Contributions of lignification, tissue arrangement patterns, and cross-sectional area to whole-stem mechanical properties in Arabidopsis thaliana.

Plant cells withstand mechanical stress originating from turgor pressure by robustly maintaining the mechanical properties of the cell wall. This applies at the organ scale as well; many plant stems act as pressurized cylinders, where the epidermis is under tension and inner tissues are under compression. The clavata3 de-etiolated3 (clv3-8 det3-1) double mutant of Arabidopsis thaliana displays cracks in its stems because of a conflict between the mechanical properties of the weak epidermis and over-proliferation of inner stem tissues. In this work, we conducted three-point bending tests on various Arabidopsis thaliana mutants, including those displaying the stem cracking phenotype, to examine the differences in their mechanical properties. The clv3-8 det3-1 double mutant exhibited reduced stem stiffness, consistent with reduced differentiation due to the clv3-8 mutation. Yet, in clv3-8, stem cross-sectional area was increased associating with the increase in vascular bundle number, and stem cross-sections displayed various shapes. To uncouple the contribution of geometry and cell-wall differentiation to the mechanical properties of the whole stems, we tested the contribution of lignified fibers to stem stiffness. In order to suppress lignin deposition in stems genetically, we generated multiple higher-order mutants by crossing clv3-8 and/or det3-1 with nst1-1 nst3-1, in which lignin deposition is suppressed. Stem stiffness was reduced markedly in all nst1-1 nst3-1 mutant backgrounds. Overall, our results suggest that stem stiffness relies on the presence of differentiated, lignified, fiber tissue as well as on the alignment or spatial distribution of vascular bundles within the stem organ.

Xylem embolism and bubble formation during freezing suggest complex dynamics of pressure in Betula pendula stems.

Freeze-thaw-induced embolism, a key limiting factor for perennial plants results from the formation of gas bubbles during freezing and their expansion during thawing. However, the ice volumetric increase generates local pressures, which can affect the formation of bubbles. To characterize local dynamics of pressure tension and the physical state of the sap during freeze-thaw cycles, we simultaneously used ultrasonic acoustic emission analysis and synchrotron-based high-resolution computed tomography on the diffuse-porous species Betula pendula. Visualization of individual air-filled vessels and the distribution of gas bubbles in frozen xylem were performed.. Ultrasonic emissions occurred after ice formation, together with bubble formation, whereas the development of embolism took place after thawing. The pictures of frozen tissues indicated that the positive pressure induced by the volumetric increase of ice can provoke inward flow from the cell wall toward the lumen of the vessels. We found no evidence that wider vessels within a tissue were more prone to embolism, although the occurrence of gas bubbles in larger conduits would make them prone to earlier embolism. These results highlight the need to monitor local pressure as well as ice and air distribution during xylem freezing to understand the mechanism leading to frost-induced embolism.

Photosynthesis, leaf hydraulic conductance and embolism dynamics in the resurrection plant Barbacenia purpurea.

The main parameters determining photosynthesis are stomatal and mesophyll conductance and electron transport rate, and for hydraulic dynamics they are leaf hydraulic conductance and the spread of embolism. These parameters have scarcely been studied in desiccation-tolerant (resurrection) plants exposed to drought. Here, we characterized photosynthesis and hydraulics during desiccation and rehydration in a poikilochlorophyllous resurrection plant, Barbacenia purpurea (Velloziaceae). Gas exchange, chlorophyll fluorescence, and leaf water status were monitored along the whole dehydration-rehydration cycle. Simultaneously, embolism formation and hydraulic functioning recovery were measured at leaf level using micro-computed tomography imaging. Photosynthesis and leaf hydraulic conductance ceased at relatively high water potential (-1.28 and -1.54 MPa, respectively), whereas the onset of leaf embolism occurred after stomatal closure and photosynthesis cessation (<-1.61 MPa). This sequence of physiological processes during water stress may be associated with the need to delay dehydration, to prepare the molecular changes required in the desiccated state. Complete rehydration occurred rapidly in the mesophyll, whereas partial xylem refilling, and subsequent recovery of photosynthesis, occurred at later stages after rewatering. These results highlight the importance of stomata as safety valves to protect the vascular system from embolism, even in a plant able to fully recover after complete embolism.

Pathogen-derived mechanical cues potentiate the spatio-temporal implementation of plant defense.

BACKGROUND: The ongoing adaptation of plants to their environment is the basis for their survival. In this adaptation, mechanoperception of gravity and local curvature plays a role of prime importance in finely regulating growth and ensuring a dynamic balance preventing buckling. However, the abiotic environment is not the exclusive cause of mechanical stimuli. Biotic interactions between plants and microorganisms also involve physical forces and potentially mechanoperception. Whether pathogens trigger mechanoperception in plants and the impact of mechanotransduction on the regulation of plant defense remains however elusive. RESULTS: Here, we found that the perception of pathogen-derived mechanical cues by microtubules potentiates the spatio-temporal implementation of plant immunity to fungus. By combining biomechanics modeling and image analysis of the post-invasion stage, we reveal that fungal colonization releases plant cell wall-born tension locally, causing fluctuations of tensile stress in walls of healthy cells distant from the infection site. In healthy cells, the pathogen-derived mechanical cues guide the reorganization of mechanosensing cortical microtubules (CMT). The anisotropic patterning of CMTs is required for the regulation of immunity-related genes in distal cells. The CMT-mediated mechanotransduction of pathogen-derived cues increases Arabidopsis disease resistance by 40% when challenged with the fungus Sclerotinia sclerotiorum. CONCLUSIONS: CMT anisotropic patterning triggered by pathogen-derived mechanical cues activates the implementation of early plant defense in cells distant from the infection site. We propose that the mechano-signaling triggered immunity (MTI) complements the molecular signals involved in pattern and effector-triggered immunity.

The shaping of plant axes and crowns through tropisms and elasticity: an example of morphogenetic plasticity beyond the shoot apical meristem.

Shoot morphogenetic plasticity is crucial to the adaptation of plants to their fluctuating environments. Major insights into shoot morphogenesis have been compiled studying meristems, especially the shoot apical meristem (SAM), through a methodological effort in multiscale systems biology and biophysics. However, morphogenesis at the SAM is robust to environmental changes. Plasticity emerges later on during post-SAM development. The purpose of this review is to show that multiscale systems biology and biophysics is insightful for the shaping of the whole plant as well. More specifically, we review the shaping of axes and crowns through tropisms and elasticity, combining the recent advances in morphogenetic control using physical cues and by genes. We focus mostly on land angiosperms, but with growth habits ranging from small herbs to big trees. We show that generic (universal) morphogenetic processes have been identified, revealing feedforward and feedback effects of global shape on the local morphogenetic process. In parallel, major advances have been made in the analysis of the major genes involved in shaping axes and crowns, revealing conserved genic networks among angiosperms. Then, we show that these two approaches are now starting to converge, revealing exciting perspectives.

Modelling the spatial crosstalk between two biochemical signals explains wood formation dynamics and tree-ring structure.

In conifers, xylogenesis during a growing season produces a very characteristic tree-ring structure: large, thin-walled earlywood cells followed by narrow, thick-walled latewood cells. Although many factors influence the dynamics of differentiation and the final dimensions of xylem cells, the associated patterns of variation remain very stable from one year to the next. While radial growth is characterized by an S-shaped curve, the widths of xylem differentiation zones exhibit characteristic skewed bell-shaped curves. These elements suggest a strong internal control of xylogenesis. It has long been hypothesized that much of this regulation relies on a morphogenetic gradient of auxin. However, recent modelling studies have shown that while this hypothesis could account for the dynamics of stem radial growth and the zonation of the developing xylem, it failed to reproduce the characteristic tree-ring structure. Here, we investigated the hypothesis of regulation by a crosstalk between auxin and a second biochemical signal, by using computational morphodynamics. We found that, in conifers, such a crosstalk is sufficient to simulate the characteristic features of wood formation dynamics, as well as the resulting tree-ring structure. In this model, auxin controls cell enlargement rates while another signal (e.g. cytokinin, tracheary element differentiation inhibitory factor) drives cell division and auxin polar transport.

Seasonal and long-term consequences of esca grapevine disease on stem xylem integrity.

Hydraulic failure has been extensively studied during drought-induced plant dieback, but its role in plant-pathogen interactions is under debate. During esca, a grapevine (Vitis vinifera) disease, symptomatic leaves are prone to irreversible hydraulic dysfunctions but little is known about the hydraulic integrity of perennial organs over the short- and long-term. We investigated the effects of esca on stem hydraulic integrity in naturally infected plants within a single season and across season(s). We coupled direct (ks) and indirect (kth) hydraulic conductivity measurements, and tylose and vascular pathogen detection with in vivo X-ray microtomography visualizations. Xylem occlusions (tyloses) and subsequent loss of stem hydraulic conductivity (ks) occurred in all shoots with severe symptoms (apoplexy) and in more than 60% of shoots with moderate symptoms (tiger-stripe), with no tyloses in asymptomatic shoots. In vivo stem observations demonstrated that tyloses occurred only when leaf symptoms appeared, and resulted in more than 50% loss of hydraulic conductance in 40% of symptomatic stems, unrelated to symptom age. The impact of esca on xylem integrity was only seasonal, with no long-term impact of disease history. Our study demonstrated how and to what extent a vascular disease such as esca, affecting xylem integrity, could amplify plant mortality through hydraulic failure.

Stem bending generates electrical response in poplar.

Under natural conditions, plants experience external mechanical stresses such as wind and touch that impact their growth. A remarkable feature of this mechanically induced growth response is that it may occur at a distance from the stimulation site, suggesting the existence of a signal propagating through the plant. In this study, we investigated the electrical response of poplar trees to a transient controlled bending stimulation of the stem that mimics the mechanical effect of wind. Stem bending was found to cause an electrical response that we called "gradual" potential, similar in shape to an action potential. However, this signal can be distinguished from the well-known plant action potential by its propagation up to 20 cm along the stem and its strong dumping in velocity and amplitude. Two hypotheses regarding the mode of propagation of the "gradual" potential are discussed.

The interplay of hydraulic failure and cell vitality explains tree capacity to recover from drought.

Global climatic models predict an increment in the frequency and intensity of drought events, which have important consequences on forest dieback. However, the mechanisms leading to tree mortality under drought conditions and the physiological thresholds for recovery are not totally understood yet. This study aimed to identify what are the key physiological traits that determine the tree capacity to recover from drought. Individuals of a conifer (Pseudotsuga menziesii M.) and an angiosperm (Prunus lusitanica L.) species were exposed to drought and their ability to recover after rehydration monitored. Results showed that the actual thresholds used for recovery from drought based on percentage loss of conductance (PLC) (i.e., 50% for conifers, 88% for angiosperms) do not provide accurate insights about the tree capacity for surviving extreme drought events. On the contrary, differences in stem relative water content (RWCStem ) and the level of electrolytes leakage (EL) were directly related to the capacity of the trees to recover from drought. This was the case for the conifer species, P. menziesii, for which higher RWCStem and lower EL values were related to the recovery capacity. Even if results showed a similar trend for the angiosperm P. lusitanica as for the conifers, differences between the two traits were much more subtle and did not allow an accurate differentiation between trees able to recover and those that were not. RWCStem and EL could work as indicators of tree capacity to recover from drought for conifers but more studies are required to confirm this observation for angiosperms.

Die hard: timberline conifers survive annual winter embolism.

During winter, timberline trees are exposed to drought and frost, factors known to induce embolism. Studies indicated that conifers cope with winter embolism by xylem refilling. We analysed the loss of hydraulic conductivity (LC) in Picea abies branch xylem over 10 years, and correlated winter embolism to climate parameters. LC was investigated by direct X-ray micro-computer tomography (micro-CT) observations and potential cavitation fatigue by Cavitron measurements. Trees showed up to 100% winter embolism, whereby LC was highest, when climate variables indicated frost drought and likely freeze-thaw stress further increased LC. During summer, LC never exceeded 16%, due to hydraulic recovery. Micro-CT revealed homogenous embolism during winter and that recovery was based on xylem refilling. Summer samples exhibited lower LC in outermost compared to older tree rings, although no cavitation fatigue was detected. Long-term data and micro-CT observations demonstrate that timberline trees can survive annual cycles of pronounced winter-embolism followed by xylem refilling. Only a small portion of the xylem conductivity cannot be restored during the first year, while remaining conduits are refilled without fatigue during consecutive years. We identify important research topics to better understand the complex induction and repair of embolism at the timberline and its relevance to general plant hydraulics.

Drought-induced lacuna formation in the stem causes hydraulic conductance to decline before xylem embolism in Selaginella.

Lycophytes are the earliest diverging extant lineage of vascular plants, sister to all other vascular plants. Given that most species are adapted to ever-wet environments, it has been hypothesized that lycophytes, and by extension the common ancestor of all vascular plants, have few adaptations to drought. We investigated the responses to drought of key fitness-related traits such as stomatal regulation, shoot hydraulic conductance (Kshoot ) and stem xylem embolism resistance in Selaginella haematodes and S. pulcherrima, both native to tropical understory. During drought stomata in both species were found to close before declines in Kshoot , with a 50% loss of Kshoot occurring at -1.7 and -2.5 MPa in S. haematodes and S. pulcherrima, respectively. Direct observational methods revealed that the xylem of both species was resistant to embolism formation, with 50% of embolized xylem area occurring at -3.0 and -4.6 MPa in S. haematodes and S. pulcherrima, respectively. X-ray microcomputed tomography images of stems revealed that the decline in Kshoot occurred with the formation of an air-filled lacuna, disconnecting the central vascular cylinder from the cortex. We propose that embolism-resistant xylem and large capacitance, provided by collapsing inner cortical cells, is essential for Selaginella survival during water deficit.

Exploring the Hydraulic Failure Hypothesis of Esca Leaf Symptom Formation.

Vascular pathogens cause disease in a large spectrum of perennial plants, with leaf scorch being one of the most conspicuous symptoms. Esca in grapevine (Vitis vinifera) is a vascular disease with huge negative effects on grape yield and the wine industry. One prominent hypothesis suggests that vascular disease leaf scorch is caused by fungal pathogen-derived elicitors and toxins. Another hypothesis suggests that leaf scorch is caused by hydraulic failure due to air embolism, the pathogen itself, and/or plant-derived tyloses and gels. In this study, we transplanted mature, naturally infected esca symptomatic vines from the field into pots, allowing us to explore xylem integrity in leaves (i.e. leaf midveins and petioles) using synchrotron-based in vivo x-ray microcomputed tomography and light microscopy. Our results demonstrated that symptomatic leaves are not associated with air embolism. In contrast, symptomatic leaves presented significantly more nonfunctional vessels resulting from the presence of nongaseous embolisms (i.e. tyloses and gels) than control leaves, but there was no significant correlation with disease severity. Using quantitative PCR, we determined that two vascular pathogen species associated with esca necrosis in the trunk were not found in leaves where occlusions were observed. Together, these results demonstrate that symptom development is associated with the disruption of vessel integrity and suggest that symptoms are elicited at a distance from the trunk where fungal infections occur. These findings open new perspectives on esca symptom expression where the hydraulic failure and elicitor/toxin hypotheses are not necessarily mutually exclusive.

The DroughtBox: A new tool for phenotyping residual branch conductance and its temperature dependence during drought.

Xylem hydraulic failure is a major driver of tree death during drought. However, to better understand mortality risk in trees, especially during hot-drought events, more information is required on both rates of residual water-loss from small branches (gres ) after stomatal closure, as well as the phase transition temperature (Tp ), beyond which gres significantly increases. Here, we describe and test a novel low-cost tool, the DroughtBox, for phenotyping gres and Tp across species. The system consists of a programmable climatically controlled chamber in which branches dehydrate and changes in the mass recorded. Test measurements show that the DroughtBox maintains stable temperature and relative humidity across a range of set points, a prerequisite for getting accurate gres and Tp values. Among a study group of four conifer and one angiosperm species, we observed a range of gres (0.44-1.64 mmol H2 O m-2 s-1 ) and Tp (39.4-43.8°C) values. Furthermore, the measured time to hydraulic failure varied between two conifers species and was shortened in both species following a heatwave event. The DroughtBox is a reliable and customizable tool for phenotyping gres and Tp , as well as for testing models of time to hydraulic failure that will improve our ability to assess climate change impacts on plants.

Over-accumulation of abscisic acid in transgenic tomato plants increases the risk of hydraulic failure.

Climate change threatens food security, and plant science researchers have investigated methods of sustaining crop yield under drought. One approach has been to overproduce abscisic acid (ABA) to enhance water use efficiency. However, the concomitant effects of ABA overproduction on plant vascular system functioning are critical as it influences vulnerability to xylem hydraulic failure. We investigated these effects by comparing physiological and hydraulic responses to water deficit between a tomato (Solanum lycopersicum) wild type control (WT) and a transgenic line overproducing ABA (sp12). Under well-watered conditions, the sp12 line displayed similar growth rate and greater water use efficiency by operating at lower maximum stomatal conductance. X-ray microtomography revealed that sp12 was significantly more vulnerable to xylem embolism, resulting in a reduced hydraulic safety margin. We also observed a significant ontogenic effect on vulnerability to xylem embolism for both WT and sp12. This study demonstrates that the greater water use efficiency in the tomato ABA overproducing line is associated with higher vulnerability of the vascular system to embolism and a higher risk of hydraulic failure. Integrating hydraulic traits into breeding programmes represents a critical step for effectively managing a crop's ability to maintain hydraulic conductivity and productivity under water deficit.

Feeling stretched or compressed? The multiple mechanosensitive responses of wood formation to bending.

Background and Aims: Trees constantly experience wind, perceive resulting mechanical cues, and modify their growth and development accordingly. Previous studies have demonstrated that multiple bending treatments trigger ovalization of the stem and the formation of flexure wood in gymnosperms, but ovalization and flexure wood have rarely been studied in angiosperms, and none of the experiments conducted so far has used multidirectional bending treatments at controlled intensities. Assuming that bending involves tensile and compressive strain, we hypothesized that different local strains may generate specific growth and wood differentiation responses. Methods: Basal parts of young poplar stems were subjected to multiple transient controlled unidirectional bending treatments during 8 weeks, which enabled a distinction to be made between the wood formed under tensile or compressive flexural strains. This set-up enabled a local analysis of poplar stem responses to multiple stem bending treatments at growth, anatomical, biochemical and molecular levels. Key Results: In response to multiple unidirectional bending treatments, poplar stems developed significant cross-sectional ovalization. At the tissue level, some aspects of wood differentiation were similarly modulated in the compressed and stretched zones (vessel frequency and diameter of fibres without a G-layer), whereas other anatomical traits (vessel diameter, G-layer formation, diameter of fibres with a G-layer and microfibril angle) and the expression of fasciclin-encoding genes were differentially modulated in the two zones. Conclusions: This work leads us to propose new terminologies to distinguish the 'flexure wood' produced in response to multiple bidirectional bending treatments from wood produced under transient tensile strain (tensile flexure wood; TFW) or under transient compressive strain (compressive flexure wood; CFW). By highlighting similarities and differences between tension wood and TFW and by demonstrating that plants could have the ability to discriminate positive strains from negative strains, this work provides new insight into the mechanisms of mechanosensitivity in plants.

PtxtPME1 and homogalacturonans influence xylem hydraulic properties in poplar.

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under- or over-express a xylem-abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under- and over-expressing aspen lines developed xylem with lower-specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.

Xylem resistance to embolism: presenting a simple diagnostic test for the open vessel artefact.

Xylem vulnerability to embolism represents an essential trait for the evaluation of the impact of hydraulics in plant function and ecology. The standard centrifuge technique is widely used for the construction of vulnerability curves, although its accuracy when applied to species with long vessels remains under debate. We developed a simple diagnostic test to determine whether the open-vessel artefact influences centrifuge estimates of embolism resistance. Xylem samples from three species with differing vessel lengths were exposed to less negative xylem pressures via centrifugation than the minimum pressure the sample had previously experienced. Additional calibration was obtained from non-invasive measurement of embolism on intact olive plants by X-ray microtomography. Results showed artefactual decreases in hydraulic conductance (k) for samples with open vessels when exposed to a less negative xylem pressure than the minimum pressure they had previously experienced. X-Ray microtomography indicated that most of the embolism formation in olive occurs at xylem pressures below -4.0 MPa, reaching 50% loss of hydraulic conductivity at -5.3 MPa. The artefactual reductions in k induced by centrifugation underestimate embolism resistance data of species with long vessels. A simple test is suggested to avoid this open vessel artefact and to ensure the reliability of this technique in future studies.

Noninvasive Measurement of Vulnerability to Drought-Induced Embolism by X-Ray Microtomography.

Hydraulic failure induced by xylem embolism is one of the primary mechanisms of plant dieback during drought. However, many of the methods used to evaluate the vulnerability of different species to drought-induced embolism are indirect and invasive, increasing the possibility that measurement artifacts may occur. Here, we utilize x-ray computed microtomography (microCT) to directly visualize embolism formation in the xylem of living, intact plants with contrasting wood anatomy (Quercus robur, Populus tremula × Populus alba, and Pinus pinaster). These observations were compared with widely used centrifuge techniques that require destructive sampling. MicroCT imaging provided detailed spatial information regarding the dimensions and functional status of xylem conduits during dehydration. Vulnerability curves based on microCT observations of intact plants closely matched curves based on the centrifuge technique for species with short vessels (P. tremula × P. alba) or tracheids (P. pinaster). For ring porous Q. robur, the centrifuge technique significantly overestimated vulnerability to embolism, indicating that caution should be used when applying this technique to species with long vessels. These findings confirm that microCT can be used to assess the vulnerability to embolism on intact plants by direct visualization.

Evidence for Hydraulic Vulnerability Segmentation and Lack of Xylem Refilling under Tension.

The vascular system of grapevine (Vitis spp.) has been reported as being highly vulnerable, even though grapevine regularly experiences seasonal drought. Consequently, stomata would remain open below water potentials that would generate a high loss of stem hydraulic conductivity via xylem embolism. This situation would necessitate daily cycles of embolism repair to restore hydraulic function. However, a more parsimonious explanation is that some hydraulic techniques are prone to artifacts in species with long vessels, leading to the overestimation of vulnerability. The aim of this study was to provide an unbiased assessment of (1) the vulnerability to drought-induced embolism in perennial and annual organs and (2) the ability to refill embolized vessels in two Vitis species X-ray micro-computed tomography observations of intact plants indicated that both Vitis vinifera and Vitis riparia were relatively vulnerable, with the pressure inducing 50% loss of stem hydraulic conductivity = -1.7 and -1.3 MPa, respectively. In V. vinifera, both the stem and petiole had similar sigmoidal vulnerability curves but differed in pressure inducing 50% loss of hydraulic conductivity (-1.7 and -1 MPa for stem and petiole, respectively). Refilling was not observed as long as bulk xylem pressure remained negative (e.g. at the apical part of the plants; -0.11 ± 0.02 MPa) and change in percentage loss of conductivity was 0.02% ± 0.01%. However, positive xylem pressure was observed at the basal part of the plant (0.04 ± 0.01 MPa), leading to a recovery of conductance (change in percentage loss of conductivity = -0.24% ± 0.12%). Our findings provide evidence that grapevine is unable to repair embolized xylem vessels under negative pressure, but its hydraulic vulnerability segmentation provides significant protection of the perennial stem.

Direct observation and modelling of embolism spread between xylem conduits: a case study in Scots pine.

Xylem embolism is one of the main processes involved in drought-related plant mortality. Although its consequences for plant physiology are already well described, embolism formation and spread are poorly evaluated and modelled, especially for tracheid-based species. The aim of this study was to assess the embolism formation and spread in Pinus sylvestris as a case study using X-ray microtomography and hydraulics methods. We also evaluated the potential effects of cavitation fatigue on vulnerability to embolism and the micro-morphology of the bordered pits using scanning electron microscopy (SEM) to test for possible links between xylem anatomy and embolism spread. Finally, a novel model was developed to simulate the spread of embolism in a 2D anisotropic cellular structure. Results showed a large variability in the formation and spread of embolism within a ring despite no differences being observed in intertracheid pit membrane anatomical traits. Simulations from the model showed a highly anisotropic tracheid-to-tracheid embolism spreading pattern, which confirms the major role of tracheid-to-tracheid air seeding to explain how embolism spreads in Scots pine. The results also showed that prior embolism removal from the samples reduced the resistance to embolism of the xylem and could result in overestimates of vulnerability to embolism.