Pit Membrane Porosity Research Articles

Drought-induced xylem pit membrane damage in aspen and balsam poplar.

Drought induces an increase in a tree's vulnerability to a loss of its hydraulic conductivity in many tree species, including two common in western Canada, trembling aspen (Populus tremuloides) and balsam poplar (Populus balsamifera). Termed 'cavitation fatigue' or 'air-seeding fatigue', the mechanism of this phenomenon is not well understood, but hypothesized to be a result of damage to xylem pit membranes. To examine the validity of this hypothesis, the effect of drought on the porosity of pit membranes in aspen and balsam poplar was investigated. Controlled drought and bench dehydration treatments were used to induce fatigue and scanning electron microscopy (SEM) was used to image pit membranes for relative porosity evaluations from air-dried samples after ethanol dehydration. A significant increase in the diameter of the largest pore was found in the drought and dehydration treatments of aspen, while an increase in the percentage of porous pit membranes was found in the dehydration treatments of both species. Additionally, the location of the largest pore per pit membrane was observed to tend toward the periphery of the membrane.

Nobody’s perfect: can irregularities in pit structure influence vulnerability to cavitation?

Recent studies have suggested that species-specific pit properties such as pit membrane thickness, pit membrane porosity, torus-to-aperture diameter ratio and pit chamber depth influence xylem vulnerability to cavitation. Despite the indisputable importance of using mean pit characteristics, considerable variability in pit structure within a single species or even within a single pit field should be acknowledged. According to the rare pit hypothesis, a single pit that is more air-permeable than many neighboring pits is sufficient to allow air-seeding. Therefore, any irregularities or morphological abnormalities in pit structure allowing air-seeding should be associated with increased vulnerability to cavitation. Considering the currently proposed models of air-seeding, pit features such as rare, large pores in the pit membrane, torus extensions, and plasmodesmatal pores in a torus can represent potential glitches. These aberrations in pit structure could either result from inherent developmental flaws, or from damage caused to the pit membrane by chemical and physical agents. This suggests the existence of interesting feedbacks between abiotic and biotic stresses in xylem physiology.

Linking irradiance‐induced changes in pit membrane ultrastructure with xylem vulnerability to cavitation

The effect of shading on xylem hydraulic traits and xylem anatomy was studied in hybrid poplar (Populus trichocarpa x deltoides, clone H11-11). Hydraulic measurements conducted on stem segments of 3-month-old saplings grown in shaded (SH) or control light (C) conditions indicated that shading resulted in more vulnerable and less efficient xylem. Air is thought to enter vessels through pores in inter-vessel pit membranes, thereby nucleating cavitation. Therefore, we tested if the ultrastructure and/or chemistry of pit membranes differed in SH and C plants. Transmission electron micrographs revealed that pit membranes were thinner in SH, which was paralleled by lower compound middle lamella thickness. Immunolabelling with JIM5 and JIM7 monoclonal antibodies surprisingly indicated that pectic homogalacturonans were not present in the mature pit membrane regardless of the light treatment. Porosity measurements conducted with scanning electron microscopy were significantly affected by the method used for sample dehydration. Drying through a gradual ethanol series seems to be a better alternative to drying directly from a hydrated state for pit membrane observations in poplar. Scanning electron microscopy based estimates of pit membrane porosity probably overestimated real porosity as suggested by the results from the 'rare pit' model.

Cell wall-degrading enzymes enlarge the pore size of intervessel pit membranes in healthy and Xylella fastidiosa-infected grapevines.

The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the PMs of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined; gold particles of known size were introduced with the water to assist in determining the size of PM pores. The effect of introducing trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), oligogalacturonides, and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the PMs, was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4- beta -glucanase) were infused into stems, and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierce's disease.

The effects of sap ionic composition on xylem vulnerability to cavitation

Recent evidence of ion-mediated changes in pit membrane porosity suggests that plants may modulate the hydraulic conductance of their xylem conduits. Under the current paradigm, membrane porosity also determines conduit vulnerability to water stress-induced cavitation. Therefore, the hypothesis of an ion-mediated regulation of xylem vulnerability to cavitation in trees was tested. Segments of five Angiosperm and two Gymnosperm species were infiltrated with ultra-pure deionized water as a reference fluid or with a 50 mM KCl solution. KCl had a strong impact on segment conductance with either a positive or a negative effect across species. When 1 mM CaCl2 was added to the reference solution, the effect of KCl was minimized for most species. By contrast, segment vulnerability to cavitation was only slightly influenced by the presence of KCl in the solution. From this it was concluded that the mechanisms controlling pit membrane permeability to water flow and its resistance to the penetration of air bubbles are largely uncoupled, which suggests that the hypothesis of a porous structure of pit membranes should be revisited.

Morphological variation of intervessel pit membranes and implications to xylem function in angiosperms

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 (r(2) = 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 (r(2) = 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.

Structure and function of bordered pits: new discoveries and impacts on whole‐plant hydraulic function

Bordered pits are cavities in the lignified cell walls of xylem conduits (vessels and tracheids) that are essential components in the water-transport system of higher plants. The pit membrane, which lies in the center of each pit, allows water to pass between xylem conduits but limits the spread of embolism and vascular pathogens in the xylem. Averaged across a wide range of species, pits account for > 50% of total xylem hydraulic resistance, indicating that they are an important factor in the overall hydraulic efficiency of plants. The structure of pits varies dramatically across species, with large differences evident in the porosity and thickness of pit membranes. Because greater porosity reduces hydraulic resistance but increases vulnerability to embolism, differences in pit structure are expected to correlate with trade-offs between efficiency and safety of water transport. However, trade-offs in hydraulic function are influenced both by pit-level differences in structure (e.g. average porosity of pit membranes) and by tissue-level changes in conduit allometry (average length, diameter) and the total surface area of pit membranes that connects vessels. In this review we address the impact of variation in pit structure on water transport in plants from the level of individual pits to the whole plant.

Scaling of angiosperm xylem structure with safety and efficiency

We tested the hypothesis that greater cavitation resistance correlates with less total inter-vessel pit area per vessel (the pit area hypothesis) and evaluated a trade-off between cavitation safety and transport efficiency. Fourteen species of diverse growth form (vine, ring- and diffuse-porous tree, shrub) and family affinity were added to published data predominately from the Rosaceae (29 species total). Two types of vulnerability-to-cavitation curves were found. Ring-porous trees and vines showed an abrupt drop in hydraulic conductivity with increasing negative pressure, whereas hydraulic conductivity in diffuse-porous species generally decreased gradually. The ring-porous type curve was not an artifact of the centrifuge method because it was obtained also with the air-injection technique. A safety versus efficiency trade-off was evident when curves were compared across species: for a given pressure, there was a limited range of optimal vulnerability curves. The pit area hypothesis was supported by a strong relationship (r2 = 0.77) between increasing cavitation resistance and diminishing pit membrane area per vessel (A(P)). Small A(P) was associated with small vessel surface area and hence narrow vessel diameter (D) and short vessel length (L)--consistent with an increase in vessel flow resistance with cavitation resistance. This trade-off was amplified at the tissue level by an increase in xylem/vessel area ratio with cavitation resistance. Ring-porous species were more efficient than diffuse-porous species on a vessel basis but not on a xylem basis owing to higher xylem/vessel area ratios in ring-porous anatomy. Across four orders of magnitude, lumen and end-wall resistivities maintained a relatively tight proportionality with a near-optimal mean of 56% of the total vessel resistivity residing in the end-wall. This was consistent with an underlying scaling of L to D(3/2) across species. Pit flow resistance did not increase with cavitation safety, suggesting that cavitation pressure was not related to mean pit membrane porosity.

Changes in pit membrane porosity due to deflection and stretching: the role of vestured pits.

The effect of increasing pressure difference (DeltaP) on intervessel pit membrane porosity was studied in two angiosperm tree species with differing pit architecture. Fraxinus americana L. possesses typical angiosperm bordered pit structure while Sophora japonica L. exhibits well-developed vestures in intervessel pit chambers. It was hypothesized (a) that large DeltaP across intervessel pits would cause the deflection of pit membranes in the stems of F. americana resulting in significant increases in porosity and thus lower cavitation thresholds, and (b) that the presence of vestures would prevent the deflection of pit membranes in S. japonica. To determine if the porosity of pit membranes increased under mechanical stress, suspensions of colloidal gold, 5 nm and 20 nm in diameter, were perfused across intervessel pit membranes at DeltaP ranging from 0.25 MPa to 6.0 MPa. The effect of increasing DeltaP on membrane porosity was also tested by comparing air seeding thresholds (Pa) in stems perfused with water or a solution with lower surface tension. Air seeding and colloidal gold experiments indicated that pit membrane porosity increased significantly with DeltaP in F. americana. In S. japonica, increases in permeability to colloidal gold with DeltaP were small and maximum pore diameters predicted from Pa were independent of DeltaP, suggesting that vestures limited the degree to which the membrane can be deflected from the centre of the pit cavity. This provides the first experimental evidence that vestures reduce the probability of air seeding through pit membranes.

Pit membrane porosity and water stress-induced cavitation in four co-existing dry rainforest tree species.

Aspects of xylem anatomy and vulnerability to water stress-induced embolism were examined in stems of two drought-deciduous species, Brachychiton australis (Schott and Endl.) A. Terracc. and Cochlospermum gillivraei Benth., and two evergreen species, Alphitonia excelsa (Fenzal) Benth. and Austromyrtus bidwillii (Benth.) Burret., growing in a seasonally dry rainforest. The deciduous species were more vulnerable to water stress-induced xylem embolism. B. australis and C. gillivraei reached a 50% loss of hydraulic conductivity at -3.17 MPa and -1.44 MPa, respectively; a 50% loss of hydraulic conductivity occurred at -5.56 MPa in A. excelsa and -5.12 MPa in A. bidwillii. To determine whether pit membrane porosity was responsible for greater vulnerability to embolism (air seeding hypothesis), pit membrane structure was examined. Expected pore sizes were calculated from vulnerability curves; however, the predicted inter-specific variation in pore sizes was not detected using scanning electron microscopy (pores were not visible to a resolution of 20 nm). Suspensions of colloidal gold particles were then perfused through branch sections. These experiments indicated that pit membrane pores were between 5 and 20 nm in diameter in all four species. The results may be explained by three possibilities: (a) the pores of the expected size range were not present, (b) larger pores, within the size range to cause air seeding, were present but were rare enough to avoid detection, or (c) pore sizes in the expected range only develop while the membrane is under mechanical stress (during air seeding) due to stretching/flexing.

Functional and ecological xylem anatomy

Abstract Cohesion-tension transport of water is an energetically efficient way to carry large amounts of water from the roots up to the leaves. However, the cohesion-tension mechanism places the xylem water under negative hydrostatic pressure ( P x ), rendering it susceptible to cavitation. There are conflicts among the structural requirements for minimizing cavitation on the one hand vs maximizing efficiency of transport and construction on the other. Cavitation by freeze-thaw events is triggered by in situ air bubble formation and is much more likely to occur as conduit diameter increases, creating a direct conflict between conducting efficiency and sensitivity to freezing induced xylem failure. Temperate ring-porous trees and vines with wide diameter conduits tend to have a shorter growing season than conifers and diffuse-porous trees with narrow conduits. Cavitation by water stress occurs by air seeding at interconduit pit membranes. Pit membrane structure is at least partially uncoupled from conduit size, leading to a much less pronounced trade-off between conducting efficiency and cavitation by drought than by freezing. Although wider conduits are generally more susceptible to drought-induced cavitation within an organ, across organs or species this trend is very weak. Different trade-offs become apparent at the level of the pit membranes that interconnect neighbouring conduits. Increasing porosity of pit membranes should enhance conductance but also make conduits more susceptible to air seeding. Increasing the size or number of pit membranes would also enhance conductance, but may weaken the strength of the conduit wall against implosion. The need to avoid conduit collapse under negative pressure creates a significant trade-off between cavitation resistance and xylem construction cost, as revealed by relationships between conduit wall strength, wood density and cavitation pressure. Trade-offs involving cavitation resistance may explain the correlations between wood anatomy, cavitation resistance, and the physiological range of negative pressure experienced by species in their native habitats.

Architecture of Branch-root Junctions in Maize: Structure of the Connecting Xylem and the Porosity of Pit Membranes

The architecture of the connecting xylem network in the vascular plexus linking branch and main root vessels has been examined using cryoSEM, and the limiting porosity of the network determined with tracers (dye, and particles of known size: latex, polystyrene and gold sols). Dye and water move freely throughout the xylem network, while particles are constrained to follow tortuous vessel-like conduits of irregularly-shaped elements linked by large-diameter perforations. These conduits end at special pit membranes (boundary pit membranes) at the periphery of main root vessels. Particles accumulate on the outer side of these filters, often filling the terminal elements of these conduits adjacent to the main root vessels. Some vessel elements within the plexus are isolated from the convoluted conduits by normal pit membranes, and often also from each other, by pit membranes and still-intact end walls in otherwise mature elements. These extra-conduit elements may be an auxiliary filtering system. The boundary pit membranes filtered out particles with mean diameters as small as 4.9±0.7nm, indicating a pore size one or two orders of magnitude smaller than most previous measurements for pit membranes, but close to pore sizes determined for hydrated primary cell walls. It is concluded that boundary pit membranes at branch-root junctions are efficient filters for microbes and particulates entering damaged branch roots. They would also restrict entry of air/water interfaces when main root xylem tension was less than approx. 100MPa.

The Relationship Between Pit Membrane Ultrastructure and Chemical Impregnability of Wood

Summary The woods of Alder and Eucalypt were examined by light microscopy before and after a chemical treatment by the Indurite process to increase the hardness of the wood. The pattern of wood cell impregnation for Alder differed significantly from Eucalypt in some respects. In Alder wood all cell types eg. vessels, fibres and rays, were impregnated in similar proportions. In comparison, in Eucalypt wood the impregnation material was largely confined to ray cells and the lumina of vessels; other cell types were either not impregnated or impregnated in very small numbers. Transmission electron microscopy of Alder and Eucalypt woods suggests that ultrastructural differences in the texture and porosity of pit membranes may be the main reason for the observed differences between these wood species with regard to their impregnability by the impregnation material used.