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(American Journal of Botany. 2009;96:409-419.) doi: 10.3732/ajb.0800248 © 2009 Botanical Society of America, Inc. |
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Anatomy and Morphology |
2 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, TW9 3DS, Surrey, UK 3 Functional Ecology Group, Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia
Received for publication 21 July 2008. Accepted for publication 25 November 2008.
ABSTRACT
Pit membranes between xylem vessels have been suggested to have functional adaptive traits because of their influence on hydraulic resistance and vulnerability to embolism in plants. Observations of intervessel pit membranes in 26 hardwood species using electron microscopy showed significant variation in their structure, with a more than 25-fold difference in thickness (70–1892 nm) and observed maximum pore diameter (10–225 nm). In some SEM images, pit membrane porosity was affected by sample preparation, although pores were resolvable in intact pit membranes of many species. A significant relationship (r2 = 0.7, P = 0.002) was found between pit membrane thickness and maximum pore diameter, indicating that the thinner membranes are usually more porous. In a subset of nine species, maximum pore diameter determined from SEM was correlated with pore diameter calculated from air-seeding thresholds (r2 = 0.8, P < 0.001). Our data suggest that SEM images of intact pit membranes underestimate the porosity of pit membranes in situ. Pit membrane porosity based on SEM offers a relative estimate of air-seeding thresholds, but absolute pore diameters must be treated with caution. The implications of variation in pit membrane thickness and porosity to plant function are discussed.
Key Words: bordered pit • cavitation • embolism • hardwoods • pit membrane • vessel element • wood anatomy • xylem
In the xylem tissue of hardwood species, water and nutrients are transferred between vessels through bordered pits. As water moves from the roots to the leaves it must traverse intervessel pit membranes hundreds or thousands of times. It has long been recognized that pit membranes play a pivotal role in maintaining the integrity of the water transport system in plants (Zimmermann and Brown, 1971
). While allowing the passage of water between xylem vessels, pit membranes must also protect the plant against the spread of gas (embolism) or pathogens through the vascular system (Tyree and Sperry, 1989; Choat et al., 2008). Thus, pit membrane structure must balance the competing functional requirements of minimizing vascular resistance, which favors thin, porous membranes, and limiting the spread of embolism and microbes, which requires robust membranes and smaller pores.
Much attention has been paid to pit membrane ultrastructure of conifer species, in which the conductive and protective functions of the membranes have been spatially resolved into the margo and torus regions (e.g., Liese and Fahnenbrock, 1952; Petty and Preston, 1969; Thomas, 1969; Bauch et al., 1972; Dute et al., 2008). The occurrence and structure of torus-margo pit membranes in angiosperms has also been the focus of several studies, although this pit membrane type is rare in angiosperms (Ohtani and Ishida, 1978; Dute and Rushing, 1987; Dute et al., 2004; Jansen et al., 2004a, 2007). Less anatomical work has focused on the "homogeneous" membranes characteristic of hardwood species, which have a relatively uniform density of microfibrils across the membrane (Schmid, 1965). Early studies emphasized differences between the ultrastructure of intervessel pit membranes and other types of pit membranes (e.g., vessel parenchyma), but indicated little variation in intervessel pit membranes of species examined (Schmid, 1965; Schmid and Machado, 1968; Yang, 1978, 1986). Because preparation of samples for transmission electron microscopy (TEM) is time consuming, most studies include only a few species. Published TEM observations of angiosperm pit membranes are indeed restricted to fewer than a dozen species of hardwoods (Wheeler, 1983). More recently, scanning electron microscope (SEM) studies by Sano (2004, 2005) revealed considerable differences in the structure of intervessel pit membranes of four hardwood species. Such variation in pit membrane structure is predicted to have important consequences for vascular function.
Physiological studies have demonstrated that pit membranes are responsible for at least 50% of the hydraulic resistance in the xylem (Wheeler et al., 2005; Choat et al., 2006; Hacke et al., 2006). Changes in the thickness and porosity of the pit membranes therefore have the potential to exert significant influence over the total hydraulic resistance in the plant. The porosity of pit membranes is also related to the vulnerability of xylem to water stress induced embolism; it is generally believed that water-stress induced cavitation occurs as a result of gas being drawn through pit membrane pores, a process known as air seeding (Zimmermann, 1983; Tyree and Sperry, 1989). Larger pit membrane pores should allow embolism to spread through the xylem at lower tensions (higher xylem water potentials) than smaller pores. Species with more porous membranes, or with more pit membrane area between vessels, will therefore be more prone to vascular dysfunction in the face of water stress. The increase in hydraulic resistance resulting from xylem embolism can impact leaf water status and gas exchange, ultimately resulting in die back of affected organs or the whole plant (Rood et al., 2000). Therefore, the influence of pit membrane structure on both hydraulic resistance and vulnerability to embolism indicate that it is an important adaptive trait with the potential to drive ecological differences between species. Despite this, few detailed examinations of pit membrane structure across a wide range of species have been undertaken.
One major concern in studies examining wood micromorphology using electron microscopy is the alteration of ultrastructural features during sample preparation (Choat et al., 2003; Pesacreta et al., 2005; Jansen et al., 2008). For SEM observations, preparation can involve harsh chemical treatments, dehydration of tissue (either air drying or critical point drying) and placement under vacuum during observation. It is reasonable to suspect that subtle features such as pit membrane porosity may have been altered by these processes. Sectioning or splitting of wood sampling may also cause damage to pit membranes. Shane et al. (2000) examined pit membranes of maize roots using cryoSEM to preserve membranes in a hydrated state and reported that pit membranes frequently developed large pores or tears as a result of even partial dehydration before observation. However, some conventional SEM studies have failed to reveal visible pores in the pit membranes of hardwood species (Wheeler, 1981, 1983; Choat et al., 2003). One possible explanation for not observing the pores in pit membranes with SEM is provided by a recent study employing atomic force microscopy (AFM), in which pit membranes were examined in nondried and dried states (Pesacreta et al., 2005). They observed that dried pit membranes had a much more compact appearance compared with that of nondried membranes in which the hydrated microfibrils were loosely arranged. The AFM observations indicate that pores present in hydrated membranes may become obscured by the contraction of microfibrils or redistribution of noncellulosic substances as the membrane dries. Thus, there is some uncertainty over the in situ porosity of pit membranes based on different methods of microscopy.
We undertook an examination of pit membrane structure in 26 hardwood species. We used SEM and TEM because the complementary information offered by these techniques allows for a greater chance of detecting artifacts that may result from preparation, particularly in SEM (Shane et al., 2000; Thorsch 2000). While preparation for the more commonly used SEM technique may cause damage to pit membranes by drying (Jansen et al., 2008), TEM provides an alternative method in which the pit membrane structure is stabilized and supported by resin during sectioning and observation. Observations with SEM and TEM were compared with air-seeding experiments, which provide an estimate of pit membrane pore size in intact and hydrated stem tissue. The species examined included plants from a range of environments including temperate deciduous hardwoods, riparian trees, and Mediterranean evergreens. The principal aims were to determine the level of variation in intervessel pit structure present in these species and to assess the reliability of electron microscopy in assessing pit membrane porosity.
MATERIALS AND METHODS
Plant material
The species studied are listed in Table 1 with reference to their family classification, origin, and collection period. All samples included fresh material from two- to four-year-old branches. All SEM and TEM observations and measurements were based on vessel elements from the current and last year’s growth ring to avoid possible degradation or changes in pit porosity and pit membrane structure caused by age or heartwood formation (Wheeler, 1981, 1983; Sano et al., 1999). Between one and three branch samples were studied per species. The few differentiating vessel elements that could be seen in the TEM material collected during spring and summer were easy to distinguish from the fully developed vessel elements with mature pit membranes by the presence of cytoplasm and more electron-dense pit membranes.
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Transmission electron microscopy
Samples were cut into 2-mm3 blocks and fixed overnight in Karnovsky’s fixative at room temperature (Karnovsky, 1965). After washing in 0.05 M phosphate buffer, the specimens were postfixed in 1% buffered osmium tetroxide for 4 h at room temperature, washed again, and dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, 100%). The ethanol was gradually replaced with LR White resin (London Resin Co., Reading, UK) over several days, with the resin changed approximately every 12 h. The resin was polymerized in a Weiss Gallenkamp (Loughborough, U.K.) vacuum oven at 60°C and 1000 mm Hg for 18–24 h. Embedded samples were trimmed with a razor blade or with a Leica EM specimen trimmer (Leica Microsystems, Vienna, Austria) and sectioned on an ultramicrotome (Ultracut, Reichert-Jung, Vienna, Austria). Transverse sections 1 and 2 µm thick were cut with a glass knife, heat-fixed to glass slides, stained with 0.5% toluidine blue O in 0.1 M phosphate buffer (O’Brien et al., 1964), and mounted in DPX (Agar Scientific, Stansted, UK). Resin-embedded material was prepared for TEM by cutting transverse, ultrathin sections between 60 and 90 nm using a diamond knife. The sections were attached to Formvar grids or 300 mesh hexagonal copper grids (Agar Scientific) and stained with uranyl acetate and lead citrate using an LKB 2168 ultrostainer (LKB-Produkter AB, Bromma, Sweden) or a Leica EM Stain Ultrostainer (Leica Microsystems, Vienna, Austria). Observations were carried out using a Jeol JEM-1210 TEM (JEOL, Tokyo, Japan) at 80 kV accelerating voltage and digital images were taken using a MegaView III camera (Soft Imaging System, Münster, Germany). A minimum of five intervessel pit fields was examined per species. Image analysis was undertaken using ImageJ software with at least 25 measurements for each quantitative pit feature. Variation in membrane thickness was analyzed with a one-way ANOVA, and differences between means were assessed with a Tukey honestly significant difference (HSD) test.
Air-seeding experiments
Branches were collected during August and September 2007 from mature trees growing at the living collections of the Royal Botanic Gardens, Kew. Air-seeding experiments were applied to Acacia pataczekii, Acer negundo, Aesculus hippocastanum, Betula pendula, Laurus nobilis, Populus fremontii, and Salix alba and were supplemented with air-seeding values for Fraxinus americana and Sophora japonica as obtained by Choat et al. (2004) using the same method. We were unable to apply this method to all species studied for microscopy (Table 1) because the vessels were either too narrow (<40 µm) or too long (>30 cm). All branch material was similar in size and age and from the same tree as the samples used for SEM and TEM. The air-seeding threshold for intervessel pit membranes was measured in individual vessels of the last two growth rings as outlined by Melcher et al. (2003) and Choat et al. (2004). Briefly, small branch segments were cut under water from lateral branches, trimmed with a razor blade at each end, and flushed with a filtered (0.22 µm) and degassed 10 mM KCl solution. Most branch segments selected were <1 cm in diameter. For each species, the maximum vessel length was estimated using the air perfusion technique. Because the air-seeding threshold probably depends on the number of end-walls penetrated, we selected segments that were shorter than the maximum vessel length to reduce the possibility that there was more than one end wall. The mean stem length varied from <8 cm in Betula pendula to >15 cm in Acacia pataczekii, Cladrastis sinensis, Laurus nobilis, and Populus fremontii. Glass microcapillaries (1 µm diameter) were pulled on a pipette puller (Pul1, World Precision Instruments, Sarasota, Florida, USA). The tip of a capillary was broken off, such that the opening of the tip was slightly narrower than the vessel diameter (40–150 µm), and inserted into the lumen of an individual vessel from the last two growth rings using a Leica Wild M8 stereozoom microscope. The tip was then glued in place using cyanoacrylic glue (Loctite 409 gel in combination with Loctite 7455 activator; Henkel Loctite Adhesives Ltd., Welwyn Gardens City, UK). Air was pushed through the vessel at low pressure (ca. 0.1 MPa). We assumed that the vessel was ending in the branch segment and that an intact pit field existed in the sample if no bubbles appeared at the distal end. Samples in which air bubbles streamed out at this pressure were discarded. The pressure threshold required to force gas across intervessel pits field was determined by attaching the glass microcapillary to a pressure source (Model 1000, PMS Instrument, Albany, New York, USA). Nitrogen gas was then applied, and the pressure was increased in 0.1 MPa steps at 1-min intervals until air bubbles appeared at the distal end of the branch as could be seen with a 10x hand lens. A constant flow of small air bubbles was interpreted as the threshold pressure for gas penetration of the intervessel pit membranes between the two vessels.
Theoretical pore diameters (DT) were calculated from air-seeding thresholds as DT = 4
cos
/Pa, where is the surface tension of water and is the contact angle at the air–water–membrane interface. The contact angle was treated as 0 because of the hydrophilic nature of pectins coating the cellulose microfibrils. Relationships between variables were plotted as linear regressions or as power laws by log-transforming data when relationships appeared nonlinear (Sokal and Rohlf, 1995).
RESULTS
A survey of quantitative pit characteristics is given in Table 2.
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Pit membrane porosity
In TEM micrographs, there were no obvious channels spanning pit membranes, although in species with very thin membranes it was difficult to determine if pores were present because of the low electron density of material in the membranes. Images obtained using SEM, however, varied greatly in the apparent porosity of pit membranes. Intact pit membranes of Laurus nobilis (Fig. 1A), Acacia pataczekii, Citrus reticulata, Olea europaea, and Quercus robur (Fig. 1B) had no visible pores. For Fraxinus americana and Arbutus uva-ursii, the majority of membranes had no resolvable pores, but pores in a few membranes were up to 30 and 70 nm in diameter, respectively. In several species, pores were of intermediate appearance, with a maximum pore diameter between 50 and 100 nm visible in many, but not all pit membranes, e.g., Sophora japonica (Fig. 1C) and Acer negundo (Fig. 1D). Acer pseudoplatanus, Adenocarpus decorticans, Aesculus hippocastanum, Betula pendula, Cordia macrostachya, Corylus avellana, Populus fremontii (Fig. 1E), Salix alba (Fig. 1F), and Ulmus procera had membranes with a fragile appearance, numerous pores, and a max pore size between 166 nm and 234 nm.
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Pit membrane thickness
The thickness of pit membranes varied by more than 25-fold difference in magnitude among the study species, with pit membranes of Acacia pataczekii having a mean (±SD) thickness of 1183 (±442) nm, while pit membranes of S. alba had an average thickness of 70 (±15) nm. Very thin pit membranes with an average thickness up to 100 nm also occurred in Aesculus hippocastanum, Betula ermanii (Fig. 3C), and Populus fremontii (Fig. 3D). The pit aperture outline could frequently be seen through the thin pit membrane when viewed under SEM (Fig. 1E, 1F). The majority of the species studied had an average thickness between 100 nm and 300 nm. An average thickness above 500 nm was recorded in Acacia pataczekii, Ailanthus altissima, Arbutus uva-ursii (Fig. 3A), Citrus reticulata (Fig. 3E), Laurus nobilis (Fig. 3B), and Olea europaea. Thinner pit membranes (e.g., Aesculus hippocastanum, Salix alba, Populus fremontii) were often almost transparent under TEM, indicating less densely packed material than in pit membranes with higher electron density (e.g., Laurus nobilis, Citrus reticulata, and Fraxinus americana; Fig. 3B, 3E, 3F), or differences in the chemical nature of pit membranes.
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Microfibrils
The outermost cellulose microfibrils in pit membranes were oriented randomly (Fig. 1B), and layers underneath usually had a more parallel arrangement (Fig. 2A, 2C). The microfibrils were frequently incrusted by amorphous material. Sometimes microfibrils seemed to extend from its pit membrane layer into its surrounding vessel wall. These microfibrils may have detached from the pit membrane and deposited on the secondary vessel wall during sample preparation. The mean microfibril thickness for all species studied based on SEM was on average 20.17 nm (±4.32). In samples of Citrus reticulata and Laurus nobilis, the microfibrils were not well defined, probably because of incrustations on the pit membrane. Electron-dense dots were consistently observed in TEM sections of transparent pit membranes of Acer pseudoplatanus, Adenocarpus decorticans, Betula ermanii (Fig. 3C), B. pendula, Cordia macrostachya, Ilex aquifolium, and Sambucus nigra. Although these dots were similar in size as the microfibrils measured using SEM, significant differences were found for Aesculus hippocastanum, Betula ermanii, and Populus fremontii, suggesting that the electron-dense dots as observed in TEM sections could either be microfibrils or granular inclusions of variable size.
Pit membrane ultrastructure
Intervessel pit membranes were composed of uniformly deposited fibrillar material, frequently with a less electron-dense inner layer than the outer layers of the pit membrane. Pit membranes with a clearly layered composition were found in most but not all intervessel pits of Fraxinus americana (Fig. 3F), Laurus nobilis (Fig. 3B), Olea europaea, Paulownia tomentosa, and Ulmus procera. All species studied had an annulus near the outer edge of the pit membrane. In general, the annulus was more electron dense, but thinner than the central area of the membrane. While intervessel pit membranes were transparent in most species, most pit membranes in Laurus nobilis (Fig. 3B) and Citrus reticulata (Fig. 3E) were more electron dense than other parts of the intervessel cell wall.
Air-seeding thresholds
Single vessel air-seeding measurements ranged from 0.9 to 2.8 MPa for nine species examined. There were correlations between air-seeding threshold (Pa) and maximum pore diameter (r2 = 0.87, P = 0.005, Fig. 4D), and mean pore diameter (r2 = 0.79, P = 0.006, Fig. 4E), and pit membrane thickness (r2 = 0.52, P = 0.03). Because air seeding will always occur at the largest pore first, pore diameters calculated from air-seeding values should represent the maximum rather than the average pore diameter. When circular pore diameters were calculated from Pa, there were significant correlations between calculated diameters and maximum (r2 = 0.8, P < 0.001, Fig. 4F) and mean pore diameter (r2 = 0.6, P < 0.0001). Calculated pore diameters were always larger than the maximum pore diameters measured from SEM images.
DISCUSSION
There was wide variation in the structure of intervessel pit membranes of the 26 angiosperm species examined, especially with respect to differences in thickness and maximum pore size. This variation is consistent with the finding of Sano (2004, 2005), who observed significant interspecific variation in the pit membrane structure of temperate hardwood species. Here we present the first record of variation in pit membrane structure in hardwood species from a range of provenances including riparian, temperate, and Mediterranean environments. This large variation in pit structure is of potentially great adaptive value given the important role of pit membranes in xylem transport (Zimmermann, 1983; Choat et al., 2008). However, because of the concerns arising from artifacts in pit membrane structure caused by sample preparation for electron microscopy, it is prudent to deal with methodological concerns before further discussing the ecological and physiological implications of our results. In particular, we emphasize the value of complimentary techniques such as SEM, TEM, and air-seeding thresholds in evaluating the fine structure of pit membranes.
Pit membrane structure and methodological concerns
In the current study, pit membranes of many species had visible pores when viewed with SEM. Some images revealed that layers of the pit membrane may be easily removed during preparation, substantially increasing the apparent membrane porosity. Previous studies have shown that there are multiple layers of microfibrils in intervessel pit membranes of hardwood species with various layers often having different orientation of microfibrils (Schmid and Machado, 1968; Sano, 2005). It appears that layers of the primary wall, which make up the pit membrane, may be easily removed when wood sections are split to expose pit fields. From this, it is tempting to conclude that many of the large pores observed with SEM are the result of layers being removed from the membrane. However, there were many instances in which pores could be seen in pit membranes that appeared intact, and in some cases, large pores were visible in pit membranes viewed through the inner aperture of the pit canal of an undamaged secondary wall (Fig. 2D). These observations indicate that pores of large diameter were not always caused by removal of cell wall material.
In comparing the TEM and SEM results of this study, pit membranes that appeared more porous in scanning electron micrographs (Aesculus hippocastanum, Betula ermanii, B. nigra, B. pendula, Corylus avellana, Populus fremontii, Salix alba, Ulmus americana, and U. procera) were also the thinnest membranes when viewed with TEM. Pores spanning the breadth of pit membranes were not discernable with TEM, perhaps because most pit membrane pores are narrower than the TEM sections (60–90 nm) (Schmitz et al., 2007). In pit membranes with a greater mean thickness than 250 nm, pores were generally not resolvable in SEM images (Fig. 4A). It is assumed that the pathway of pores through thicker pit membranes is more complex than those through thin membranes because of the differing orientation of successive layers of microfibrils and that pores of thicker membranes are therefore easily obscured when the membrane is dried. Moreover, the width of the microfibrils as measured using SEM does not seem to be associated with pit membrane thickness, indicating that the pit membrane thickness is mainly determined by the number of microfibril layers.
Air-seeding thresholds should measure the pressure difference at which gas will penetrate pit membranes separating an embolized and functional vessel under natural conditions. The correlation of Pa with maximum pore diameter and mean pit membrane thickness shows that pit membrane characteristics observed with SEM and TEM bear a consistent relationship to the ability of pit membranes to resist air seeding. The relationship between maximum and theoretical pore sizes (Fig. 4F) suggests that SEM observations of porosity bear a consistent relationship with in situ porosity of membranes. However, maximum pore diameters observed with SEM were always 2–4 times smaller than theoretical maximum pore diameters, particularly in species with thicker membranes and no resolvable pores in SEM images. This finding suggests that (1) pores become enlarged by stretching during air seeding due to the pressure difference across the membrane and/or (2) that the large pores responsible for air seeding are rare and not often detected with SEM (Choat et al., 2003, 2004). Therefore, SEM and TEM measurements of membrane structure appear to provide reliable information on the relative ease with which gas can penetrate between vessels, i.e., thinner pit membranes will be more easily damaged or are more likely to have naturally occurring large pores than thick membranes. However, the absolute values for pore diameter derived from SEM should be treated as suspect and complemented with other techniques such as air seeding and particle perfusion experiments (Choat et al., 2005). It would be interesting to know the appearance of the pit membrane after the air-seeding experiments. Previous experiments did not reveal any evidence that pit membranes of Fraxinus americana and Sophora japonica ruptured when exposed to high pressures (6 MPa), suggesting that at least in these species deflection and stretching of the pit membranes does not result in irreversible changes in pit membrane porosity (Choat et al., 2004).
Physiological and ecological implications of variation in pit structure
The large variation in pit membrane thickness and average pit membrane pore diameter observed in this study is expected to have a significant effect on the area-specific hydraulic resistance of the pit membrane (rmem). Given that pit resistance is estimated to account for half of total xylem resistance, this variation could influence hydraulic efficiency of the xylem significantly. However, recent evidence from physiological studies has suggested that the trade-off between vulnerability to embolism and hydraulic resistance does not take place at the pit level (Wheeler et al., 2005). Across a range of species, there was no correlation between rmem and vulnerability to embolism; species with lower rmem, suggesting higher average porosity, were not necessarily more vulnerable to embolism (Hacke et al., 2006). An explanation for this result is based on the hypothesis that large pit membrane pores responsible for air seeding are rare, i.e., they only occur in one of the many pit membranes that connect two vessels (Choat et al., 2003). In this situation, vulnerability to embolism scales with the total surface area of pit membrane connecting vessels, with the chance of a large pore occurring increasing in a stochastic fashion with increasing surface area. The "pit area" hypothesis is supported by a strong relationship between pit membrane area between vessels and vulnerability to embolism (Wheeler et al., 2005).
In light of new evidence demonstrating large variation in pit membrane structure across species, it seems unlikely that pit structure is playing a negligible role in this trade-off. In species with thinner, more porous membranes, there should be a greater chance of having a large pore for a given area of membrane overlap compared with species that have thicker membranes. Therefore, we would expect significant variation in vulnerability to embolism between species with different membrane thickness and porosity even if overlap area of pit membrane is constant. In support of this, plots of vulnerability to embolism vs. pit overlap area show considerable variation in vulnerability to embolism for a given pit area (Hacke et al., 2006), suggesting that variation in pit structure explains significant differences in vulnerability to embolism within a narrow band of pit area. However, caution must be exercised when using pit anatomical measurements to directly infer physiological function. For instance, previous measurements indicate that Laurus nobilis is more vulnerable to embolism than Acer negundo, which has thinner and more porous pit membranes (Hacke and Sperry, 2003). It is now apparent that vulnerability to embolism results from the interaction of both tissue level properties (vessel diameter, length, and overlap area) and pit level structure (porosity, thickness, and aperture dimensions). Further work incorporating hydraulic measurements is required to unravel the relative importance of pit level and tissue level structure in the ability of plants to persist in differing environments and to assess the adaptive value of the large variation observed in pit membrane structure in this study.
Differences in thickness are likely to influence the degree to which pit membranes are damaged or altered structurally by mechanical deformation. Deflection and stretching of pit membranes has been shown to result from large pressure differences that develop between embolized and functional xylem vessels (Choat et al., 2004). The thickness of pit membranes is expected to play a significant role in the degree to which pit membranes would be susceptible to rupture or enlargement of existing pores. The relationship between Pa and pit membrane thickness suggests that there is little gain in resistance to air seeding after a certain thickness has been reached; there was no difference in Pa between Laurus nobilis and Acacia patazcekii despite a twofold difference in thickness. This observation may be because there was a larger area of pit membrane (more pits) between vessels of Acacia patazcekii and therefore a greater chance of a large pore being present (see earlier pit area hypothesis). The size of the pit aperture is also likely to be important in determining the likelihood of pit membrane damage due to stretching, because a larger aperture will leave more of the pit membrane unsupported. Thus, species such as Salix alba that have thin membranes and wide pit apertures should be more easily damaged than species with thick membranes and small pit apertures such as Laurus nobilis.
The correlation between pit membrane thickness and secondary cell wall thickness provides some insights into the structural basis of the strong correlation between vulnerability to cavitation and wood density (Hacke et al., 2001; Cochard et al., 2008). Conduits with thicker secondary walls for a given lumen diameter (thickness to span ratio) are more resistant to implosion resulting from pressure differences that develop between embolized and functional conduits. Hacke et al. (2001) demonstrated that cavitation generally occurs via air seeding before the pressure difference across the pitted wall is great enough to cause implosion. Some structural features must underlie the scaling between air-seeding thresholds and vessel implosion, lowering the risk of permanent damage to xylem conduit. If thinner pit membranes are associated with increased vulnerability to cavitation, a structural link is provided for the correlative relationship between thickness to span ratio and vulnerability to cavitation. Of course, other structural features such as pit overlap area (Wheeler et al., 2005) and fiber wall thickness (Jacobsen et al., 2005; Pratt et al., 2007) must also play a role in what is likely to be a complex relationship between vulnerability to cavitation and wood density.
Vestured pits occur in the three legume species studied (Acacia patazcekii, Adenocarpus decorticans, and Sophora japonica). In addition, sparsely developed vestures occur in Fraxinus americana (Fig. 3F), while all other species examined had nonvestured pits (Jansen et al., 2001). No apparent correlation is found between the presence of vestures and structural aspects such as pit membrane thickness. However, there is a significant relationship (P = 0.0012) between the air-seeding threshold and the distribution of vestures: air-seeding thresholds in Acacia patazcekii, Sophora japonica, Fraxinus americana, and two additional legume species (Cladrastis sinensis, Robinia pseudoacacia; S. Jansen, personal observation) have an average air-seeding threshold of 2.39 MPa ± 0.61 (N = 30), while the average air-seeding threshold for all nonvestured pit samples tested in this study is 1.71 MPa ± 0.88 (N = 28). Interestingly, intervessel pit membranes in Cladrastis sinensis and Robinia pseudoacacia are relatively thin (on average 119 ± 25 nm and 119 ± 35 nm, respectively; SJ personal observation), indicating that vestures may support the pit membrane and reduce the degree to which the pit membrane can be deflected, which in turn increases the air-seeding value (Zweypfenning, 1978; Jansen et al., 2003, 2004b; Choat et al., 2004). Species without vestured pits, however, seem to require a thick pit membrane to provide a high air-seeding value. Further study on a larger number of species with vestured pits is required to generalize the hydraulic safety that vestured pits may provide.
FOOTNOTES
1 The authors thank D. Chatelet and T. Rockwell for collecting plant material. This study was supported by a grant from the Royal Society (2006/Rl) and NERC (NE/E001122/1), and a Lennox Boyd fellowship (RBG, Kew). S.J. and B.C. contributed equally to the publication. 
4 Author for correspondence (e-mail: s.jansen{at}kew.org.uk)
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40 pits per species). Dmaxdata could not be obtained for species in which pores were not resolvable under SEM. Pit membrane thickness (Tm) and total intervessel cell wall thickness (Tw) were measured from TEM micrographs (N
