Structural Variation and Polysaccharide Profiling of Intervessel Pit Membranes

Functional roles of intervessel pit membrane (PM) depend on its structure and polysaccharide composition, which are mostly unknown or lack of accurate information. This study uses grapevine as a model plant and an immunogold-scanning electron microscopy technique to simultaneously analyze structures and polysaccharide compositions of intervessel PMs in relation to their functions. Intervessel PMs with different structural integrity were found in functional xylem with about 90 % of them being intact with a smooth or relatively smooth surface and the rest 10 % with progressively degraded structures. The results also elucidated details of the removal process of wall materials from surface toward its depth during the natural intervessel PM degradation. Four groups of pectic and hemicellulosic polysaccharides were present in intervessel PMs but displayed differential spatial distributions and quantities: weakly methyl-esterified homogalacturonans abundant in the surficial layers, heavily methyl-esterified homogalacturonans and xylans mostly in deep layers, and fucosylated xyloglucans relatively uniform in presence at different depths of an intervessel PM. This information is crucial to reveal the polysaccharide profiling of primary cell wall and to understand intervessel PM's roles in the safety and regulation of water transport as well as the plant susceptibility to vascular diseases.

The Widened Pipe Model of plant hydraulic evolution

Shaping global water and carbon cycles, plants lift water from roots to leaves through xylem conduits. The importance of xylem water conduction makes it crucial to understand how natural selection deploys conduit diameters within and across plants. Wider conduits transport more water but are likely more vulnerable to conduction-blocking gas embolisms and cost more for a plant to build, a tension necessarily shaping xylem conduit diameters along plant stems. We build on this expectation to present the Widened Pipe Model (WPM) of plant hydraulic evolution, testing it against a global dataset. The WPM predicts that xylem conduits should be narrowest at the stem tips, widening quickly before plateauing toward the stem base. This universal profile emerges from Pareto modeling of a trade-off between just two competing vectors of natural selection: one favoring rapid widening of conduits tip to base, minimizing hydraulic resistance, and another favoring slow widening of conduits, minimizing carbon cost and embolism risk. Our data spanning terrestrial plant orders, life forms, habitats, and sizes conform closely to WPM predictions. The WPM highlights carbon economy as a powerful vector of natural selection shaping plant function. It further implies that factors that cause resistance in plant conductive systems, such as conduit pit membrane resistance, should scale in exact harmony with tip-to-base conduit widening. Furthermore, the WPM implies that alterations in the environments of individual plants should lead to changes in plant height, for example, shedding terminal branches and resprouting at lower height under drier climates, thus achieving narrower and potentially more embolism-resistant conduits.

Artifactual lipid coatings on intervessel pit membranes in dried xylem tissues of some angiosperms

Intervessel pit membranes are recognized as key structures for influencing water flow/embolism resistance. The mechanisms remain largely unclear owing to difficulties in examining them intact in nature. This study investigates ethanol-extractable pit membrane incrustations (PMIs), which were previously reported in certain angiosperms and may affect their water conduction. The presence of PMIs was determined for 40 angiosperms by field-emission scanning electron microscopy (FE-SEM). Candidate components of PMIs were determined by chemical analyses of wood extracts, and their distributions in the xylem were examined by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Cryo-TOF-SIMS and cryo-FE-SEM were also performed to clarify the native distribution of PMIs. PMIs were observed in 11 species. Some of them were categorized as fat trees, which are known to store abundant lipids. Tilia japonica sapwood displaying PMIs contained large amounts of lipids, which were distributed in the dried xylem tissue, consistent with the distribution of the PMIs. In the frozen samples of T. japonica, however, the distributions were restricted to the parenchyma. In conclusion, PMIs consist of an artifactual coating of lipids originated from the parenchyma in dried samples at room temperature. Researchers performing surface analyses of plant cell walls should take strong precautions against such self-coating by these intrinsic chemicals.

Hydraulic consequences of enzymatic breakdown of grapevine pit membranes

Xylella fastidiosa (Xf) is the xylem-dwelling bacterial agent associated with Pierce’s Disease (PD), which leads to significant declines in productivity in agriculturally important species like grapevine (Vitis vinifera). Xf spreads through the xylem network by digesting the pit membranes between adjacent vessels, thereby potentially changing the hydraulic properties of the stem. However, the effects of Xf on water transport varies depending on the plant host and the infection stage, presenting diverse outcomes. Here, we investigated the effects of polygalacturonase, an enzyme known to be secreted by Xf when it produces biofilm on the pit membrane surface, on stem hydraulic conductivity and pit membrane integrity. Experiments were performed on six grapevine genotypes with varying levels of PD resistance, with the expectation that pit membrane resistance to degradation by polygalacturonase may play a role in PD-resistance. Our objective was to study a single component of this pathosystem in isolation to better understand the mechanisms behind reported changes in hydraulics, thereby excluding the biological response of the plant to the presence of Xf in the vascular system. Pit membrane damage only occurred in stems perfused with polygalacturonase. Although the damaged pit membrane area was small (2-9% of the total pit aperture area), membrane digestion led to significant changes in the median air-seeding thresholds, and most importantly, shifted frequency distribution. Finally, enzyme perfusion also resulted in a universal reduction in stem hydraulic conductivity, suggesting the development of tyloses may not be the only contributing factor to reduced hydraulic conductivity in infected grapevine.

Three-dimensional imaging of xylem at cell wall level through near field nano holotomography

Detailed imaging of the three-dimensionally complex architecture of xylary plants is important for studying biological and mechanical functions of woody plants. Apart from common two-dimensional microscopy, X-ray micro-computed tomography has been established as a three-dimensional (3D) imaging method for studying the hydraulic function of wooden plants. However, this X-ray imaging method can barely reach the resolution needed to see the minute structures (e.g. pit membrane). To complement the xylem structure with 3D views at the nanoscale level, X-ray near-field nano-holotomography (NFH) was applied to analyze the wood species Pinus sylvestris and Fagus sylvatica. The demanded small specimens required focused ion beam (FIB) application. The FIB milling, however, influenced the image quality through gallium implantation on the cell-wall surfaces. The measurements indicated that NFH is appropriate for imaging wood at nanometric resolution. With a 26 nm voxel pitch, the structure of the cell-wall surface in Pinus sylvestris could be visualized in genuine detail. In wood of Fagus sylvatica, the structure of a pit pair, including the pit membrane, between two neighboring fibrous cells could be traced tomographically.

Theoretical determination of pit membrane natural frequency for destruction by resonance effect

The low permeability of many wood species causes significant problems during processing. Industrial methods used for increasing wood permeability reduce strength properties, are energy consuming, and are not viable economically. Destruction of pit membranes in wood cell walls can provide an increase in wood permeability without affecting wood strength properties. It can be accomplished using resonance applied to the pit membranes. Theoretical analysis and calculations have been performed to determine pit membrane (torus and margo) natural frequency. Membrane natural frequencies of bordered pits of Norway spruce are in the range of 3 to 11 MHz. Water in the pit chamber did not have a significant effect on the resonant frequency of the membrane. The main limitation of the amplitude of membrane fluctuations inside the pit chamber was the width of the chamber. Two methods to initiate resonance frequency for pit membrane destruction have been suggested, namely, alternating electric field application and microwave energy pulsation.

Characterization and comparison of the wood anatomical traits of plantation grown Quercus acutissima and Quercus variabilis

Oaks are important tree species, providing essential biomaterial for the wood industry. We characterize and compare wood anatomical traits of plantation grown Quercus acutissima Carruth. and Q. variabilis Blume to provide more detailed information to understand xylem radial growth, structure, and function, as well as differences between sapwood and heartwood, to provide data relevant for tree breeding and value-added wood utilization of oak plantations in China. In this study, radial strips were collected at breast height from the main trunk of the two species. Latewood percentage and growth ring width were investigated by X-ray densitometry and a Tree Ring Analysis System, respectively. Vessel and fibre lumen diameter, vessel and fibre wall thickness, vessel density, fibre wall thickness/diameter ratio, tissue proportions, and pit membrane thickness in between vasicentric tracheids were observed with light microscopy and electron microscopy and quantified. There were significant differences in a few wood anatomical traits between the two species: vessel wall thickness and vessel lumen diameter were higher in Q. acutissima than in Q. variabilis, while higher axial parenchyma proportion in sapwood was found in Q. variabilis than in Q. acutissima. More abundant tyloses were found in heartwood than in sapwood of both species. Our work showed the intraspecific and interspecific variation of the two species. Most differences between sapwood and heartwood must be attributed to differences in cambial age during their formation.

Xylem network connectivity and embolism spread in grapevine(Vitis vinifera L.)

Xylem networks are vulnerable to the formation and spread of gas embolisms that reduce water transport. Embolisms spread through interconduit pits, but the three-dimensional (3D) complexity and scale of xylem networks means that the functional implications of intervessel connections are not well understood. Here, xylem networks of grapevine (Vitis vinifera L.) were reconstructed from 3D high-resolution X-ray micro-computed tomography (microCT) images. Xylem network performance was then modeled to simulate loss of hydraulic conductivity under increasingly negative xylem sap pressure simulating drought stress conditions. We also considered the sensitivity of xylem network performance to changes in key network parameters. We found that the mean pit area per intervessel connection was constant across 10 networks from three, 1.5-m stem segments, but short (0.5 cm) segments fail to capture complete network connectivity. Simulations showed that network organization imparted additional resistance to embolism spread beyond the air-seeding threshold of pit membranes. Xylem network vulnerability to embolism spread was most sensitive to variation in the number and location of vessels that were initially embolized and pit membrane vulnerability. Our results show that xylem network organization can increase stem resistance to embolism spread by 40% (0.66 MPa) and challenge the notion that a single embolism can spread rapidly throughout an entire xylem network.

Intervessel pit membrane thickness best explains variation in embolism resistance amongst stems of Arabidopsis thaliana accessions

Background and Aims The ability to avoid drought-induced embolisms in the xylem is one of the essential traits for plants to survive periods of water shortage. Over the past three decades, hydraulic studies have been focusing on trees, which limits our ability to understand how herbs tolerate drought. Here we investigate the embolism resistance in inflorescence stems of four Arabidopsis thaliana accessions that differ in growth form and drought response. We assess functional traits underlying the variation in embolism resistance amongst the accessions studied using detailed anatomical observations. Methods Vulnerability to xylem embolism was evaluated via vulnerability curves using the centrifuge technique and linked with detailed anatomical observations in stems using light microscopy and transmission electron microscopy. Key Results The data show significant differences in stem P50, varying 2-fold from −1.58 MPa in the Cape Verde Island accession to −3.07 MPa in the woody soc1 ful double mutant. Out of all the anatomical traits measured, intervessel pit membrane thickness (TPM) best explains the differences in P50, as well as P12 and P88. The association between embolism resistance and TPM can be functionally explained by the air-seeding hypothesis. There is no evidence that the correlation between increased woodiness and increased embolism resistance is directly related to functional aspects. However, we found that increased woodiness is strongly linked to other lignification characters, explaining why mechanical stem reinforcement is indirectly related to increased embolism resistance. Conclusions The woodier or more lignified accessions are more resistant to embolism than the herbaceous accessions, confirming the link between increased stem lignification and increased embolism resistance, as also observed in other lineages. Intervessel pit membrane thickness and, to a lesser extent, theoretical vessel implosion resistance and vessel wall thickness are the missing functional links between stem lignification and embolism resistance.