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

Jose Manuel Torres Ruiz, Hervé Cochard, Brendan Choat, Steven Jansen, Rosa Ana Lopez Rodriguez, Ivana Tomášková, Carmen M. Padilla-Díaz, Eric Badel, Régis Burlett, Andrew King, et al.

► To cite this version:

Jose Manuel Torres Ruiz, Hervé Cochard, Brendan Choat, Steven Jansen, Rosa Ana Lopez Rodriguez, et al.. Xylem resistance to embolism: presenting a simple diagnostic test for the open vessel artefact. New Phytologist, 2017, 215 (1), pp.489-499. 10.1111/nph.14589. hal-01518575

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<figure><img src="image_1.png" /></figure>
1 Xylem resistance to embolism: presenting a simple diagnostic test
2 for the open vessel artefact

3
4 Torres-Ruiz JM*¹, Cochard H², Choat B³, Jansen S⁴, López R²,³, Tomášková I⁵, Padilla-Díaz CM⁶, Badel E², Burlett R¹, King A⁷, Lenoir N⁸, Martin-StPaul NK⁹ and Delzon S¹.

6
7 *Author for correspondence:
8 José M. Torres-Ruiz
9 email: torresruizjm@gmail.com; telephone: +00330540006973.

10
11 ¹ BIOGECO, INRA, Univ. Bordeaux, 33615 Pessac, France.
12 ²PIAF, INRA, Univ. Clermont-Auvergne, 63100 Clermont-Ferrand, France.
13 ³Western Sydney University, Hawkesbury Institute for the Environment, Richmond, NSW
14 2753, Australia
15 ⁴Ulm University, Institute of Systematic Botany and Ecology, Albert-Einstein-Allee 11, 89081
16 Ulm, Germany

17 ⁵Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Kamýcká 129,
18 165 00 Praha 6 – Suchdol, Czech Republic

19 ⁶Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS, CSIC), Avenida Reina
20 Mercedes, 10, 41012, Sevilla, Spain

21 ⁷Synchrotron SOLEIL, L'Orme de Merisiers, Saint Aubin-BP48, Gif-sur-Yvette CEDEX,
22 France.

23 ⁸CNRS, University of Bordeaux, UMS 3626 Placamat F-33608 Pessac, France.

24 ⁹INRA, UR629 Ecologie des Forêts Méditerranéennes (URFM), Avignon, France.

25
26
27 Number of words main body: 5257

28 Tables: 1 // Figures: 6 (3 of them in colour) // Support. Information: 1 figure.

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## 31 Summary

32 Xylem vulnerability to embolism represents an essential trait for evaluating the
33 impact of hydraulics in plant function and ecology. The standard centrifuge
34 technique is widely used for constructing vulnerability curves although its
35 accuracy when applied to long-vesseled species remains under debate.

36 We developed a simple diagnostic test to determine if the open-vessel artefact
37 influences centrifuge estimates of embolism resistance. Xylem samples from
38 three species with differing vessel lengths were exposed to less negative xylem
39 pressures via centrifugation than the minimum pressure the sample had
40 previously experienced. Additional calibration was obtained from non-invasive
41 measurement of embolism on intact olive plants by X-Ray microtomography.

42 Results showed artefactual decreases in hydraulic conductance (k) for segments
43 with open vessels when exposed to a less negative xylem pressure than the
44 minimum pressure the sample had previously experienced. X-Ray
45 microtomography indicated that most of the embolism formation in olive occurs
46 at xylem pressures below -4.0 MPa, reaching 50% loss of hydraulic conductivity
47 at -5.3 MPa.

48 The artefactual reductions in k induced by centrifugation underestimate
49 embolism resistance data of long-vesseled species. A simple test is suggested to
50 avoid this open vessel artefact and to ensure the reliability of this technique in
51 future studies.

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59 **Keywords:** Artefact, centrifuge technique, embolism, micro-CT, olive, xylem.

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## Introduction

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The continuous water column that connects the soil and the upper portions of the plants through the xylem is exposed to negative pressures induced by the water evaporation at the leaf surface. Water can be pulled up from the soil through the entire plant system thanks to the strong cohesive forces between water-molecules (Dixon, 1914). Under water deficit, the relative water content of the soil decreases, inducing a more negative soil water potential, and the negative pressure inside the water conducting xylem tissue may become interrupted by gas bubbles. These bubbles (emboli) can expand to block conduits and reduce long-distance vascular transport of water. As the number of gas-filled conduits increases, the xylem hydraulic conductance ($k$, kg s⁻¹ MPa⁻¹) decreases until water flow stops, causing desiccation of plant tissues and eventually plant death under severe and prolonged droughts. Failure of the plant hydraulic system is now considered to be the principal mechanism of drought-induced plant mortality (Brodribb & Cochard 2009; Brodribb et al., 2010; Urli et al., 2013; Salmon et al. 2015; Anderegg et al., 2016). Given the narrow hydraulic safety margins within which many plants operate (Choat et al., 2012), evaluating the resistance to embolism across plant species is essential to predict future mortality events and shifts in species distribution ranges due to climate change with more intense and severe drought events (IPCC, 2014).

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The resistance to xylem embolism is typically assessed by constructing vulnerability curves, which represent the variation in specific hydraulic conductivity ($K_s$, kg s⁻¹ MPa⁻¹ m⁻¹) as a function of xylem water potential (Sperry et al., 1988; Cochard et al., 2009; Torres-Ruiz et al. 2017). During the last few decades, a variety of methods have been developed to determine the loss of $K_s$ in xylem samples exposed to increasing levels of water stress (see Cochard et al., 2013 for a review). Although there is general consensus about centrifuge methods to construct vulnerability curves for species with short conduits (e.g., conifer species), the application of appropriate methods for long-vesseled species remains a subject of debate, especially with regards to the standard centrifuge technique (Alder et al., 1997; Jacobsen & Pratt 2012; McElrone et al. 2012; Tobin et al., 2013; Torres-Ruiz et al., 2014; Hacke et al., 2015). This technique is based on spinning xylem samples in a custom-built rotor that induces a wide range of pressures at the centre of a sample. In contrast to the Cavitron method (Cochard et al., 2005), no water flow is induced through the sample during spinning in the standard centrifuge technique. Each pressure applied may induce a certain loss of

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conductivity that is determined gravimetrically with a custom built device (see e.g., Torres-Ruiz et al. 2012) or with a commercial instrument relying on Coriolis type mass-flow meters (e.g., Xyl'em embolism meter; Bronkhorst, Montigny-les-Cormeilles, France). The debate is centred on high losses of Ks that the centrifuge technique induces at relatively mild xylem pressures for long-vesseled species, which results in exponential vulnerability curves (the so called 'r-shaped' curves). These curves have been interpreted as a methodological artefact resulting from the presence of open vessels, i.e. vessels that have no vessel end within the stem segment (Choat et al., 2010; Cochard et al., 2010; Torres-Ruiz et al., 2014). However, other authors consider these curves to be a reliable estimate of embolism resistance in these species, i.e. the occurrence of significant embolism within very moderate ranges of water stress (Christman et al. 2012; Sperry et al., 2012; Hacke et al., 2015). The discrepancy in these two interpretations impedes our progress in understanding the role of hydraulics in plant function (Delzon & Cochard, 2014) and generates confusion regarding the results and conclusions reached in the field of plant hydraulics. Much efforts have been devoted to clarify whether there is an open-vessel artefact with the standard-centrifuge technique, but contradictory conclusions have been reached even when working with the same plant species. For example, contrasting vulnerability curves and resistances to embolism have been observed for Olive (Olea europaea) depending on the technique used for assessing the degree of embolism. While observations with high-resolution computed tomography indicate that embolism does not occur in Olive stems until a certain threshold in xylem pressure is reached (approx. -3.0MPa) and that vulnerability curves of this species show sigmoidal shapes (i.e., a so called 's-shaped' curve; Torres-Ruiz et al., 2014), a recent study reported significant decreases in Ks at relatively high xylem water potentials (Hacke et al., 2015), suggesting r-shaped behaviour and the high embolism vulnerability of this species.

Contrasting conclusions regarding the reliability of the standard centrifuge technique have also been attributed to differences in the centrifuge rotor design used for the measurements (Hacke et al. 2015). Briefly, there are two custom rotor designs in use, the original rotor of Alder et al. (1997) and the Cavitron rotor (Cochard et al., 2005). These designs differ mainly in the way samples are secured and how sample ends are immersed in solution during the spinning. The possible effects of the rotor design on vulnerability curves have been evaluated by Torres-Ruiz et al. (2014), who
concluded that both designs produce biased results (overestimated vulnerability to embolism) when samples contain open vessels. Samples in Torres-Ruiz et al. (2014) were cut in two pieces for measuring Ks gravimetrically after spinning, and the appropriateness of this protocol has been questioned by some authors (Hacke et al. 2015). Although some intraspecific variation in resistance to embolism could be expected between studies due to the use of different plant material, varieties, seasonality, growth conditions, etc., the large discrepancies observed for species such as olive warrant further investigation.

Typically, resistance to embolism has been assessed by inducing decreasing xylem pressures and determining the corresponding losses in k. However, when using a centrifuge method with stem or root segments, it is not possible to determine with certainty that such losses in k reflect patterns of embolism that occur *in situ* or if they come from an artefact due to the presence of open-vessels (Fig. 1). By using alternative techniques that allow the direct observation of xylem embolism in a non-invasive manner, such as MRI (Holbrook *et al.*, 2001; Kaufmann *et al.*, 2009; Choat *et al.*, 2010) and X-ray microtomography (micro-CT; Cochard *et al.*, 2015; Torres-Ruiz *et al.*, 2015a, 2016; Bouche *et al.*, 2016; Choat *et al.*, 2016), the open-vessel artefact can be avoided and the process of embolism formation evaluated with greater certainty. As access to some of these non-invasive techniques can be restricted, we propose here a simple alternative test to diagnose the probability of an open vessel artefact occurring and thus ensuring the accuracy of the standard centrifuge technique for a given species. This consists of spinning xylem samples to a less negative pressure than the minimum xylem pressure (i.e. water potential) experienced previously under native conditions or during the progressive dehydration of the plant (Fig. 1). As it is unlikely that embolism will be induced at a less negative pressure, any decrease in Ks before and after the spinning of the sample should represent an experimental artefact.

The aim of this study was to test if Ks values resulting from the standard centrifuge technique are influenced by the presence of open vessels in stem samples with a different vessel anatomy. For this, xylem samples from three vessel-bearing species with contrasting proportions of open vessels were progressively dehydrated to a given xylem pressure. After measuring their Ks gravimetrically, they were exposed to a less negative xylem pressure by using the standard centrifuge technique, and their Ks measured again. Measurements were undertaken with both rotor designs ('original
design' Alder *et al.*, 1997 and 'Cavitron design' Cochard *et al.*, 2005) to evaluate the effects of the rotor type on the results. In addition, embolism resistance in olive trees was determined in intact plants by using micro-CT and compared to earlier centrifuge measurements for this species.

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## 165 Material and methods

### 166 Plant material

Three species with different vessel length distributions were used in this study. Two relatively long-vesseled species: *Olea europaea* Linnaeus and *Quercus palustris* Muenchh, and one relatively short-vesseled species: *Betula pendula* Roth (Ennajeh *et al.*, 2011; Torres-Ruiz *et al.*, 2014). Samples from *Q. palustris* and *B. pendula* were collected from mature individuals located near the campus of University of Bordeaux (France) for measurements with the Cavitron rotor design, and at the Blue Mountains Botanic Garden (NSW, Australia) for measurements with the Alder rotor design. For *O. europaea*, two to three year old saplings were ordered, repotted into 1.5-L pots and grown for two months under well-watered conditions and at ambient light and temperature at the University of Bordeaux (France) and at the Hawkesbury Institute for the Environment (NSW, Australia). A subset of these olive saplings was transported to the SOLEIL synchrotron (Paris, France) in April 2015 for direct micro-CT visualization of embolism formation while the saplings were progressively dehydrated.

### 180 Presence of open vessels

The presence of open vessels was determined in similar samples (i.e. similar length, diameter, age and position in the tree) collected from the same branches (for *B. pendula* and *Q. palustris*) or similar saplings (for *O. europaea*) to those used for evaluating the open-vessel artefact in both rotor designs (i.e. 15 or 40 cm long depending on the rotor design). Samples were injected with air at low pressure (50-100 kPa) while its apical end was immersed in water (Greenidge 1952; Zimmerman & Jeje, 1981) to check for the presence of open vessels. In those species for which a continuous flow of air bubbles was detected, the percentage of open vessel was determined by applying the silicone injection method (Hacke *et al.*, 2007). New samples, and not the ones used for evaluating the open-vessel artefact, were used to perform these measurements to avoid the effect of any potential damage to the vascular system caused by the centrifugation
itself could have on the final results. Xylem samples were first flushed basipetally with 10 mM KCl and 1 mM CaCl₂ solution at 0.15 MPa for 45 min to remove embolism. Samples were then injected under 30-50 kPa pressure overnight with a 10:1 silicone:hardener mix (RTV-141; Rhodia, Cranbury, NJ, USA). A fluorescent optical brightener (Ciba Uvitex OB; Ciba Specialty Chemicals, Tarrytown, NY, USA) mixed with chloroform 1% (w/w) was added to the silicone (one drop per gram) for the detection of silicone-filled vessels in transverse sections under fluorescent microscopy (NanoZoomer; Hamamatsu Photonics, Japan). After 2-3 days allowing the silicone to harden, the two sample ends were sectioned with a sliding microtome at 5 mm from each end. As the silicone mixture does not penetrate pit membranes but fills entirely the vessel lumina starting from the injection site, the amount of open vessels through the sample was determined by counting the fraction of silicone-filled vessels at both sample ends.

## 205 Evaluation of the open-vessel artefact

Since embolism can theoretically not be induced via air-seeding by exposing the xylem to less negative water potentials than the minimum water potential previously experienced, the open-vessel artefact was evaluated by spinning xylem samples at a velocity that corresponded to a less negative pressure than the pressure reached by stem branches during a previous drought treatment. Two different rotor designs, the original rotor design (Alder et al. 1997) and the Cavitron (Cochard et al. 2005), were used to check if the type of rotor could have an effect on the results. Several branches (≥ 2.0 m in length) of *B. pendula* and *Q. palustris* were collected in the early morning and transported to the lab where they were allowed to dehydrate progressively (up to 5 hours, depending on the species). For *O. europaea* (var. arbequina for the Cavitron design and manzanilla for the original one), saplings were exposed to increasing water stress by withholding irrigation. The xylem pressure of each branch and sapling was monitored regularly by measuring the leaf water potential with a Scholander-type pressure chamber (DGMeca, Gradignan, France) on two leaves per branch or sapling that were previously bagged up (at least 1h before taking the water potential measurements). Once the xylem pressure reached values of -1.8 MPa or lower, a xylem sample was immediately collected for $K_s$ measurements. Samples were collected according to the experimental protocol of Torres-Ruiz et al. (2015a) to avoid the excision artefact described by Wheeler et al. (2013). Briefly, the entire branch or
sapling was progressively recut under water so that the xylem samples were excised once the xylem pressure was released. Depending on the species and the diameter of the rotors used, different sample lengths were used to avoid/ensure the presence of open vessels in the samples, which allowed us to check their effect on Ks after spinning. Thus, for the long-vesseled species (O. europaea and Q. palustris) excised samples were 15 cm long for both rotor designs, whereas for the short-vesseled one, B. pendula, samples were 40 cm long for the Cavitron design and 15 cm long for the original design (40-cm-diameter original rotor not available). Ks for each sample was determined by attaching the samples to a tubing apparatus (Sperry et al. 1988; Torres-Ruiz et al. 2012) filled with a degassed and ultrapure 10 mM KCl and 1 mM CaCl2 solution. All samples were about 3 to 9 mm in xylem diameter (i.e. without bark) and they included growth rings from previous years. However, no air-seeding fatigue is expected since embolised conduits were not flushed. Before taking measurements, both sample ends were debarked and slightly trimmed under water with a sharp razor blade to adjust the desired length and to clear any accidentally blocked vessels. To account for possible passive uptake of water by the sample, Ks was calculated as the slope of the flow, which was calculated from the linear regression of the pressure gradient for three different pressure heads (between 2.0 and 3.5 kPa) as described by Torres-Ruiz et al. (2012). Previous tests showed that the pressure gradients used for our measurements were low enough to avoid displacing air from embolised vessels open at both ends of the segment. Samples were installed in a custom-built rotor of the Alder et al. (1997) design (15 cm diameter) or the Cochard et al., (2005) Cavitron design (40 cm). They were then spun for 5 min, with their ends immersed in the same solution as used for Ks measurements, at a velocity equivalent to a xylem pressure of -1.5 MPa and without inducing any flow through the samples during spinning. To avoid desiccation and to keep the solution in contact with the sample ends when the rotor was stopped, foam pads soaked in the solution used for measurements were placed in the reservoirs of both rotor designs (Tobin et al., 2013). After spinning, samples were removed from the rotor and Ks was measured again. Samples were spun only once ('single-spin centrifuge method', Hacke et al., 2015) to avoid any effect associated with repeated spinning on the same sample. Sapwood area-specific conductivity (Ks) instead of percentage loss of hydraulic conductivity (PLC) values was reported to avoid any possible drift by errors when calculating maximum Ks (Sperry et al., 2012). The native Ks of five extra olive saplings
258 was also measured in order to test for possible changes in Ks at relative high water
259 potential.

260 Checking for possible embolism repair during xylem relaxation

261 The relaxation of xylem pressure carried out to avoid the excision artefact could
262 favour embolism repair in some species (Trifilò et al. 2014). If this was the case for our
263 species, Ks measured for the dehydration treatment would be higher than Ks measured
264 after centrifugation only because partial refilling had occurred with xylem relaxation
265 treatment. In this case lower Ks after spinning could be erroneously interpreted as an
266 artefact. In order to eliminate this possibility we performed an additional phloem
267 girdling treatment, which has been reported to be an effective method to inhibit xylem
268 refilling (Trifilò et al. 2014). Ks from girdled and non-girdled samples was measured
269 after xylem relaxation following the same protocol described for the evaluation of the
270 open-vessel artefact. Thus, once the plant material reached a xylem pressure of -1.8
271 MPa or lower, a group of 5 to 6 samples per species were girdled for their entire length
272 by removing 5-10 mm wide bark rings at 10-15 cm intervals, while a second group of 5
273 to 8 samples were left intact (control). To prevent desiccation, the exposed wood was
274 immediately covered with a thin layer of silicone grease. For collecting the girdled and
275 control samples, the entire branches were progressively recut under water and the
276 samples excised once the xylem pressure was released. Samples were then attached to
277 the tubing apparatus and Ks measured gravimetrically as described above to check for
278 possible repairing processes (i.e. higher Ks values) occurring in the non-girdled samples.

279 To determine if the process of connecting and disconnecting or perfusing the
280 samples with the measurement solution could account for a possible decline in
281 conductance after centrifugation, Ks was measured for one of the long-vesseled species
281 (Olea europaea) before (Ks1) and after (Ks2) disconnecting the samples. For this
282 purpose, samples were kept in water for a similar time as the duration of the centrifuge
283 spinning (i.e., 5 min) and then reconnected to the tubing of the conductivity apparatus.
284

285 Resistance to embolism in intact olive saplings: X-ray microtomography.

286 Synchrotron based computed X-ray microtomography (micro-CT) was used to visualize
287 embolised and water filled conduits in the current-year shoots of intact olive saplings
287 (Olea europaea, var. arbequina). A total of 15 olive saplings were scanned between 8
288 and 12 April 2015 at the French synchrotron facility SOLEIL (Paris, France) using the
289
290 micro-CT beamline (PSICHE). Two weeks prior to the scans, irrigation was withheld
291 progressively in different saplings in order to generate a wide range of xylem water
292 potentials at the time of the scanning.

293 Once at the synchrotron and 1.5 hours before each scan, two leaves, located 20
294 mm below the scanned area, were wrapped in a plastic bag and covered with aluminium
295 foil to prevent transpiration and allow the equilibrium between leaf and stem water
296 potential. Water potential was then measured on these bagged leaves right before the
297 scan with a Scholander-type pressure chamber (Precis 2000, Gradignan, France).
298 Current-year shoots were scanned using a high flux (3.1011 photons mm⁻²) 25 keV
299 monochromatic X-ray beam. The projections were recorded with a Hamamatsu Orca
300 Flash sCMOS camera equipped with a 250 µm thick LuAG scintillator and visible light
301 optics providing an effective pixel size of 3 microns. The complete tomographic scan
302 included 1,500 projections, 50 ms each, for a 180° rotation. Samples were exposed for
303 75 s to the X-ray beam. Tomographic reconstructions were performed using PyHST2
304 software (Mirone et al., 2014) using the Paganin method (Paganin, 2006).

305 The loss of theoretical conductance *k* was determined from a transverse 2D
306 micro-CT slice taken from the centre of the scan volume by estimating the theoretical *k*
307 of each sapling stem based on the conduit dimensions of embolised and functional
308 conduits. A final scan was carried out on each shoot after cutting it in air at
309 approximatively 5 mm above the scan area. Because the xylem was still under negative
310 pressure, we therefore induced embolism in all remaining functional vessels in order to
311 create a reference scan ('cutting artefact', Wheeler *et al.*, 2013; Cochard *et al.*, 2015;
312 Torres-Ruiz *et al.*, 2015a; Choat *et al.*, 2016), while distinguishing at the same time a
313 small amount of living vessels (i.e., not fully developed), which were excluded from our
314 calculations. The maximum theoretical *k* (*k*<sub>max</sub>, kg s⁻¹ MPa⁻¹) of each sapling was then
315 calculated according to the Hagen-Poiseuille equation:

$$k_{\text{max}} = \frac{\Sigma\pi D^4}{128\eta} \quad (1)$$

316 where *D* is the conduit diameter and *η* is the viscosity of water. The *k* for each
317 sapling and xylem pressure was calculated by subtracting the total *k* of all the embolised
318 vessels from its *k*<sub>max</sub>, being the percent loss of *k* (PLC) calculated as:

$$PLC = 100 \times (1 - k / k_{\text{max}})$$
To account for a small amount of embolised vessels that are typically observed at high xylem water potentials, the vulnerability curve was fitted with a modified Weibull function (Neufeld et al., 1992) to include an additional independent parameter (Torres-Ruiz et al., 2015b):

$$PLC = (100 - y_0)(1 - e^{-(\frac{x}{b})^c}) + y_0$$

with being $x$ the xylem pressure, $b$ the xylem pressure for a PLC of 63%, $c$ a dimensionless parameter controlling the shape of the curve and $y_0$ the PLC at a xylem water potential of 0 MPa. The air entry pressure ($P_e$), indicating the threshold xylem pressure at which loss of conductivity begins to increase rapidly (Meinzer et al., 2009), was computed from the $x$-intercept of the tangent through the midpoint of the vulnerability curve.

## Statistics

Differences within a species in mean $K_s$ values measured both in girdled and non-girdled (control) samples, and before and after spinning were tested with paired t-tests after testing for normality and homogeneity of variances. The tests were made at a probability level of 5%. All analyses were performed using SigmaPlot (SPSS Inc., Chicago, IL, USA).

## Results

The air-injection method confirmed the presence of open vessels in 15-cm long samples for the two long-vesseled species (*Q. palustris* and *O. europaea*), whereas no open vessels were detected for *B. pendula* in the 15 cm and 40 cm-long stem segments. Results from the silicone injection technique showed that the percentage of open vessels for the 15 cm-long samples of *Q. palustris* and *O. europaea* (Fig. 2) was 70.7±7.3% ($n = 4$) and 27.0±8.4% ($n = 4$), respectively.

The progressive dehydration induced mean xylem pressures between -1.8 and -2.5 MPa before taking $K_s$ measurements (Table 1) for the three species studied. A significant decrease in $K_s$ was observed for the two long-vesseled species when samples were exposed in a centrifuge to a less negative xylem pressure than the minimum pressure previously experienced by the entire plant or large branches (i.e. -1.5 MPa, Fig. 2).
351 3), independent of the rotor design. Thus, *O. europaea* showed a mean decrease in *K*<sub>s</sub> of
352 46.8±8.1% and 54.3±4.9% using the Cavitron and the original rotor, respectively,
353 whereas for *Q. palustris* this decrease reached 66.6±9.1% and 61.4±6.7%, respectively.
354 In contrast, samples of the short-vesseled species *B. pendula* showed similar
355 conductances before and after spinning for both rotor types.

356 There was no evidence that the relaxation of the xylem pressure carried out to
357 avoid the excision artefact resulted in embolism repair; similar Ks values were observed
358 in girdled and control xylem-relaxed samples for the three species evaluated (Fig. 4a).
359 Differences in Ks values reported in Fig. 2 and 4 are due to the fact that both sets of
360 measurements were carried out at a different time. No differences in Ks were found
361 between control samples that were kept in water for 5 minutes and then reconnected to
362 the conductivity apparatus (Fig. 4b).

363 Micro-CT images from drought stressed olive saplings showed that the PLC
364 remained relatively low (12.6%) when xylem pressure decreased from -0.4 to -4.0 MPa
365 (Fig. 5). However, images showed a significant loss of hydraulic conductance (PLC =
366 75.7%) when xylem pressure reached -6.2 MPa. Thus, the vulnerability curve for olive
367 obtained by direct micro-CT observation showed only a slight increase in PLC up to -
368 4.0 MPa (Fig. 6). As the Pe values indicated, at xylem pressures below -4.0 MPa the
369 loss of k started to increase steeply, reaching 50% loss of k (P50) at -5.3 MPa.

## Discussion

370
All xylem samples with some proportion of their vessels open at both cut ends
371
showed a decrease in Ks when they were exposed to a less negative xylem pressure than
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the minimum pressure previously experienced during the progressive dehydration of the
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branch samples (*Q. palustris*) or the intact plant (*O. europaea*). This observation was
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found by applying the standard centrifuge for both the Alder and Cavitron rotor designs.
375
Thus, *Q. palustris* and *O. europaea* showed a mean decrease in Ks of 70.8 and 49.6%,
376
respectively, when using the Alder rotor design, and 61.4 and 54.3%, respectively,
377
when using the Cavitron design. As embolism should not be induced by exposing the
378
xylem to less negative pressures, the observed reductions in Ks are likely due to
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experimental artefact. Indeed, the control test showed that such reductions were not
380
caused by wounding responses, perforation plate morphology, clogging or embolism
381
induction while samples were loaded and unloaded in the tubing conductivity apparatus,
382
or by other artefacts during handling and sample preparation. Also, direct micro-CT
observations show little embolism formation in intact plants at a xylem pressure of -1.5
MPa providing further evidence that this artefact was the main cause of reductions in Ks
after centrifugation. Samples of B. pendula that did not contain any open vessels
exhibited no change in Ks before and after being spun in both rotors. These results are
consistent with the hypothesis that the experimental artefact causing significant
embolism at xylem pressures near zero is associated with the presence of open xylem
vessels in the test segment. This artefact was previously reported when applying the
standard centrifuge technique to long-vesseled species (Choat *et al.*, 2010; McElrone *et
al.*, 2012; Choat *et al.*, 2016), with evidence showing that the shape of the vulnerability
curves obtained with this method varied with the amount of open vessels (Torres-Ruiz
*et al.*, 2014). A possible explication of the observed decreases in Ks after spinning could
be the ease with which water can drain from open vessels during spinning due to
asymmetric vessel morphology. In particular, it is well known that the inner diameter of
vessel lumina shows considerable variation along its axis (Akachuku, 1987; Balaz *et al.*,
2016). Moreover, xylem vessels are not the ideal 'pipes' running perfectly straight but
show some tortuosity (Braun 1959; Tyree and Zimmermann 2002). Therefore, it is
likely that during the spinning minor pressure differences occur between both ends, with
air entering one end, and water draining out of the other one.

402

The present study provides additional evidence that the standard centrifuge
technique is prone to underestimating resistance to embolism in samples with open
vessels, regardless of rotor design. This is consistent with a survey of more than 1,200
vulnerability curves (Cochard et al., 2013), illustrating that exponential-shaped curves
are remarkably frequent for species with long xylem vessels when they were obtained
with a centrifuge methods, i.e. both the standard-centrifuge and the Cavitron method.

Our findings contrast with the conclusion that the standard centrifuge technique
accurately measures vulnerability curves of long-vesseled species such as grapevine and
olive (Jacobsen and Pratt, 2012; Hacke et al., 2015; Pratt et al., 2015). This conclusion
was based on the agreement between native Ks and PLC values from dehydration-based
measurements and from single-spin centrifugations (Fig. 6 in Hacke et al., 2015).
However, no native Ks values or direct observations were reported in Hacke et al.
(2015) for xylem pressures between 0.0 and -1.0 MPa, i.e. the pressures at which the
largest changes in $K_s$ are expected according to their exponential-shaped curve. Also, their measurements were conducted in September and October, i.e. late summer. Since the trees sampled by Hacke et al. (2015) could have been exposed to lower water potentials during mid-summer, they could have experienced some embolism that would not correspond with the water potential measured at the time of the $K_s$ measurements. These underestimated $K_s$ values at relatively high water potentials could therefore agree with the reduced centrifuge $K_s$ values resulting from an open-vessel artefact and, therefore, be erroneously considered as an evidence for rejecting the occurrence of an open-vessel artefact. Moreover, no effect of the amount of open vessels on the shape of the curve was reported when using 13.5 and 27cm-long samples with different amounts of open vessels (Fig. 4 in Hacke et al., 2015). If the amount of open vessels is relatively high (i.e. >50%) for both stem sizes, vulnerability curves based on samples can be equally biased (Fig. 3 in Hacke et al., 2015). Moreover, a control experiment with samples showing 0% of open vessels is required to confirm that the amount of open vessels has no effect on a vulnerability curve that is based on centrifuge techniques. Similarly low resistances to embolism were observed for 1-yr-old grapevine stems with different percentages of open vessels (Jacobsen & Pratt, 2012). Although some differences in $P_{50}$ could be expected between varieties of grapevine, independent studies carried out on intact grapevine plants by micro-CT have reported considerably higher stem resistances to embolism for Vitis vinifera (McElrone et al. 2012; Charrier et al. 2016).

It is important to highlight that special attention was paid to address previous criticisms from similar tests on the open-vessel artefact, following the latest recommendations and accounting for various sources of error in hydraulic measurements: the use of foam pads to avoid the draining of open vessels (Tobin et al., 2013); single centrifugation of samples ('single-spin' protocol, Hacke et al., 2015); accounting for passive water uptake by samples (Torres-Ruiz et al., 2012); releasing the xylem pressure before excising samples (Wheeler et al., 2013; Torres-Ruiz et al., 2015a); and hydraulic quantification based on $K_s$ values instead of PLC (Sperry et al., 2012). Also, no evidence for embolism repair was detected in any of the three species evaluated, showing similar $K_s$ values between girdled (i.e., samples with their possible refilling mechanism inhibited, see Trifilò et al. 2014) and non-girdled (control) samples after relaxing their xylem pressure. Indeed, there is growing evidence from various
449 research groups that the water transport system of plants is generally resistant to air entry for the normal, daily range of pressures the xylem experiences (McElrone *et al.*, 2012; Martin-StPaul *et al.*, 2014; Bouche *et al.*, 2016; Brodribb *et al.*, 2016; Choat *et al.*, 2016; Lens *et al.*, 2016). A challenge ahead, however, which is at least as important as technical progress in quantifying xylem embolism resistance, includes a better understanding of the actual mechanisms behind drought- and frost-induced embolism formation in plant xylem, in particular air-seeding (Charrier *et al.*, 2014; Jansen and Schenk, 2015; Schenk *et al.*, 2016).

457 Our results do not undermine the standard centrifuge technique, but highlight the importance of conducting simple checks on plant material in order to ensure that embolism resistance is measured accurately. These checks are not necessary for tracheid bearing species but are advisable for angiosperm species, particularly those known to have long xylem vessels. Only when $K_s$ remains similar before and after spinning at a less negative pressure than the minimum pressure previously experienced by the material can vulnerability curves based on the centrifuge technique be considered as accurate. In fact, the test proposed here would probably have avoided the discrepancy about the shape of the response of $k$ to xylem pressure for olive (Torres-Ruiz *et al.*, 2014; Hacke *et al.*, 2015). The direct observation of embolism formation in intact olives trees carried out in this study, however, confirms that embolism formation occurs when xylem water potential falls below a threshold value that is reached under severe drought conditions, supporting its designation as a drought-tolerant species (LoGullo & Salleo, 1988; Fernández & Moreno, 1999; Connor & Fereres, 2005; Diaz-Espejo *et al.*, 2012) and the sigmoidal shape of the vulnerability curves of this species (Torres-Ruiz *et al.*, 2014), which is consistent with the high-embolism-resistance paradigm proposed by Delzon & Cochard (2014).

474 The use of different varieties, growing conditions, plant sizes, sampling period, etc. is likely to explain some degree of intraspecific variability in $P_{50}$ between studies (this study; Ennajeh *et al.*, 2008; Torres-Ruiz *et al.*, 2013), but not the observation of dramatically contrasting embolism resistance strategies (Torres-Ruiz *et al.*, 2014). Indeed, the low variability in $P_{50}$ typically observed within a single species (Martinez-Vilalta *et al.*, 2004; Lamy *et al.*, 2014) and the fact that ‘exponential’ curves are typically associated with centrifuge methods and long-vesseled angiosperms species
481 (Cochard *et al.*, 2013) casts further doubt over measurements indicating a low resistance
to embolism for olive.
482

483 **Conclusions**

484 This study demonstrates that the standard centrifuge technique does not accurately
485 measure resistance to embolism in xylem samples with some proportion of open
486 vessels, regardless of the rotor design used. Wood samples with open-vessels showed a
487 significant decrease in Ks after spinning at a less negative xylem pressure than the
488 minimum pressure previously experienced by the plant material, which was not due to
489 embolism formation. Micro-CT observations of intact olive plants confirmed the high-
490 embolism-resistance strategy for Olive, with almost no embolism formation until water
491 potential reaches a certain threshold value. Overall, the findings from this study are in
492 line with earlier evidence that embolism resistance in plants, especially for long-
493 vesseled species, is not as low as previously suggested.
494

494

495 **Acknowledgments**

496 We thank the Experimental Unit of Pierroton, UE 0570, INRA, 69 route d'Arcachon,
497 33612 CESTAS (France), and the Blue Mountains Botanic Garden staff and Robert
498 Spooner-Hart for providing material and technical support. For fluorescent microscopy,
499 we thank the Bordeaux Imaging Center (CNRS-INSERM) and Bordeaux University.
500 Sébastien Marais is acknowledged for technical support with fluorescent microscopy.
501 CMP-D was supported by a scholarship from the Spanish Ministry of Economy and
502 Competitiveness (EEBB-I-15-09191). We thank the PSICHE beamline (SOLEIL
503 synchrotron facility, project 20141229). This work was supported by the programme
504 'Investments for the Future' ANR-10-EQPX-16, XYLOFOREST) from the French
505 National Agency for Research. BC was supported by an Australian Research Council
506 (Future Fellowship grant no. FT130101115). RL was supported by a Marie Curie
507 fellowship (no. 624473) of the EU FP7 Programme.
508

508 **Author contributions**

509 JMT-R designed the experiment, carried out the measurements, performed the data
510 analysis and wrote the first manuscript draft. JMT-R, IT, RL, SD, EB and CMP-D
511 collected the plant material and carried out some of the measurements. JMT-R, HC, SD,
512 BC, SJ, EB, RB, AK, NL and NKS-P assisted with setting up the Synchrotron scans.

513 JMT-R, SJ, BC, HC and SD contributed ideas and assisted substantially with

514 manuscript development.

515

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717 Tables

Table 1: Overview of the species studied with reference to their sample length, mean native xylem water potentials before taking specific hydraulic conductivity measurements, and the pressure induced by centrifugation using the Cavitron or the original rotor design (i.e. Alder *et al.*, 1997 design). Values indicate mean ± standard error.

<table><thead><tr><th rowspan="2">Species</th><th colspan="2">Sample length (cm)</th><th colspan="2">Native xylem water potentials (MPa, mean ± SE; n)</th><th rowspan="2">Induced pressure (MPa)</th></tr><tr><th>Cavitron</th><th>Original</th><th>Cavitron</th><th>Original</th></tr></thead><tbody><tr><td><i>Betula pendula</i></td><td>40</td><td>15</td><td>-2.1 ± 0.1; 6</td><td>-1.8 ± 0.0; 6</td><td>-1.5</td></tr><tr><td><i>Olea europaea</i></td><td>15</td><td>15</td><td>-2.4 ± 0.1; 7</td><td>-2.0 ± 0.1; 6</td><td>-1.5</td></tr><tr><td><i>Quercus palustris</i></td><td>15</td><td>15</td><td>-2.5 ± 0.2; 6</td><td>-2.0 ± 0.1; 6</td><td>-1.5</td></tr></tbody></table>

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736
737 **Figure Legends**

738 **Fig. 1.** Two contrasting curves showing the predicted loss in $K_s$ according to the ‘s-shaped’ and ‘r-shaped’ scenario. Resistance to xylem embolism has typically been 739 evaluated by inducing lower xylem pressures progressively and determining their 740 corresponding losses in $K_s$ (closed circles), which makes it difficult to distinguish 741 between real and artefactual loss in $K_s$ (closed circle ‘a’ to ‘b’). By exposing a sample to 742 a less negative pressure, i.e. nearer to zero, than the minimum pressure previously 743 experienced, a decrease in $K_s$ can be considered as artefactual (open circle ‘a’ to ‘b’) 744 since less negative pressures cannot induce embolism (open circle ‘a’ to ‘c’). 745

746 **Fig. 2** Percentage of open vessels (i.e., open at both sample ends) in 15cm-long samples 747 of *Olea europaea* and *Quercus palustris*. There are no data for *Betula pendula* since no 748 open vessels were detected in either the 15 cm-long samples or the 40 cm-long ones for 749 this species. Each column is the average of four samples per species and vertical bars 750 represent ± the standard error.

751 **Fig. 3.** Mean specific hydraulic conductivity ($K_s$) for *Betula pendula*, *Olea europaea* 752 and *Quercus palustris* xylem samples before (NP, ‘native xylem pressure) and after 753 spinning in a rotor according to the Cavitron (panels on the left) or the original rotor 754 design (Alder et al. 1997; panels on the right) to induce a less negative xylem pressure 755 (-1.5 MPa, AS, ‘after spinning’) than the minimum pressure previously experienced by 756 the plant. Each column is the average of *n* samples per species and vertical bars 757 represent the standard error. Significant differences between NP and AS are indicated as 758 $p<0.05$ or $p<0.01$; *n.s.* indicates no significant differences.

759 **Fig. 4 a)** Mean specific hydraulic conductivity ($K_s$) for *Betula pendula*, *Olea europaea* 760 and *Quercus palustris* girdled and control (i.e. non-girdled) xylem samples after xylem 761 pressure relaxation. **b)** Specific hydraulic conductivity ($K_s$) for *Olea europaea* measured 762 before ($K_{s1}$) and after ($K_{s2}$) disconnecting the samples, leaving them to sit in water for 763 an equivalent time as those that were spun (i.e. 5 min) and reconnecting them to the 764 tubing conductivity apparatus. Each column is the average of *n* samples per species and 765 vertical bars represent the standard error. *n.s.* indicates no significant differences (i.e. 766 $p>0.05$).

767
**Fig. 5** Transverse micro-CT images of *Olea europaea* at different xylem water potentials ($Ψ_{xyl}$, bottom of each picture). Embolised vessels are observed as black. The percentage loss of hydraulic conductivity (PLC) is indicated for each sample. Scale bar = 500 µm.

**Fig. 6** Vulnerability curve obtained by direct observation (micro-CT) from intact olive plants (full, black dots correspond to individual PLC measurements based on scans, and the solid black line to the Weibull fitting). The grey dashed line represents the tangent through the midpoint of the vulnerability curve and its x-intercept represents the air entry pressure ($P_e$) according to Meinzer *et al.* (2009).

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<figure><img src="image_2.png" /></figure>

Fig. 1. Two contrasting curves showing the predicted loss in Ks according to the 's-shaped' and 'r-shaped' scenario. Resistance to xylem embolism has typically been evaluated by inducing lower xylem pressures progressively and determining their corresponding losses in Ks (closed circles), which makes it difficult to distinguish between real and artefactual loss in Ks (closed circle 'a' to 'b'). By exposing a sample to a less negative pressure, i.e. nearer to zero, than the minimum pressure previously experienced, a decrease in Ks can be considered as artefactual (open circle 'a' to 'b') since less negative pressures cannot induce embolism (open circle 'a' to 'c').

210x296mm (150 x 150 DPI)
<figure><img src="image_3.png" /><figcaption>Fig. 2. Percentage of open vessels (i.e., open at both sample ends) in 15cm-long samples of *Olea europaea* and *Quercus palustris*. There are no data for *Betula pendula* since no open vessels were detected in either the 15 cm-long samples or the 40 cm-long ones for this species. Each column is the average of four samples per species and vertical bars represent ± the standard error.</figcaption></figure>

116x89mm (300 x 300 DPI)
<figure><img src="image_4.png" /><figcaption>Fig. 3. Mean specific hydraulic conductivity ($K_s$) for *Betula pendula*, *Olea europaea* and *Quercus palustris* xylem samples before (NP, 'native xylem pressure') and after spinning in a rotor according to the Cavitron (panels on the left) or the original rotor design (Alder et al. 1997; panels on the right) to induce a less negative xylem pressure (-1.5 MPa, AS, 'after spinning') than the minimum pressure previously experienced by the plant. Each column is the average of *n* samples per species and vertical bars represent the standard error. Significant differences between NP and AS are indicated as *p*<0.05 or *p*<0.01; *n.s.* indicates no significant differences.</figcaption></figure>

210x296mm (150 x 150 DPI)
<figure><img src="image_5.png" /><figcaption>Fig. 4. a) Mean specific hydraulic conductivity ($K_s$) for *Betula pendula*, *Olea europaea* and *Quercus palustris* girdled and control (i.e. non-girdled) xylem samples after xylem pressure relaxation. b) Specific hydraulic conductivity ($K_s$) for *Olea europaea* measured before ($K_{s1}$) and after ($K_{s2}$) disconnecting the samples, leaving them to sit in water for an equivalent time as those that were spun (i.e. 5 min) and reconnecting them to the tubing conductivity apparatus. Each column is the average of *n* samples per species and vertical bars represent the standard error. *n.s.* indicates no significant differences (i.e. *p*>0.05).</figcaption></figure>

175x298mm (300 x 300 DPI)
<figure><img src="image_6.png" /><figcaption>Fig. 5. Transverse micro-CT images of *Olea europaea* at different xylem water potentials (Ψ<sub>xyl</sub>, bottom of each picture). Embolised vessels are observed as black. The percentage loss of hydraulic conductivity (PLC) is indicated for each sample. Scale bar = 500 µm.</figcaption></figure>

91x254mm (150 x 150 DPI)
<figure><img src="image_7.png" /><figcaption>Fig. 6. Vulnerability curve obtained by direct observation (micro-CT) from intact olive plants (full, black dots correspond to individual PLC measurements based on scans, and the solid black line to the Weibull fitting). The grey dashed line represents the tangent through the midpoint of the vulnerability curve and its x-intercept represents the air entry pressure (P<sub>e</sub>) according to Meinzer *et al.* (2009).</figcaption></figure>

100x77mm (300 x 300 DPI)