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In conifers, xylogenesis produces during a growing season 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 characterised 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 hypothesised that much of this regulation relies on a morphogenetic gradient of auxin. However, recent modelling works 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 a regulation by a crosstalk between auxin and a second biochemical signal, using dynamical modelling. 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, TDIF) drives cell division and auxin polar transport.HighlightA dynamical model proves that two interacting signals (auxin, plus a cytokinin or the TDIF peptide) can drive wood formation dynamics and tree-ring structure development in conifers.

International audience; Lycophytes are the earliest diverging extant lineage of vascular plants, sister to all other vas-cular 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 (K shoot) 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 K shoot , with a 50% loss of K shoot occurring at À1.7 and À2.5 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 À3.0 and À4.6 MPa in S. haematodes and S. pulcherrima, respectively. X-ray microcomputed tomogra-phy images of stems revealed that the decline in K shoot 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.

International audience; 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 (g(res)) after stomatal closure, as well as the phase transition temperature (T-p), beyond which g(res) significantly increases. Here, we describe and test a novel low-cost tool, the DroughtBox, for phenotyping g(res) and T-p 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 g(res) and T-p values. Among a study group of four conifer and one angiosperm species, we observed a range of g(res) (0.44-1.64 mmol H2O m(-2) s(-1)) and T-p (39.4-43.8 degrees 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 g(res) and T-p, as well as for testing models of time to hydraulic failure that will improve our ability to assess climate change impacts on plants.

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 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 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.One sentence summaryPoplar stem bending induces an electrical response with high speed and strong decrement.

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.

Knowledge on variations of drought resistance traits are needed to predict the potential of trees to acclimate to coming severe drought events. Xylem vulnerability to embolism is a key parameter related to such droughts, and its phenotypic variability relies mainly on environmental plasticity. We investigated the structural determinants controlling the plasticity of vulnerability to embolism, focusing on the key elements involved in the air bubble entry in vessels, especially the intervessel pits. Poplar saplings (Populus tremula x alba (Aiton) Sm., 1804) grown in contrasted water availability or light exposure exhibited differences in the vulnerability to embolism (P50) in a range of 0.76 MPa. We then characterized the structural changes in features related to pit quantity and pit structure, from the pit ultrastructure to the organization of xylem vessels, using different microscopy techniques (transmission electron microscopy, scanning electron microscopy, light microscopy). A multispectral combination of X-ray microtomography and light microscopy analysis allowed measuring the vulnerability of each single vessel and testing some of the relationships between structural traits and vulnerability to embolism inside the xylem. The pit ultrastructure did not change, whereas the vessel dimensions increased with the vulnerability to embolism and the grouping index and fraction of intervessel cell wall both decreased with the vulnerability to embolism. These findings hold when comparing between trees or between the vessels inside the xylem of an individual tree. These results evidenced that plasticity of vulnerability to embolism in hybrid poplar occurs through changes in the pit quantity properties such as pit area and vessel grouping rather than changes on the pit structure.

International audience; Abstract 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.

International audience; Shoot morphogenetic plasticity is crucial to the adaptation of plants to their fluctuating environments. Major insights on shoot morphogenesis have been gathered 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 on the morphogenetic control by 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 feed-forward and feedback effects of the global shape on the local morphogenetic process. In parallel, major advances have been made in the analysis the major genes involved in the shaping of axes and crowns, revealing conserved genic networks among angiosperms. Then, we show that these two approaches are now starting to converge, revealing exciting perspectives.

International audience; 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.

Bio-based composites with lignosulfonates as a matrix and corn cob particles as reinforcement were developed. Their mechanical strength in compression mode was evaluated with determination of Young modulus (E) and ultimate compressive strength (σ max). The influence of fiber content, fibers particle size, and compaction pressure applied on the composite during processing was investigated with a design of experiments approach. The model enabled to calculate an optimum for σ max equal to 18 MPa for a corresponding Young modulus of 0.27 GPa. Particle size was found to be the most influent parameter on mechanical properties responses. In order to understand this dependency, an automated optical particle analysis was performed to measure size and shape of corn cob particles. Distribution leading to the highest σ max was the one with the less smooth surface of particles (low convexity shape parameter), providing a better mechanical adhesion between fiber and matrix. Increasing compaction pressure and fiber content was in favor of compressive strength. Interface between lignosulfonates and corn cob inside the composites was observed with X-ray microtomography technique. It was noticed that interlocking occurred between the two phases.

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 analysed 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 behaviour 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.

Leaf hydraulic conductance (Kleaf) and capacitance (Cleaf) are among the key parameters in plant-water regulation. Understanding the responses of these hydraulic traits to drought conditions remains a challenge for describing comprehensive plant-water relationships. The ability of an organism to resist and/or tolerate embolism events, which may occur at high negative pressure caused by hydric stress, relies on how well it can sustain a hydraulic system in a dynamic equilibrium. Populus deltoides is a water-saving tree species with a stomatal conductance that declines rapidly with reduced water availability. Under unfavorable conditions, the stomatal control of transpiration is known to be closely coordinated with a loss of plant hydraulic functioning that can ultimately result in hydraulic failure through xylem embolism, notably in leaves. The effects of drought on leaf hydraulics are also related to regulation in water permeases such as the aquaporins. To describe the responses linked to leaf hydraulics under severe drought and rewatering conditions, water-stressed poplars were monitored daily on an ecophysiological and a molecular scale. A structural and expression analysis on a set of aquaporins was carried out in parallel by in situ hybridization analysis and quantitative PCR. In complement, water distribution in water-challenged leaves was investigated using X-ray microtomography. A general depression of leaf hydraulic conductance and relative water content occurred under drought, but was reversed when plants were rewatered. More interestingly, (i) extreme leaf water deficiency led to marked xylem and lamina embolism, but a degree of hydric integrity in the midrib extraxylem territories and the bundle sheath of the minor veins was maintained, and (ii) the sub-tissue water allocation correlated well with an over-accumulation of several PIP and TIP aquaporins. Our multi-facet molecular ecophysiological approach revealed that leaves were able to secure a certain level of hydric status, in particular in cell territories near the “living ribs”, which provided rapid hydric adjustment responses once favorable conditions were restored. These findings contribute to an integrated approach to leaf hydraulics, thus favoring a better understanding of the cell mechanisms involved in tree vulnerability to climate changes.

Centrifuge-based techniques to assess xylem vulnerability to embolism are increasingly being used, although we are yet to reach a consensus on the nature and extent of artefactual embolism observed in some angiosperm species. In particular, there is disagreement over whether these artefacts influence both the spin (Cavitron) and static versions of the centrifuge technique equally. We tested two methods for inducing embolism: bench dehydration and centrifugation. We used three methods to measure the resulting loss of conductivity: gravimetric flow measured in bench-dehydrated and centrifuged samples (static centrifuge), in situ flow measured under tension during spinning in the centrifuge (Cavitron) and direct imaging using X-ray computed microtomography (microCT) observations in stems of two species of Hakea that differ in vessel length. Both centrifuge techniques were prone to artefactual embolism in samples with maximum vessel length longer than, or similar to, the centrifuge rotor diameter. Observations with microCT indicated that this artefactual embolism occurred in the outermost portions of samples. The artefact was largely eliminated if flow was measured in an excised central part of the segment in the static centrifuge or starting measurements with the Cavitron at pressures lower than the threshold of embolism formation in open vessels. The simulations of loss of conductivity in centrifuged samples with a new model, CAVITOPEN, confirmed that the impact of open vessels on the vulnerability to embolism curve was higher when vessels were long, samples short and when embolism is formed in open vessels at less negative pressures. This model also offers a robust and quantitative tool to test and correct for artefactual embolism at low xylem tensions.

The frequencies of free oscillations of plants, or plant parts, depend on their geometries, stiffnesses, and masses. Besides direct biomechanical interest, free frequencies also provide insights into plant properties that can usually only be measured destructively or with low-throughput techniques (e.g., change in mass, tissue density, or stiffness over development or with stresses). We propose here a new high-throughput method based on the noncontact measurements of the free frequencies of the standing plant. The plant is excited by short air pulses (typically 100 ms). The resulting motion is recorded by a high speed video camera (100 fps) and processed using fast space and time correlation algorithms. In less than a minute the mechanical behavior of the plant is tested over several directions. The performance and versatility of this method has been tested in three contrasted species: tobacco (Nicotiana benthamian), wheat (Triticum aestivum L.), and poplar (Populus sp.), for a total of more than 4000 data points. In tobacco we show that water stress decreased the free frequency by 15%. In wheat we could detect variations of less than 1 g in the mass of spikes. In poplar we could measure frequencies of both the whole stem and leaves. The work provides insight into new potential directions for development of phenotyping.

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.

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.

Key message: Direct, non-invasive X-ray microtomography and optical technique observations applied in stems and leaves of intact seedlings revealed that laurel is highly resistant to drought-induced xylem embolism. Contrary to what has been brought forward, daily cycles of embolism formation and refilling are unlikely to occur in this species and to explain how it copes with drought. Context: There has been considerable controversy regarding xylem embolism resistance for long-vesselled angiosperm species and particularly for the model species for refilling (Laurus nobilis L.). Aims: The purpose of this study was to resolve the hydraulic properties of this species by documenting vulnerability curves of different organs in intact plants. Methods: Here, we applied a direct, non-invasive method to visualize xylem embolism in stems and leaves of intact laurel seedlings up to 2-m tall using X-ray microtomography (microCT) observations and the optical vulnerability technique. These approaches were coupled with complementary centrifugation measurements performed on 1-m long branches sampled from adult trees and compared with additional microCT analyses carried out on 80-cm cut branches. Results: Direct observations of embolism spread during desiccation of intact laurels revealed that 50% loss of xylem conductivity (Psi(50)) was reached at - 7.9 +/- 0.5 and - 8.4 +/- 0.3 MPa in sterns and leaves, respectively, while the minimum xylem water potentials measured in the field were - 4.2 MPa during a moderate drought season. Those findings reveal that embolism formation is not routine in Laurus nobilis contrary to what has been previously reported. These Psi(50) values were close to those based on the flow-centrifuge technique (- 9.2 +/- 0.2 MPa), but at odds with microCT observations of cut branches (- 4.0 +/- 0.5 MPa). Conclusion: In summary, independent methods converge toward the same conclusion that laurel is highly resistant to xylem embolism regardless its development stage. Under typical growth conditions without extreme drought events, this species maintains positive hydraulic safety margin, while daily cycles of embolism formation and refilling are unlikely to occur in this species.

Plants have developed different tropisms: in particular, they re-orient the growth of their branches towards light (phototropism) or upwards (gravitropism). How these tropisms affect the shape of a tree crown remains unanswered. We address this question by developing a propagating front model of tree growth. This model being length-free, it leads to self-similar solutions, independent of the initial conditions, at long time. Varying the intensities of each tropism, different self-similar shapes emerge, including singular ones. Interestingly, these shapes bear similarities with existing tree species. It is concluded that the core of specific crown shapes in trees relies on the balance between tropisms., 11 pages, 7 figures

Plants can be highly segmented organisms with an independently redundant design of organs. In the context of plant hydraulics, leaves may be less embolism resistant than stems, allowing hydraulic failure to be restricted to distal organs that can be readily replaced. We quantified drought-induced embolism in needles and stems of Pinus pinaster using high-resolution computed tomography (HRCT). HRCT observations of needles were compared with the rehydration kinetics method to estimate the contribution of extra-xylary pathways to declining hydraulic conductance. High-resolution computed tomography images indicated that the pressure inducing 50% of embolized tracheids was similar between needle and stem xylem (P50 needle xylem = -3.62 MPa, P50 stem xylem = -3.88 MPa). Tracheids in both organs showed no difference in torus overlap of bordered pits. However, estimations of the pressure inducing 50% loss of hydraulic conductance at the whole needle level by the rehydration kinetics method were significantly higher (P50 needle = -1.71 MPa) than P50 needle xylem derived from HRCT. The vulnerability segmentation hypothesis appears to be valid only when considering hydraulic failure at the entire needle level, including extra-xylary pathways. Our findings suggest that native embolism in needles is limited and highlight the importance of imaging techniques for vulnerability curves.

International audience; 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.

Torres-Ruiz, J.M. et al... .--11 páginas.-- 6 figuras.-- 1 tabla.-- 63 referencias, 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, Was supported by a scholarship from the Spanish Ministry of Economy and Competitiveness (EEBB-I-15-09191). We thank the PSICHE beamline (SOLEIL synchrotron facility, project 20141229). This work was supported by the programme ‘Investments for the Future’ (ANR-10-EQPX-16, XYLOFOREST) from the French National Agency for Research. B.C. was supported by the Australian Research Council (Future Fellowship grant no. FT130101115). R.L. was supported by a Marie Curie fellowship (no. ) of the EU FP7 Programme.

In recent years, the validity of embolism quantification methods has been questioned, especially for long-vesseled plants. Some studies have suggested that cutting xylem while under tension, even under water, might generate artificial cavitation. Accordingly, a rehydration procedure prior to hydraulic measurements has been recommended to avoid this artefact. On the other hand, concerns have been raised that xylem refilling might occur when samples are rehydrated. Here, we explore the potential biases affecting embolism quantification for grapevine (Vitis vinifera L.) petioles harvested under tension or after xylem relaxation. We employ direct visualization of embolism through X-ray micro-computed tomography (microCT) to test for the occurrence of fast refilling (artifactually low per cent loss of conductivity (PLC) due to rehydration prior to sample harvest) as well as excision-induced embolism (artifactually high embolism due to air introduction during harvest). Additionally, we compared the response functions of both stomatal regulation and xylem embolism to xylem pressure (Psix). Short-time (20 min) xylem tension relaxation prior to the hydraulic measurement resulted in a lower degree of embolism than found in samples harvested under native tensions, and yielded xylem vulnerability curves similar to the ones obtained using direct microCT visualization. Much longer periods of hydration (overnight) were required before xylem refilling was observed to occur. In field-grown vines, over 85% of stomatal closure occurred at less negative Psix than that required to induce 12% PLC. Our results demonstrate that relaxation of xylem tension prior to hydraulic measurement allows for the reliable quantification of native embolism in grapevine petioles. Furthermore, we find that stomatal regulation is sufficiently conservative to avoid transpiration-induced cavitation. These results suggest that grapevines have evolved a strategy of cavitation resistance, rather than one of cavitation tolerance (diurnal cycles of embolism and repair).

Virot et al. [E. Virot et al., Phys. Rev. E 93, 023001 (2016)] assert that the critical wind speed at which \ensuremath{\geqslant}50% of all trees in a population break is \ensuremath{\approx}42 m/s, regardless of tree characteristics. We show that empirical data do not support this assertion, and that the assumptions underlying the theory used by Virot et al. are inconsistent with the biomechanics of trees.

The vascular system of grapevine has been reported as being highly vulnerable, even though grapevine regularly experiences seasonal drought. Stomata would consequently 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 overestimation of vulnerability. The aim of this study was to provide an unbiased assessment of (i) the vulnerability to drought-induced embolism in perennial and annual organs, and (ii) the ability to refill embolized vessels in two Vitis species. X-ray micro-CT observations on intact plants indicated that both V. vinifera and V. riparia were relatively vulnerable, with the pressure inducing 50% loss of stem hydraulic conductivity (Ψ50Stem) = -1.7 and -1.3MPa, respectively. In V. vinifera, both the stem and petiole had similar sigmoidal vulnerability curves, but differed in Ψ50 (-1.7 and -1.0MPa 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): P=-0.11{plus minus}0.02MPa; ∆PLC=0.02{plus minus}0.01%). However, positive xylem pressure was observed at the basal part of the plant (P=0.04{plus minus}0.01MPa), leading to recovered conductance (∆PLC=-0.24{plus minus}0.12%). Our findings provide evidence that grapevine is unable to repair embolized xylem vessels under negative pressure, but its hydraulic vulnerability segmentation provides a significant protection of the perennial stem.

Highlight The dynamics of water fluxes and cavitation events induced by ice formation were analyzed by visualization (X-ray microtomography and MRI), and detected with a microdendrometer and ultrasonic acoustic emissions., Freeze–thaw cycles induce major hydraulic changes due to liquid-to-ice transition within tree stems. The very low water potential at the ice–liquid interface is crucial as it may cause lysis of living cells as well as water fluxes and embolism in sap conduits, which impacts whole tree–water relations. We investigated water fluxes induced by ice formation during freeze–thaw cycles in Juglans regia L. stems using four non-invasive and complementary approaches: a microdendrometer, magnetic resonance imaging, X-ray microtomography, and ultrasonic acoustic emissions analysis. When the temperature dropped, ice nucleation occurred, probably in the cambium or pith areas, inducing high water potential gradients within the stem. The water was therefore redistributed within the stem toward the ice front. We could thus observe dehydration of the bark’s living cells leading to drastic shrinkage of this tissue, as well as high tension within wood conduits reaching the cavitation threshold in sap vessels. Ultrasonic emissions, which were strictly emitted only during freezing, indicated cavitation events (i.e. bubble formation) following ice formation in the xylem sap. However, embolism formation (i.e. bubble expansion) in stems was observed only on thawing via X-ray microtomography for the first time on the same sample. Ultrasonic emissions were detected during freezing and were not directly related to embolism formation. These results provide new insights into the complex process and dynamics of water movements and ice formation during freeze–thaw cycles in tree stems.

Direct visualization shows enhanced embolism of xylem samples when they are collected under tension.

Plant lignocellulosic biomass, mostly composed of cell walls, is one of the largest, mostly untapped, reserves of renewable carbon feedstock on the planet. Energy-rich polysaccharide polymers of plant cell walls can be broken down to produce fermentable sugars used to produce bioethanol. However, the complex structure of plant cell walls, and in particular, the presence of lignin, makes them recalcitrant to enzymatic degradation. Reducing this recalcitrance represents a major technological challenge. Brachypodium distachyon is an excellent model to identify parameters underlying biomass quality of energy grasses. In this work, we identified a mutant line spa1 with a so far undescribed phenotype combining brittleness with increased elasticity of the internodes. Mutant cell walls contain less crystalline cellulose and changes in hemicellulose and lignin quality and quantity. Using a dedicated reactor to follow in real-time, the evolution of straw particle size and sugar release during enzymatic digestion, we show that, despite the increased lignin content, the spa1 mutant has a dramatic reduced recalcitrance to saccharification compared to the WT. These observations demonstrate that other parameters besides lignin content are relevant for the improvement of biomass recalcitrance in energy grasses.

MEDLINE:24918205; International audience; Background and Aims Various correlations have been identified between anatomical features of bordered pits in angiosperm xylem and vulnerability to cavitation, suggesting that the mechanical behaviour of the pits may play a role. Theoretical modelling of the membrane behaviour has been undertaken, but it requires input of parameters at the nanoscale level. However, to date, no experimental data have indicated clearly that pit membranes experience strain at high levels during cavitation events.Methods Transmission electron microscopy (TEM) was used in order to quantify the pit micromorphology of four tree species that show contrasting differences in vulnerability to cavitation, namely Sorbus aria, Carpinus betulus, Fagus sylvatica and Populus tremula. This allowed anatomical characters to be included in a mechanical model that was based on the Kirchhoff–Love thin plate theory. A mechanistic model was developed that included the geometric features of the pits that could be measured, with the purpose of evaluating the pit membrane strain that results from a pressure difference being applied across the membrane. This approach allowed an assessment to be made of the impact of the geometry of a pit on its mechanical behaviour, and provided an estimate of the impact on air-seeding resistance.Key Results The TEM observations showed evidence of residual strains on the pit membranes, thus demonstrating that this membrane may experience a large degree of strain during cavitation. The mechanical modelling revealed the interspecific variability of the strains experienced by the pit membrane, which varied according to the pit geometry and the pressure experienced. The modelling output combined with the TEM observations suggests that cavitation occurs after the pit membrane has been deflected against the pit border. Interspecific variability of the strains experienced was correlated with vulnerability to cavitation. Assuming that air-seeding occurs at a given pit membrane strain, the pressure predicted by the model to achieve this mechanical state corresponds to experimental values of cavitation sensitivity (P50).Conclusions The results provide a functional understanding of the importance of pit geometry and pit membrane structure in air-seeding, and thus in vulnerability to cavitation.

20 páginas, 4 figuras, 1 tabla, 47 referencias, Different methods have been devised to analyze vulnerability to cavitation of plants. Although a good agreement between them is usually found, some discrepancies have been reported when measuring samples from long-vesseled species. The aim of this study was to evaluate possible artefacts derived from different methods and sample sizes. Current-year shoot segments of mature olive trees (Olea europaea), a long-vesseled species, were used to generate VCs by bench dehydration, pressure collar and both static- and flow-centrifuge methods. For the latter, two different rotors were used to test possible effects of the rotor design on the curves. Measurements of native embolism and High- Resolution Computed Tomography images were used in order to validate the VC methods. The pressure collar and the two centrifugal methods showed greater vulnerability to cavitation than the dehydration method. The shift in vulnerability thresholds in centrifuge methods was more pronounced in shorter samples, supporting the open vessels artefact hypothesis since a higher proportion of vessels was open in short samples. The two different rotor designs used for the flow-centrifuge method revealed similar vulnerability to cavitation. Only the bench dehydration method or x-ray methods produced VCs that agreed with native levels of embolism measured in the field and measured water potentials., We thank the Spanish Ministry of Science and Innovation (research projects AGL2006-04666/AGR and AGL2009-11310/AGR) and the Austrian Science Fund (FWF) (P20852- B16 and L 556-B16) for funding this work. J.M. Torres-Ruiz held a doctoral fellowship from the Spanish Ministry of Science and Innovation (BES-2007-17149, MCINN).

Mechanical cues are essential signals regulating plant growth and development. In response to wind, trees develop a thigmomorphogenetic response characterized by a reduction in longitudinal growth, an increase in diameter growth, and changes in mechanical properties. The molecular mechanisms behind these processes are poorly understood. In poplar, PtaZFP2, a C2H2 transcription factor, is rapidly up-regulated after stem bending. To investigate the function of PtaZFP2, we analyzed PtaZFP2-overexpressing poplars (Populus tremula 9 Populus alba). To unravel the genes downstream PtaZFP2, a transcriptomic analysis was performed. PtaZFP2-overexpressing poplars showed longitudinal and cambial growth reductions together with an increase in the tangent and hardening plastic moduli. The regulation level of mechanoresponsive genes was much weaker after stem bending in PtaZFP2-overexpressing poplars than in wild-type plants, showing that PtaZFP2 negatively modulates plant responsiveness to mechanical stimulation. Microarray analysis revealed a high proportion of down-regulated genes in PtaZFP2-overexpressing poplars. Among these genes, several were also shown to be regulated by mechanical stimulation. Our results confirmed the important role of PtaZFP2 during plant acclimation to mechanical load, in particular through a negative control of plant molecular responsiveness. This desensitization process could modulate the amplitude and duration of the plant response during recurrent stimuli.

Under drought conditions, the xylem of trees that conducts ascending sap produces ultrasonic emissions whose exact origin is not clear. We introduce a new method to record simultaneously both acoustic events and optical observation of the xylem conduits within slices of wood that were embedded in a transparent material setting a hydric stress. In this article, we resolved the rapid development of all cavitation bubbles and demonstrated that each ultrasound emission was linked to the nucleation of one single bubble, whose acoustic energy is an increasing function of the size of the conduit where nucleation occurred and also of the hydric stress. We modelled these observations by the fact that water columns in conduits store elastic energy and release it into acoustic waves when they are broken by cavitation bubbles. Water columns are thus elastic, and not rigid, ‘wires of water’ set under tension by hydric stresses. Cavitation bubbles are at the origin of an embolism, whose development was followed in our experiments. Such an embolism of sap circulation can result in a fatal condition for living trees. These findings provide new insights for the non-destructive monitoring of embolisms within trees, and suggest a new approach to study porous media under hydric stress.

Times Cited: 0; Understanding drought tolerance mechanisms requires knowledge about the induced weakness that leads to tree death. Bud survival is vital to sustain tree growth across seasons. We hypothesized that the hydraulic connection of the bud to stem xylem structures was critical for its survival. During an artificial drastic water stress, we carried out a census of bud metabolic activity of young Populus nigra L. trees by microcalorimetry. We monitored transcript expression of aquaporins (AQPs; plasma membrane intrinsic proteins (PIPs), X intrinsic proteins (XIPs) and tonoplast membrane intrinsic proteins (TIPs)) and measured local water status within the bud and tissues in the bearer shoot node by nuclear magnetic resonance (NMR) imaging. We found that the bud respiration rate was closely correlated with its water content and decreased concomitantly in buds and their surrounding bearer tissues. At the molecular level, we observed a modulation of AQP pattern expressions (PIP, TIP and XIP subfamilies) linked to water movements in living cells. However, AQP functions remain to be investigated. Both the bud and tree died beyond a threshold water content and respiration rate. Nuclear magnetic resonance images provided relevant local information about the various water reservoirs of the stem, their dynamics and their interconnections. Comparison of pith, xylem and cambium tissues revealed that the hydraulic connection between the bud and saturated parenchyma cells around the pith allowed bud desiccation to be delayed. At the tree death date, NMR images showed that the cambium tissues remained largely hydrated. Overall, the respiration rate (Rco2) and a few AQP isoforms were found to be two suitable, complementary criteria to assess the bud metabolic activity and the ability to survive a severe drought spell. Bud moisture content could be a key factor in determining the capacity of poplar to recover from water stress.

Times Cited: 0; Xylem cavitation resistance has profound implications for plant physiology and ecology. This process is characterized by a 'vulnerability curve' (VC) showing the variation of the percentage of cavitation as a function of xylem pressure potential. The shape of this VC varies from 'sigmoidal' to 'exponential'. This review provides a panorama of the techniques that have been used to generate such a curve. The techniques differ by (i) the way cavitation is induced (e.g. bench dehydration, centrifugation, or air injection), and (ii) the way cavitation is measured (e.g. percentage loss of conductivity (PLC) or acoustic emission), and a nomenclature is proposed based on these two methods. A survey of the literature of more than 1200 VCs was used to draw statistics on the usage of these methods and on their reliability and validity. Four methods accounted for more than 96% of all curves produced so far: bench dehydration-PLC, centrifugation-PLC, pressure sleeve-PLC, and Cavitron. How the shape of VCs varies across techniques and species xylem anatomy was also analysed. Strikingly, it was found that the vast majority of curves obtained with the reference bench dehydration-PLC method are 'sigmoidal'. 'Exponential' curves were more typical of the three other methods and were remarkably frequent for species having large xylem conduits (ring-porous), leading to a substantial overestimation of the vulnerability of cavitation for this functional group. We suspect that 'exponential' curves may reflect an open-vessel artefact and call for more precautions with the usage of the pressure sleeve and centrifugation techniques.

Leaves, the distal section of the soil-plant-atmosphere continuum, exhibit the lowest water potentials in a plant. In contrast to angiosperm leaves, knowledge of the hydraulic architecture of conifer needles is scant. We investigated the hydraulic efficiency and safety of Pinus pinaster needles, comparing different techniques. The xylem hydraulic conductivity (k(s)) and embolism vulnerability (P(50)) of both needle and stem were measured using the cavitron technique. The conductance and vulnerability of whole needles were measured via rehydration kinetics, and Cryo-SEM and 3D X-ray microtomographic observations were used as reference tools to validate physical measurements. The needle xylem of P. pinaster had lower hydraulic efficiency (k(s) = 2.0 × 10(-4) m(2) MPa(-1) s(-1)) and safety (P(50) = - 1.5 MPa) than stem xylem (k(s) = 7.7 × 10(-4) m(2) MPa(-1) s(-1); P(50) = - 3.6 to - 3.2 MPa). P(50) of whole needles (both extra-vascular and vascular pathways) was - 0.5 MPa, suggesting that non-vascular tissues were more vulnerable than the xylem. During dehydration to - 3.5 MPa, collapse and embolism in xylem tracheids, and gap formation in surrounding tissues were observed. However, a discrepancy in hydraulic and acoustic results appeared compared with visualizations, arguing for greater caution with these techniques when applied to needles. Our results indicate that the most distal parts of the water transport pathway are limiting for hydraulics of P. pinaster. Needle tissues exhibit a low hydraulic efficiency and low hydraulic safety, but may also act to buffer short-term water deficits, thus preventing xylem embolism.

Wood provides water transport and mechanical support of trees. Sap is transported under negative pressure in plant xylem conduits, which can be subject to embolism during severe drought. Typically, denser woods show thicker cell walls and stronger mechanical properties. Ten transgenic poplar lines modified for expression of genes involved in lignin metabolism were produced from the female clone 717-1B4 of Populus tremula × Populus alba to test the hypothesis of a possible trade-off between hydraulic and mechanical functions. Poplar lines underexpressed genes encoding for cinnamoyl alcohol dehydrogenase (CAD), cinnamoyl CoA reductase (CCR) and caffeic acid 3-O- methyltransferase (COMT), while new poplar lines underexpressed the CAD genes or overexpressed the MYB308 gene, encoding for a transcription factor repressing the phenylpropanoid metabolism. To maximize the contrast between line behaviors, these plants were grown under two different water regimes, and the impact on their hydraulic traits and xylem properties was analyzed to test for a link between water condition and mechanical and hydraulic properties. Our results show that the resistance to xylem cavitation was lower for the transgenic lines than for the control line 717-1b4 while they show neither a positive nor a negative tendency for the longitudinal Young's modulus between the transgenic lines and the control line. ASOMT10b and ASOMT2b, which possessed a down-regulated expression for all the genes, showed a lower value of the resistance to implosion index (t/b)2. No difference for xylem hydraulic conductivity between the lines was found. The changes in lignin metabolism in these transgenic lines did not affect the water transport, despite the change in the lignin content. Our data on the transgenic poplar lines do not therefore support the mechanical vs. hydraulic trade-off hypothesis and we point out that angiosperm trees have numerous ways to acclimate their internal structure in order to adjust their mechanical properties without hydraulic coupling. Moreover, we observed an acclimation to water stress for P50 but not for the Young's modulus. MYB308-25.1 showed better mechanical properties and vulnerability to cavitation than the control line 717-1b4. Finally, we present evidence that lignins are involved in the vulnerability to cavitation, probably through modifications of pit structure and behavior.

Mention d'édition : P. Wotjaszek (ed); Mechanosensitive control of plant growth is a major process shaping how terrestrial plants acclimate to the mechanical challenges set by wind, self-weight, and autostresses. Loads acting on the plant are distributed down to the tissues, following continuum mechanics. Mechanosensing, though, occurs within the cell, building up into integrated signals; yet the reviews on mechanosensing tend to address macroscopic and molecular responses, ignoring the biomechanical aspects of load distribution to tissues and reducing biological signal integration to a "mean plant cell." In this chapter, load distribution and biological signal integration are analyzed directly. The Sum of Strain Sensing model S 3 m is then discussed as a synthesis of the state of the art in quantitative deterministic knowledge and as a template for the development of an integrative and system mechanobiology.

Xylem vulnerability to cavitation differs between tree species according to their drought resistance, more xerophilous species being more resistant to xylem cavitation. Variability in xylem vulnerability to cavitation is also found within species, especially between in situ populations. The origin of this variability has not been clearly identified. Here we analyzed the response of xylem hydraulic traits of Populus tremula xPopulus alba trees to three different soil water regimes. Stem xylem vulnerability was scored as the xylem water potential causing 12, 50 and 88% loss of conductivity (P(12), P(50) and P(88)). Vulnerability to cavitation was found to acclimate to growing conditions under different levels of soil water content, with P(50) values of -1.82, -2.03 and -2.45 MPa in well-watered, moderately water-stressed and severely water-stressed poplars, respectively. The value of P(12), the xylem tension at which cavitation begins, was correlated with the lowest value of midday leaf water potential (epsilon m) experienced by each plant, the difference between the two parameters being approximately 0.5 MPa, consistent with the absence of any difference in embolism level between the different water treatments. These results support the hypothesis that vulnerability to cavitation is a critical trait for resistance to drought. The decrease in vulnerability to cavitation under growing conditions of soil drought was correlated with decreased vessel diameter, increased vessel wall thickness and a stronger bordered pit field (t/b)2. The links between these parameters are discussed.