistance has been this feat by utilizing the hydrogen bonding between water largely ignored in models describing the movement of water molecules to maintain a column of liquid water under tension through plants (West et al., 1999). Currently, empirical data are (Zimmermann, 1983). However, water under tension is available from studies comparing lumen and pit membrane susceptible to spontaneous phase changes from liquid to vapor resistances using a variety of indirect methods. For example, (cavitation), which results in embolism that can severely limit Schulte and Gibson (1988) estimated pit membrane resistivity water transport. To counteract this potentially lethal problem, of six tracheid-bearing species by dissolving pit membranes plants employ a highly compartmentalized transport system of with cellulase and reported that pit membrane resistivity xylem conduits that allows failures in the integrity of the water accounted for 14–84% of the total resistivity. Using scaled-up column to be isolated from functioning conduits. Conduits are physical models, Lancashire and Ennos (2002) calculated the separated by partially hydrolyzed primary cell walls known as resistance of conifer bordered pits at 29% of total resistance for pit membranes that permit the movement of water from one a ‘‘typical’’ tracheid. More recently, values for resistance of conduit to the next but limit the movement of gas and pathogens. angiosperm pit membranes have been provided by models For pit membranes to perform their protective function, pore (Sperry and Hacke, 2004) and measurements on progressively sizes must be small; average pore diameters in the pit membranes shortened stems (Sperry et al., 2005; Wheeler et al., 2005). of dicotyledonous tree species are estimated to be 5–20 nm Sperry et al. (2005) found that end wall resistivity averaged (Choat et al., 2003). However, such small pore sizes generate 54% of total resistivity across a range of xylem structures, and significant hydraulic resistance. Thus, water moving through the this result was confirmed in a broader range of vessel-bearing xylem encounters two principal resistances, the resistance due to species (Wheeler et al., 2005; Hacke et al., 2006). the conduit walls as it moves along the conduit longitudinally The fact that pit membranes may account for a high proportion of vascular resistance is particularly important in the context of (lumen resistance) and the resistance due to pitted walls and pit the ion mediated ‘‘hydrogel’’ response attributed to pit membrane membranes as it moves laterally between conduits (Fig. 1). polysaccharides. Zwieniecki et al. (2001b) demonstrated that While the resistance of vessels with simple perforation plates xylem hydraulic resistance could be significantly altered by can be accurately predicted from the Hagan–Poiseuille changes in the ionic concentration of xylem sap. A high pit equation (Zwieniecki et al., 2001a), the resistance of pits and membrane resistance would facilitate hydrogel regulation of vascular resistance, allowing plants to respond to short-term 1 Manuscript received 13 January 2006; revision accepted 18 April 2006. environmental variation. This would be particularly advanta- The authors thank J. Wilson for assistance with measurements and R. geous in the complex branching canopies of mature trees in which Schalek for help with electron microscopy. This research was supported by grants from the Andrew W. Mellon Foundation, the USDA (NRICGP hydraulic resistance and evaporative demand may vary greatly. 2001-35100-10615) and the National Science Foundation (IBN 0078155). While a range of techniques has been employed to obtain 4 Author for correspondence (e-mail: [email protected]); present estimates of pit membrane or end wall resistance, pit resistance address: Department of Viticulture and Enology, University of has not been directly measured. We used the microcapillary California, Davis, Davis, California 95616 USA; phone: (530) 752- technique of Zwieniecki et al. (2001a) to measure the hydraulic 0380; fax: (530) 752-0382 resistance of intervessel pit membranes in two ring porous tree 993 994 AMERICAN JOURNAL OF BOTANY [Vol. 93

Fig. 2. Experimental technique used to measure pitted wall and pit membrane hydraulic resistance. Perfusing solution was driven across a section of pitted wall (shown in grey) between two adjacent xylem vessels. Flow rates across the wall were measured as the mass of liquid coming onto an analytical balance. The ratio of pit membrane to pitted wall area (a) was used to calculate area-specific resistance of pit membranes.

Fig. 1. Simplified representation of vessel connections in angio- sperms. As water moves through the vascular system, it encounters two Cambridge, Massachusetts, between June and August of 2004. Branches 1–2 principal resistances: resistance due to the lumen of the vessel and m in length were cut from mature trees using extension shears and returned to resistance due to movement through pitted walls that connect vessels. the laboratory in sealed plastic bags. In the laboratory, smaller stem segments of Vessels are connected end on end in a longitudinal file. The total length of 2–4 cm in length were cut from the branches in the 2-yr age zone. The ends of each vessel (L) is subdivided into the length of overlap with another vessel these segments were shaved with a fresh razor blade and flushed with a filtered (l) and the length of unconnected vessel lumen plus one overlap (L0). The 10 mmol/L KCl solution at 100 kPa to remove any emboli present in the stem. length unit L0 gives the average distance traveled by a water molecule as it The transverse section of the cut stem was viewed using a stereomicroscope, and a glass microcapillary was inserted into an earlywood vessel in the passes from one vessel to the next in the file. The length of overlap l is outermost growth ring. The vessels selected were directly adjacent to used to calculate the area of pitted wall and pit membrane connecting two a neighboring vessel in cross section, with which it shared a section of pitted vessels in the file. wall. The capillary was glued in place using cyanoacrylic glue (Locite 409, Henkle Corp., Rocky Hill, Connecticut, USA) and a dilute safranin dye solution (0.01% w/v in deionized water) was perfused through the vessel at species, Fraxinus americana Marsh. and Ulmus americana L. a low pressure (’50 kPa) to identify the vessel at the distal end of the segment. These species were selected because the large diameters of Measurements made during protocol development indicated that the safranin their earlywood xylem vessels (60–120 lm) facilitate measure- dye solution did not increase the hydraulic resistance of the pitted wall. The ments on individual vessels using the microcapillary technique. segment was then trimmed with a razor blade from the distal end until a direct connection between the two neighboring vessels could be seen. The vessel into In addition, the two species provide a contrast in pit structure, which the microcapillary was glued was distinguished from its connected as Fraxinus has small pits with thick pit membranes (Sano, neighbor by injecting air into the vessel while observing the distal cut end 2005), while Ulmus has large pits with thinner membranes. To under the stereomicroscope. A second microcapillary was then glued into the place our measurements of pit resistance in their hydraulic neighboring vessel at the distal end. Fluid could then be driven across the section of pitted wall between the neighboring vessels (Fig. 2). context, we compared them with lumen resistance for vessels A reservoir of perfusing solution (10 mmol/L KCl, filtered to 0.2 lm) was of average dimensions to determine the relative contribution of placed into a pressure chamber (Model 600, Moisture Stress, Corvallis, each component to the total hydraulic resistance. Oregon, USA) and a section of PEEK tubing with one end submerged in the solution was connected to the proximal microcapillary by a swagelok fitting. The distal microcapillary was connected by water-filled tubing to a beaker on an analytical balance (0.01 mg resolution) and a pressure between 0.2 to 0.5 MATERIALS AND METHODS MPa was applied to the pressure chamber to drive flow across the segment. The 3 hydraulic resistance of the segment (Rh MPasm ) was calculated as Rh ¼ DP/ Pit membrane hydraulic resistance—Branches of Fraxinus americana and F, where F is the flow rate of solution onto the balance in m3s1 and DP is the Ulmus americana were collected from the Harvard University campus, pressure drop across the segment in MPa. Preliminary experiments indicated July 2006] CHOAT ET AL.—DIRECT MEASUREMENTS OF PIT MEMBRANE HYDRAULIC RESISTANCE 995 that the hydraulic resistance of the glass microcapillaries was negligible Partitioning of vascular resistance—The hydraulic resistance of vessel compared with the resistance of the pitted walls. The surface area of wall lumen and pit membrane components was compared on the basis of average connecting the neighboring vessels was measured after cutting serial transverse vessel dimensions and the values of rmem obtained experimentally. sections (30 lm thick) every 120 lm along the stem segment with a sliding The Hagen–Poiseuille equation was used to calculate the resistance of the microtome. Images of the stained vessels were then collected at 2003 lumen: magnification and analyzed using ImageJ software (National Institute of 0 Health, Bethesda, Maryland, USA). The perimeter of the pitted wall that 128gL R ¼ ð1Þ connected the two vessels was measured in each section, and the surface area of lum pD4 the pitted wall between each section was calculated as the area of a trapezoid. The total area of pitted wall was calculated as the sum of these sections. The where g is the dynamic viscosity of water at 208C (1.002 3 109 MPas) and D average fraction of pit membrane area per pitted wall area (a) was measured is the diameter of the conduit. L0 was used for the length of the conduit because, from longitudinal sections through the earlywood xylem vessels of 2-yr-old on average, water will travel only this distance as it moves through the vessel branches from the same trees (Fig. 2). The surface area of pit membrane from one end wall to the next (Fig. 1): Hagen–Poiseuille flow involves no net 2 connecting the two vessels (Amem,inm ) was calculated as the product of pitted movement of water in the transverse plane (Wilcox, 1997) and a finite element wall area and pit membrane fraction. The area-specific resistance of the pit analysis of flow through an oblique perforation plate by Schulte and Castle 1 membrane (rmem, in MPasm ) was then calculated as rmem ¼ Rh Amem. The (1993) showed deviations from this pattern only in the immediate neighbor- resistance of an individual pit (Rind) was calculated as Rind ¼ rmem/Aind, where hood of the plate. The pit membrane resistance (Rmem) was calculated as the Aind is the surface area of an individual pit membrane. The resistance due to pit area-specific resistance (rmem) divided by the area of pit membrane between canals (Rcan) was calculated from measurements of pit canal length and radius ‘‘typical’’ vessels (Amem). The area of membrane between two vessels was using the Hagan–Poiseuille equation. Four measurements of pit resistance were calculated as Amem ¼ aDl. In this case, the diameter of the vessel is used made for each species, with each replicate coming from a separate mature tree. because the vessels take on a more elliptical form while connected to each Pit membrane structure was observed in each species using scanning other, with the length of the straight connecting wall equaling the initial electron microscopy. Samples branches were collected from the same trees on diameter of the vessels (Fig. 1). The resistance of the vessel was calculated which pit resistance measurements were undertaken and sections were cut from using the Hagen–Poiseuille formula for a vessel with the same cross-sectional 2-yr-old zone. Sections were split through the current year growth of earlywood area. More elaborate corrections are possible (Lewis, 1992) but of little effect vessels and air dried before being mounted on aluminum stubs with carbon when the deviations from a circular vessel cross-section are small, as here tape. Samples were coated with platinum/palladium for 20 s at 30 mA using (Sperry et al., 2005). Though a vessel tapers in the region of overlap, in this a sputter coater, and the sides of the wood samples were coated with colloidal region an increasing proportion of the flux is carried in the connecting vessel as carbon paste. This approach was designed to minimize the degree to which the the resistivity increases, so it was assumed that this would contribute little to the coating could obscure the fine features of the pit membranes. Samples were overall resistance. The total resistance (Rtot) of the conduit was then calculated observed with a field emission scanning electron microscope (FESEM, FEI, by adding the lumen and pit membrane resistances in series: Hillsboro, Oregon, USA) at an accelerating voltage of 2.5 to 3.0 kV. 0 128gL rmem Rtot ¼ 4 þ ð2Þ Vessel dimensions—The mean diameter of earlywood vessels was pD Amem measured from transverse sections of 2-yr-old branches and trunks of the four These calculations were made for a vessel of average length, diameter, and trees used in pit resistance measurements. For 2-yr-old branches, sections were overlap area to find the ratio of pit membrane resistance to total resistance taken from branches used for pit resistance measurements, with one section (R /R ). taken from each branch and at least 100 vessels measured in each section. For mem tot We also examined how the total resistivity (resistance per unit length R /L) trunks, cores were taken with an increment borer at breast height and 50–100 tot of the conduit varied with vessel length, assuming that the proportion of vessel vessels were measured in sections taken from these cores. overlap (l l/L) remained constant with the vessel length. In this case, the The mean vessel length for each species was measured by perfusing stem f ¼ diameter D, the fraction of pit membrane area per wall area a, and the overlap segments with a silicon elastomer (Rhodorsil RTV 141, Rhodia, Cranbury, New Jersey, USA) colored red with a pigment (LSDR-11, Dow Corning, fraction lf were set at the average value for each species, while the vessel length Kendallville, Indiana, USA) following methods outlined in Sperry et al. (2005). varied. Substituting for Amem and replacing l with lfL then gives Branch samples 1–2 m in length were cut from trees and brought to the Rtot 128gð1 lf Þ rmem laboratory. Branches were cut in the 2-yr-old growth region and flushed with ¼ 4 þ 2 ð3Þ filtered deionized water for 15 min at 100 kPa to remove emboli. Branches L pD alf DL were then placed into the pressure chamber and injected with the elastomer for Because vessel lengths could not be measured directly on the trunk, we 1 h at 0.5 MPa and cured in an oven for 1 h at 708C (Wheeler et al., 2005). Mean vessel length was estimated from the number of vessel endings in a given estimated vessel lengths from measurements of vessel diameter based on how length of stem. This approach, which is much less labor intensive than determining vessel length would scale with diameter if the ratio pit membrane resistance to the entire distribution of vessel lengths, was selected because our goal was to lumen resistance remained constant. If c ¼ Rmem/Rlum is the ratio of pit estimate partitioning between end wall and lumen resistance based on measured membrane to lumen resistances values of r and mean vessel length. Stem segments were cut close to the 2–3- 3 mem rmempD year terminal scar and injected in either the acropetal or basipetal direction. c ¼ 0 ð4Þ The number of vessel endings in a segment was calculated as the number of al128gL elastomer-filled vessels at the point of injection minus the number of filled vessels Substituting for L0 and l and solving for L then gives 10 cm from the injection point. We counted only the large earlywood vessels  because these were the vessels used in pit conductivity measurements. The mean 1 pr 1=2 vessel length was calculated as the length of the segment divided by the fraction of L ¼ mem D3=2 ð5Þ vessel endings within the segment. For each species, three stem segments were c lf ð1 lf Þa128g injected in the acropetal direction and three were injected in the basipetal direction. The average area of pitted wall overlap between vessels was measured using This shows that vessel length would have to scale with vessel diameter to the methods described by Wheeler et al. (2005). The fraction of the vessel length power of 1.5 to give the same ratio of pit membrane to lumen resistance in the along which two adjacent vessels overlapped was calculated as the ratio of trunk as in the branches. grouped vessels to the total of solitary and grouped vessels (with each cluster of vessels accounting for one group). The ratio L0/L was calculated as: L0/L ¼ 1 (length fraction)/2; the length fraction is divided by 2 because half of the total RESULTS overlap per vessel should be in one end of the vessel (Fig. 1). The actual length of overlap between two adjacent vessels (l) was calculated from measured mean vessel lengths as l ¼ L L0. Measurements were made on the same transverse Vascular anatomy—Earlywood vessels from 2-yr-old sections used for measurement of vessel diameter. For each species, four branches were both longer and wider in Fraxinus than in sections from 2-yr-old branches and four sections from trunks were analyzed. Ulmus (Table 1). Mean vessel diameter (6SE) was 79.9 (60.9) 996 AMERICAN JOURNAL OF BOTANY [Vol. 93

TABLE 1. Dimensions of earlywood xylem vessels and pits in 2-yr-old images suggested that membranes of Ulmus were generally branches of Fraxinus americana and Ulmus americana. Values are more porous and thinner than those of Fraxinus. means 6 SE (N ¼ 4–6) for xylem vessel diameter (D), vessel length (L), overlap length of pitted wall between vessels (l), fraction of pit Pit membrane hydraulic resistance—The area-specific pit membrane area in pitted wall (a), and surface area of individual pit membrane resistance differed between the two species with membranes (Aind). Parameters are for large earlywood vessels only. rmem being twice as great in Fraxinus as in Ulmus (Table 2). Dimension Fraxinus americana Ulmus americana The combination of low rmem and larger pits resulted in Rind of Ulmus pit membranes being an order of magnitude lower than D (lm) 79.9 (60.13) 72.5 (60.90) for pit membranes of Fraxinus. The magnitude of resistance 1 1 L (m) 5.30 (60.95) 3 10 1.65 (60.10) 3 10 values for individual pits indicated that the vast majority of l (m) 7.30 (60.87) 3 102 4.67 (60.27) 3 102 a 0.49 (60.03) 0.67 (60.02) resistance was in the pit membranes and that the geometry of 2 Aind (lm ) 12.5 (60.43) 53.5 (60.14) the pit canal and chamber had a negligible effect on the resistance values. The calculated resistance of pit canals (Rcan) for both species was three orders of magnitude less than values calculated for Rind. lminFraxinus and 72.5 (60.9) lminUlmus. In the trunk, mean vessel diameters were more than double that of 2-yr-old Partitioning of resistance—Based on the Rlum calculated for branches with 210.4 (614.2) lminFraxinus and 170.8 a vessel of mean length and diameter and the Rmem calculated (614.0) lminUlmus. In this study, the narrow latewood from the mean area of pit membrane overlap between vessels, vessels have been ignored for two reasons. First, all measure- the hydraulic resistance of pit membranes was 87% of the total ments of pit membrane resistance were made on earlywood resistance in Ulmus and 80% of the total in Fraxinus (Table 2). vessels, and there is evidence that pit membrane structure may The relationship of vessel resistivity to vessel length differ between earlywood and latewood vessels of ring porous demonstrates how the constraints placed on resistivity by pit trees (Jansen et al., 2004). Second, up to 95% of flow takes membrane resistance and lumen resistance change with length (Fig. 5). At shorter lengths, resistivity is high and dominated by place through earlywood vessels during the growing season; pit membrane resistance because of the larger area of pit therefore, they are of much greater physiological relevance membrane relative to connecting vessels. As vessel length during this period (Ellmore and Ewers, 1986). increases, resistivity is increasingly controlled by the lumen The mean vessel length for 2-yr-old branches of Fraxinus resistance until it approaches an asymptote (Hagen–Poiseuille was 0.53 (60.09) m, while the mean length of Ulmus vessels flow). Measured vessel lengths were well below the saturating was 0.16 (60.01) m. Vessels observed in transverse sections of lengths in both species. They were also below the vessel Fraxinus were more often solitary than in the wood of Ulmus lengths at which Rmem and Rlum become equal, those being 1.06 and this was reflected in the higher pitted wall overlap fractions m for Fraxinus and 0.43 m for Ulmus. This analysis assumes (l) measured for Ulmus (Table 1). The dimensions of pits that vessel diameter remains constant with changing vessel differed between the species, with pits of Fraxinus being length. In fact, vessel diameter increases with length, both smaller than those of Ulmus. Pit membranes of Fraxinus had within one region of the tree (wide earlywood vessels are a mean diameter of 3.9 lm, while membranes of Ulmus were longer than narrow latewood vessels) and when comparing 8.3 lm in diameter. Both species possess alternate pitting, with different age regions of the tree (vessel diameters from the the ratio of pit membrane area to pitted wall area (a) being trunk are twice as great as in 2-yr-old branches). Thus, the greater in Ulmus than in Fraxinus (Table 1). relationships shown in Fig. 5 are intended only to illustrate where vessels with the average dimensions measured for each Pit membrane ultrastructure—Pit membranes of earlywood species fit in the spectrum of possible vessel resistivities. vessels from both species were homogeneous in structure as is Equation 5 demonstrates that vessel length must increase at typical for hardwood species (Figs. 3, 4). Although some the power of 1.5 with vessel diameter in Fraxinus and Ulmus in Ulmus species are known to possess pit membranes with order for the ratio of Rmem/Rlum to remain constant. If the ratio a torus-margo structure, this was not the case in earlywood of Rmem/Rlum is assumed to be the same in the trunk as in 2-yr- vessels observed here (Fig. 4). Cellulose microfibrils were old branches, then mean vessel lengths calculated from the randomly arrayed in a tight meshwork in pit membranes of average diameters and overlap lengths observed in trunks are both species. Porosity varied between individual membranes; 2.24 m for Fraxinus and 0.67 m for Ulmus. However, it is possible that R /R is not constant between the young in some membranes openings of 10–50 nm were apparent, mem lum branches and the trunk, which would affect calculated values of while in others no openings could be detected. In general, pit mean vessel length. For instance, if Rmem and Rlum were equal membranes of Fraxinus were less likely to have detectable in the trunk then calculated mean vessel lengths would be 4.98 openings than pit membranes of Ulmus, and some membranes m and 1.74 m for Fraxinus and Ulmus, respectively. were encrusted with material that covered the cellulose microfibrils (Fig. 3c, d). Most membranes of Ulmus contained detectable openings within the range of 10–50 nm in diameter DISCUSSION indicating that cellulose microfibrils were less densely arrayed than in membranes of Fraxinus (Fig. 4b, c). In some cases, The area-specific hydraulic resistance of pit membranes was pores were obviously an artifact of preparation because the a significant component of vascular resistance in earlywood membrane had been deflected across the chamber and both vessels of Fraxinus americana and Ulmus americana, with 3 1 tearing and enlarged openings were visible (Fig. 4d). Despite rmem being 5.32 3 10 (60.39) MPasm for Fraxinus and evidence of these artifactual increases in membrane pore size, 2.56 3 103 (60.69) MPasm1 for Ulmus. The fact that pit July 2006] CHOAT ET AL.—DIRECT MEASUREMENTS OF PIT MEMBRANE HYDRAULIC RESISTANCE 997

Fig. 3. Scanning electron micrographs of intervessel pit membranes from earlywood vessels of Fraxinus americana. (a) Secondary wall has been removed to reveal pit membranes (arrow) between two vessels. (b) Pit membrane with some opening of 10–50 nm in diameter. (c) Pit membrane with no visible openings between microfibrils. (d) Pit membrane with encrusting material (arrow) partially covering cellulose microfibrils.

membranes of Fraxinus had a significantly higher rmem than observed with scanning electron microscopy may differ from those of Ulmus indicates that there were intrinsic differences in pit membranes in a hydrated state and thus caution must be the structure of the pit membranes between the two species in used when interpreting data from scanning electron micro- terms of thickness or porosity. Scanning electron micrographs graphs (Choat et al., 2003). revealed that pit membranes of Ulmus were generally more Estimates of individual pit resistance were four to five orders porous than those of Fraxinus. This is consistent with the of magnitude higher in Fraxinus and Ulmus than values given observations of Sano (2004, 2005), who noted that pit in previous studies from physical (Lancashire and Ennos, membranes of Fraxinus mandshurica were thicker than those 2002) and mathematical models (Sperry and Hacke, 2004). of three other dicot species he observed. It is also apparent Large differences in Rind could be expected between conifer from TEM observations that pit membranes of Fraxinus are margo-torus type pit membranes and homogeneous membranes thicker than those of Ulmus (B. Choat and S. Jansen [Royal of angiosperms because of the wider pore diameters found in Botanical Gardens, Kew, United Kingdom], unpublished data). the margo region and the larger size of conifer tracheid pit If pit membranes of Ulmus are thinner and more porous than membranes (Hacke et al., 2004). The area-specific resistance of those of Fraxinus, this would explain the lower rmem of Ulmus pit membranes was also higher than modeled or measured measured in this study. However, it must also be noted that the values reported for homogeneous angiosperm pit membranes structure and porosity of pit membranes in dried samples (Sperry and Hacke, 2004, Sperry et al., 2005, Wheeler et al., 998 AMERICAN JOURNAL OF BOTANY [Vol. 93

Fig. 4. Scanning electron micrographs of intervessel pit membranes from earlywood vessels of Ulmus americana. (a) Pit membranes with openings evident in most membranes (b) Pit membrane with many opening of 10–50 nm in diameter. (c) Pit membrane with fewer visible openings. (d) Deflection of a pit membrane across the pit chamber has resulted in tears and enlarged pores (arrow) in the area over the pit aperture.

2005). Modeled values of area-specific resistance for homo- is balanced against higher Rlum. This is supported by data geneous angiosperm pit membranes presented by Sperry and showing that mean area-specific resistance of pit membranes is Hacke (2004) were two to four orders of magnitude below higher for ring porous species than for diffuse porous species values measured here in Fraxinus and Ulmus. The area-specific (Hacke et al., 2006). pit resistances obtained empirically by Wheeler et al. (2005) Based upon measurements of mean earlywood vessel were higher than modeled values, ranging from 31–626 diameter and length and the average overlap of adjacent xylem MPasm1, but still lower than those measured in the present vessels, the resistance due to water moving through pit study. The higher values of rmem measured in Fraxinus and membranes was 80–87% of the total resistance for water flow Ulmus may relate to differences in xylem anatomy. With the through 2-yr-old branches. This is at the upper margin of exception of Vitis vinifera, all of the angiosperm species previous estimates, with Schulte and Gibson (1988) finding examined by Wheeler et al. (2005) were diffuse porous plants a range of 14–84% and Sperry et al. (2005) finding between with short vessels. Both Fraxinus and Ulmus are ring porous 31% and 76%. The higher ratio of Rmem/Rtot found in the species and have earlywood xylem vessels of much greater present study was mainly attributable to the higher values of lengths than those in diffuse porous trees (Zimmermann and rmem measured for Fraxinus and Ulmus. Estimates of Rmem Jeje, 1981). The higher rmem in these species is permissible were also influenced by the average length of overlap because of the higher Rlum due to long vessels, i.e., higher rmem calculated from solitary and grouped vessels in transverse July 2006] CHOAT ET AL.—DIRECT MEASUREMENTS OF PIT MEMBRANE HYDRAULIC RESISTANCE 999

TABLE 2. Parameters for pit membrane hydraulic resistance for 2-yr-old branches of Fraxinus americana and Ulmus americana. Empirical values are given for the area-specific resistance of pit membranes (rmem) and resistance of individual pits (Rind). Values of pit canal resistance (Rcan) were calculated using the Hagan–Poiseuille equation. Values for resistance of pit membranes (Rmem) and vessel lumen (Rlum) were calculated for vessels of average length, diameter, and wall overlap area using eq. 2. Values of rmem and Rind are means with SE in parentheses (N ¼ 4).

Hydraulic resistance Fraxinus americana Ulmus americana

1 3 3 rmem (MPasm ) 5.32 (60.39) 3 10 2.56 (60.69) 3 10 3 14 13 Rind (MPasm ) 4.26 (60.31) 3 10 4.74 (61.27) 3 10 3 11 10 Rcan (MPasm ) 3.68 (60.05) 3 10 2.80 (60.04) 3 10 3 9 9 Rmem (MPasm ) 1.79 3 10 1.13 3 10 3 8 8 Rlum (MPasm ) 4.55 3 10 1.79 3 10 Rmem/Rtot (%)80 87

Fig. 5. The relationship between total vessel hydraulic resistivity sections. Given the convoluted nature of vascular networks in (resistance per unit length) and vessel length (L) for vessels of mean angiosperm trees, the representation of vessel overlap (Fig. 1) diameter and overlap fraction in Fraxinus americana and Ulmus and the techniques used to estimate them may significantly americana. Hollow circles represent the mean vessel lengths measured underestimate the complexity of the actual flow path. for each species and filled circles represent the length at which pit The method used for measuring pit membrane resistance membrane and lumen resistivities are equal. The inset shows the here provides a rough estimate of overlap between two vessels percentage of total resistance made up by the lumen resistance with because the stem segment was cut back until two connected increasing vessel length. vessels could be seen at each end of the segment. The overlap of adjacent vessels was generally between 0.003 and 0.010 m another in the vertical series and that half of these connections in length, while the mean overlap lengths estimated from transverse sections were 0.073 m and 0.047 m for Fraxinus and would be below the vessel in question and half above. Ulmus, respectively. The difference between the observed and However, it is clear that further detailed analysis of plant estimated lengths of overlap suggests that many lateral vascular networks is required to test this assumption. connections exist along the length of earlywood vessels. This As noted in recent studies, when pit membrane and lumen is supported by reconstructions of the vascular network in resistances are considered together, resistivity (or conductivity) Fraxinus and Ulmus species (Zimmermann and Tomlinson, asymptotes at a certain vessel length and further decreases in 1967; Burggraaf, 1972; Newbanks et al., 1983; Kitin et al., resistivity are minimal (Sperry and Hacke, 2004; Sperry et al., 2004). 2005). Lancashire and Ennos (2002) demonstrated that when The contribution of lateral overlaps to water movement conduit length is constrained, the optimal diameter at which between vessels depends on the relative positions of the resistance is minimized is reached when pit and lumen overlapping vessels in the vertical file. If connections occur resistance are exactly co-limiting. This poses the question as between vessels in the same vertical plane, then no pressure to why vessels lengths are constrained below the point at which gradient will exist between them and therefore little exchange resistivity approaches an asymptote. From the perspective of of water will take place. However, if there is enough vertical reducing resistance along the xylem pathway, the optimal displacement between vessels with lateral connections, then vessel would be long, wide and have maximum pit membrane exchange of water will occur there as well as across the end overlap area with adjacent vessels. However, lowering walls. Unfortunately, no vessel endings were recorded in resistance of the conduit must be balanced against the previous three-dimensional (3-D) reconstructions of Fraxinus increasing vulnerability to embolism. The longer and wider species, so lateral overlaps cannot be compared with vessel an individual vessel, the greater the proportion of transport endings (Burggraaf, 1972, Kitin et al., 2004). In a reconstruc- capacity that will be lost if the vessel becomes embolized. In tion of Ulmus americana wood, many vessel endings can be addition, increasing surface area of pit membranes has been seen as well as lateral connections; both vessel endings and shown to increase the probability of air seeding occurring lateral connections run between 1 and 6 mm (Newbanks et al., between vessels (Choat et al., 2005; Wheeler et al., 2005). 1983, Zimmermann, 1983). Based on evidence from 3-D reconstructions, the overlap area estimated from transverse Wheeler et al. (2005) reported a significant inverse relationship sections is likely to more closely represent the total overlap between pit area per vessel and the tension at which a 50% loss between one vessel along its length and all surrounding vessels of hydraulic conductivity occurred in 15 angiosperm species. rather than simply the end wall connections between two This relationship occurs because the large pit membrane pores vessels directly above and below one another in a vertical file. responsible for air seeding are apparently rare and the chance Thus, the vessel configuration illustrated in Fig. 1 is of having a pit membrane with a large pore increases with a simplified form of the vascular network that does not increasing surface area of interface between two vessels (Choat represent the complexity of the actual network. For the et al., 2004; Wheeler et al., 2005). Therefore, increases in purposes of the analysis in this study, we assumed that all conduit dimensions worsens the impact of an embolism on connections between vessels, whether lateral or true end walls, conductivity, while increases in pit membrane interface area were interfaces at which water is transferred from one vessel to increase the probability of water-stress-induced embolism 1000 AMERICAN JOURNAL OF BOTANY [Vol. 93 occurring, as well as the probability of embolism propagating HACKE, U. G., J. S. SPERRY,J.K.WHEELER, AND L. CASTRO. 2006. Scaling through the system. of angiosperm xylem structure with safety and efficiency. Tree The analysis on which Fig. 5 is based assumes a constant Physiology 26: 619–701. diameter for xylem vessels as vessel length increases. Average JANSEN, S., B. CHOAT,S.VINCKIER,F.LENS,P.SCHOLS, AND E. SMETS. 2004. vessel diameters increase in the basipetal direction for most tree Intervascular pit membranes with a torus in the wood of Ulmus species (Zimmermann, 1983) and vessel diameters in the trunks (Ulmaceae) and related genera. New Phytologist 163: 51–59. KITIN, P. B., T. FUJII,H.ABE, AND R. FUNADA. 2004. Anatomy of the vessel of Fraxinus and Ulmus were more than double those in 2-yr- network within and between tree rings of Fraxinus lanuginosa old branches. The idea that lumen and end wall resistances may (). American Journal of Botany 91: 779–788. be equal in hardwood species has been discussed in the LANCASHIRE, J. R., AND A. R. ENNOS. 2002. Modelling the hydrodynamic literature for some time (Zimmermann and Brown, 1971); resistance of bordered pits. Journal of Experimental Botany 53: Hacke et al. (2006) reported that, on average, pit and lumen 1485–1493. resistances were co-limiting in diffuse and ring porous tree LEWIS, A. M. 1992. Measuring the hydraulic diameter of a pore or conduit. species although there was significant spread around the 1:1 American Journal of Botany 79: 1158–1161. line. Equal partitioning of resistance was not observed in 2-yr- NEWBANKS, D., A. BOSCH, AND M. H. ZIMMERMANN. 1983. Evidence for old branches of Fraxinus and Ulmus although it is possible that xylem dysfunction by embolization in dutch elm disease. Phytopa- the resistance is partitioned differently in other regions of the thology 73: 1060–1063. tree. Mean vessel lengths calculated for the trunk, assuming SANO, Y. 2005. Inter- and intraspecific structural variations among that / remained constant, were 2.5 m for and intervascular pit membranes as revealed by field-emission scanning Rmem Rlum Fraxinus electron microscopy. American Journal of Botany 92: 1077–1084. 0.67 m for Ulmus; for lumen and pit membrane resistances to SANO, Y. Z. 2004. Intervascular pitting across the annual ring boundary in be equal, the vessel lengths calculated from mean diameters Betula platyphylla var. japonica and Fraxinus mandshurica var. and wall overlaps in the trunk were 4.4 m and 1.4 m for japonica. International Association of Wood Anatomists Journal 25: Fraxinus and Ulmus, respectively. 129–140. Zimmermann and Jeje (1981) measured vessel lengths in SCHULTE, P. J., AND A. C. GIBSON. 1988. Hydraulic conductance and Fraxinus americana trees and found that in the trunk, vessels tracheid anatomy in 6 species of extant seed plants. Canadian Journal were commonly 5–10 m in length. Unfortunately, similar of Botany 66: 1073–1079. vessel length data does not exist for Ulmus, but Newbanks et SCHULTE, P. J., AND A. L. CASTLE. 1993. Water-flow through vessel al. (1983), referring to unpublished data, stated that the perforation plates—a fluid mechanical approach. 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