Eric Badel

X-ray microtomography (micro-CT): a reference technology for high-resolution quantification of xylem embolism in trees

As current methods for measuring xylem embolism in trees are indirect and prone to artefacts, there is an ongoing controversy over the capacity of trees to resist or recover from embolism. The debate will not end until we get direct visualization of the vessel content. Here, we propose desktop X-ray microtomography (micro-CT) as a reference direct technique to quantify xylem embolism and thus validate more widespread measurements based upon either hydraulic or acoustic methods. We used desktop micro-CT to measure embolism levels in dehydrated or centrifuged shoots of laurel - a long-vesseled species thought to display daily cycles of embolism formation and refilling. Our direct observations demonstrate that this Mediterranean species is highly resistant to embolism and is not vulnerable to drought-induced embolism in a normal range of xylem tensions. We therefore recommend that embolism studies in long-vesseled species should be validated by direct methods such as micro-CT to clear up any misunderstandings on their physiology.

Poplar vulnerability to xylem cavitation acclimates to drier soil conditions

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 x Populus 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 (psi 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.

Vulnerability to cavitation in Olea europaea current-year shoots: further evidence of an open-vessel artifact associated with centrifuge and air-injection techniques

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 artifacts 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 vulnerability curves (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. Indeed, high-resolution computed tomography (HRCT) images were used to evaluate the functional status of xylem at different water potentials. Measurements of native embolism were used to validate the methods used. 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-vessel artifact hypothesis as a higher proportion of vessels were open in short samples. The two different rotor designs used for the flow-centrifuge method revealed similar vulnerability to cavitation. Only the bench dehydration or HRCT methods produced VCs that agreed with native levels of embolism and water potential values measured in the field.

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

X-ray computed microtomography provides a high-resolution, noninvasive method to evaluate vulnerability to drought-induced embolism in living, intact plants. 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.

Are needles of Pinus pinaster more vulnerable to xylem embolism than branches? New insights from X‐ray computed tomography

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.

Evidence for hydraulic vulnerability segmentation and lack of xylem refilling under tension

Direct, noninvasive observations of embolism formation and repair reveal a lack of refilling under negative pressure and a xylem hydraulic vulnerability segmentation in grapevine. 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.

Arabidopsis thaliana as a model species for xylem hydraulics: does size matter?

While Arabidopsis thaliana has been proposed as a model species for wood development, the potential of this tiny herb for studying xylem hydraulics remains unexplored and anticipated by scepticism. Inflorescence stems of A. thaliana were used to measure hydraulic conductivity and cavitation resistance, whereas light and electron microscopy allowed observations of vessels. In wild-type plants, measured and theoretical conductivity showed a significant correlation (R 2 = 0.80, P < 0.01). Moreover, scaling of vessel dimensions and intervessel pit structure of A. thaliana were consistent with structure–function relationships of woody plants. The reliability and resolution of the hydraulic methods applied to measure vulnerability to cavitation were addressed by comparing plants grown under different photoperiods or different mutant lines. Sigmoid vulnerability curves of A. thaliana indicated a pressure corresponding to 50% loss of hydraulic conductance (P 50) between –3 and –2.5MPa for short-day and long-day plants, respectively. Polygalacturonase mutants showed a higher P 50 value (–2.25MPa), suggesting a role for pectins in vulnerability to cavitation. The application of A. thaliana as a model species for xylem hydraulics provides exciting possibilities for (1) exploring the molecular basis of xylem anatomical features and (2) understanding genetic mechanisms behind xylem functional traits such as cavitation resistance. Compared to perennial woody species, however, the lesser amount of xylem in A. thaliana has its limitations.

Hydraulic efficiency and safety of vascular and non-vascular components in Pinus pinaster leaves

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.

Integrative Mechanobiology of Growth and Architectural Development in Changing Mechanical Environments

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.

Ultrasonic emissions reveal individual cavitation bubbles in water-stressed wood

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.