Dr Rosana López
email: rosana.lopez@upm.es

Markus Nolf

Remko A Duursma

Eric Badel

Richard J Flavel

Hervé Cochard

Brendan Choat

Forestal Etsi Montes

Mitigating the open vessel artefact in centrifuge based measurement of embolism resistance

Keywords: Xylem Embolism, Drought Resistance, vulnerability to cavitation, cavitron, centrifuge technique, x-ray micro CT Vulnerability to embolism, xylem embolism, drought, centrifuge technique, Cavitron, X-Ray microCT

words max) 20

Centrifuge-based techniques to assess xylem vulnerability to embolism are increasingly being used, 21 although we are yet to reach a consensus on the nature and extent of artefactual embolism 22 observed in some angiosperm species. In particular, there is disagreement over whether these 23 artefacts influence both the spin (Cavitron) and static versions of the centrifuge technique equally. 24

We tested two methods for inducing embolism: bench dehydration and centrifugation. We used 25 three methods to measure the resulting loss of conductivity: gravimetric flow measured in bench-26 dehydrated and centrifuged samples (static centrifuge), in situ flow measured under tension during 27 spinning in the centrifuge (Cavitron), and direct imaging using X-ray microCT observations in stems 28 of two species of Hakea that differ in vessel length. 29

Both centrifuge techniques were prone to artefactual embolism in samples with maximum vessel 30 length longer, or similar, to the centrifuge rotor diameter. Observations with microCT indicated that 31 this artefactual embolism occurred in the outer most portions of samples. The artefact was largely 32 eliminated if flow was measured in an excised central part of the segment in the static centrifuge or 33 starting measurements with the Cavitron at pressures lower than the threshold of embolism 34 formation in open vessels. The simulations of loss of conductivity in centrifuged samples with a new 35 model, CAVITOPEN, confirmed that the impact of open vessels on the vulnerability to embolism 36 curve was higher when vessels were long, samples short and when embolism is formed in open 37 vessels at less negative pressures. This model also offers a robust and quantitative tool to test and 38 correct for artefactual embolism at low xylem tensions. 39

Introduction 44

Xylem water transport is dependent upon water held in a metastable state of water; evaporation of 45 water from the leaf cell walls generates tension, which is transmitted through the water column to 46 the roots. Water under tension is prone to cavitation, i.e. the abrupt transition from a metastable 47 liquid to a gas, resulting in the formation of gas emboli that block the xylem conduits and impairs 48 water transport [START_REF] Tyree | Do woody plants operate near the point of catastrophic xylem 691 dysfunction caused by dynamic water stress? : answers from a model[END_REF]. As tension in the xylem sap increases, for example during 49 drought, so does the probability of embolism formation. During severe or prolonged droughts, 50 hydraulic failure can result in the complete loss of hydraulic conductance in the xylem and 51 subsequent canopy dieback, or whole plant death (Brodribb and Cochard 2009;[START_REF] Nardini | Shoot desiccation and hydraulic failure in temperate woody 643 angiosperms during an extreme summer drought[END_REF]52 Rodríguez-Calcerrada et al. 2017;[START_REF] Urli | Xylem embolism threshold for 696 catastrophic hydraulic failure in angiosperm trees[END_REF][START_REF] Venturas | Chaparral shrub 698 hydraulic traits, size, and life history types relate to species mortality during California's 699 historic drought of 2014[END_REF]. Hydraulic failure is now 53 considered a principal cause of drought-induced plant mortality and forest die off (Choat et al. 2012;54 Sala et al. 2010). The projected rise in global mean temperature and frequency of extreme climate 55 events over the next century will impact forest ecosystems and shift species distribution ranges. In 56 this sense, resistance to embolism has emerged as a crucial parameter to understanding species 57 ecology, differences in water use strategies, and for predicting future mortality events (Brodribb 58 2017). 59

Xylem resistance to embolism is usually characterized with a vulnerability curve, showing the 60 decrease in hydraulic conductivity as a function of the xylem tension. Since the publication of the 61 first vulnerability curves for woody plants were published in 1985 [START_REF] Sperry | Xylem embolism in the palm Rhapis excelsa[END_REF] and 1986 (Tyree and 62 Dixon 1986), a number of techniques that allow for more rapid measurement of vulnerability have 63 been introduced (see Cochard et al. (2013) for a detailed review). However, although the time 64 required for construction of a vulnerability curve has been dramatically reduced, recent work 65 suggests that some of these methods are prone to experimental artefact (Choat et al. 2010;Cochard 66 et al. 2010;[START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF][START_REF] Torres-Ruiz | Vulnerability to cavitation in Olea europaea current-year shoots: further 677 evidence of an open-vessel artifact associated with centrifuge and air-injection techniques[END_REF]). This has led to re-examination of 67 methodology used to measure vulnerability to embolism [START_REF] Jansen | Current controversies and challenges in applying plant hydraulic 638 techniques[END_REF]. 68 patterns of embolism spread through the sample may differ from a naturally dehydrated sample (Cai 96 et al. 2010). The tension profile in the centrifuged segment is highest in the axis of rotation (i.e. in 97 the middle section of the segment) and declines towards the segment ends (Cochard et al. 2005), 98 while during natural dehydration the tension profile across the segment is expected to remain 99 approximately constant (Cai et al. 2010). Nevertheless, the vulnerability curves generated by 100 centrifugation agree well with the bench-top method in conifers and short-vesseled angiosperm 101 species (Alder et al. 1997;Cochard et al. 2005;Cochard et al. 2010;[START_REF] Li | Evaluation of centrifugal methods for measuring 640 xylem cavitation in conifers, diffuse-and ring-porous angiosperms[END_REF]. In contrast, 102 inconsistent results have been obtained for species with long vessels, specifically those in which a 103 significant number of vessels in the sample are longer than the centrifuge rotor (Choat et al. 2010;104 Jacobsen and Pratt 2012;[START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF][START_REF] Torres-Ruiz | Vulnerability to cavitation in Olea europaea current-year shoots: further 677 evidence of an open-vessel artifact associated with centrifuge and air-injection techniques[END_REF]. 105

Since 2005 the number of vulnerability curves constructed by centrifugation has increased 106 exponentially (see Fig. 3 in Cochard et al. (2013)). Accordingly, considerable effort has been devoted 107 to testing and validation of centrifuge techniques, whether measuring the flow gravimetrically after 108 spinning (static centrifuge method), or while centrifuging (Cavitron method). However, we are yet to 109 reach a consensus on the nature and extent of artefactual embolism observed with centrifuge 110 techniques. In particular, there is disagreement over whether these artefacts influence both spin 111 (Cavitron rotor) and static versions of the centrifuge technique equally [START_REF] Hacke | The standard 631 centrifuge method accurately measures vulnerability curves of long-vesselled olive stems[END_REF]Sperry et 112 al. 2012). In recent years, the application of x-ray computed microtomography (microCT) to the 113 study of plant hydraulics has emerged as a potentially powerful tool to validate hydraulic 114 techniques. In addition to providing a non-invasive assay of xylem function, it allows for analyses of 115 spatial and temporal patterns of embolism formation (Brodersen et al. 2013;Choat et al. 2016;116 Dalla-Salda et al. 2014;Torres-Ruiz et al. 2016). 117 In this study we evaluated the performance of both centrifuge techniques against bench 118 dehydration in order to examine possible discrepancies associated with each technique. First, we 119 F o r P e e r R e v i e w 6 tested two methods for inducing embolism: bench dehydration and centrifugation. We then tested 120 three ways of measuring the resulting loss of conductivity: gravimetric flow measured in bench-121 dehydrated and centrifuged samples (static centrifuge), in situ flow measured under tension during 122 spinning in the centrifuge (Cavitron), and direct imaging using X-ray microCT observation. All 123 experiments were carried out with two species of the genus Hakea that differ in vessel length. H. 124 dactyloides is a short vesseled species with maximum vessel length shorter than 14 cm, whereas H. 125 leucoptera has longer vessels and maximum vessel length is ca. 25 cm. Additionally, we compared 126 results obtained using two rotor diameters (14 and 27 cm) to assess the effect of sample length, and 127 measured hydraulic flow both in the whole, spun segments and excised middle sections. Spatial 128 patterns of embolism within samples were visualized with X-ray microCT after centrifugation in 129 order to provide further insight into potential discrepancies. Finally, a new model, CAVITOPEN was 130 developed to simulate the effect of vessel and sample lengths on centrifuge estimates of embolism 131 resistance. We hypothesized that i) both centrifuge techniques, the static centrifuge and the 132 cavitron, are prone to similar artefacts when constructing vulnerability curves of long-vesseled 133 species; ii) the shape of the vulnerability curve of centrifuged samples will depend on the amount of 134 cut open vessels; iii) image techniques and standard flow measurements will produce similar 135 vulnerability curves. 136

Material and methods 137

Plant Material 138

Experiments were carried out on branch material of two diffuse-porous species of the same genus 139 exhibiting different vessel lengths, Hakea dactyloides (Gaertn.) Cav. and Hakea leucoptera R. Br. 140 Branches were sampled from natural populations of H. dactyloides at Mount Banks (33° 34' 46'' S,141 150° 21' 56'' E;NSW,Australia) and H. leucoptera at Binya State Forest (34° 11' 16'' S,146° 16' 13'' 142 E;NSW,Australia) from May to September 2016 (late autumn-winter in the South Hemisphere). Sun 143 exposed branches of 1.5-2.0 m length were collected in the field in the early morning and 144 2016. Two leaves of five plants per species were covered with aluminium foil and sealed with a 149 plastic bag 1 hour before excision and measurement with a pressure chamber (PMS Instrument Co.,150 Albany, OR, USA). 151

Native embolism was determined in current-year, one-year and two-year old segments of 5 152 branches per species to ensure that the effects of previous natural water stress were minimised. 153

Note that segments containing 1-year and 2-year-old growth were necessary to fit in the 27 cm rotor 154 of the centrifuge. Measuring native embolism we also wanted to control for sample collection date 155 because branches were cut at different times during late autumn-winter 2016 to avoid long storage. 156 Branch proximal end was cut underwater to release tension for 30 min [START_REF] Torres-Ruiz | Direct X-ray microtomography observation confirms the induction 684 of embolism upon xylem cutting under tension[END_REF]157 Wheeler et al. 2013) and then the branch was progressively recut under water to segments 50 mm 158 long. Note that at least twice the maximum vessel length was removed from the cut end after 159 tension relaxation. Thereafter, the edges of these segments were trimmed using a razor blade. Initial 160 conductivity (K h ) was measured in 50 mm long segments with filtered, degassed 2 mmol KCl solution 161 at low pressure (≤ 4 kPa) with a liquid flowmeter (LiquiFlow L13-AAD-11-K-10S; Bronkhorst High-162 Tech B.V., Ruurlo, the Netherlands). The segments were then flushed with the same solution at a 163 minimum of 0.20 MPa for 15 min to remove embolism and subsequently determine maximum 164 hydraulic conductivity (K max ). The native percentage loss of conductivity (PLC) was calculated for 165 each segment as: 166 PLC = 100 x (1-K h / K max ) (equation 1) 167

Maximum vessel length and vessel length distribution 170

Ten branches per species were sampled from the same plants as used for hydraulic measurements 171 to determine maximum vessel length with the air perfusion technique [START_REF] Ewers | Variation in vessel length and diameter in stems of six tropical and 629 subtropical lianas[END_REF]. Once 172 in the lab, 60 cm long segments were flushed for 1 h with degassed, filtered 2 mmol KCl solution at 173 0.18-0.20 MPa to remove any embolism. Then each segment was infiltrated with compressed air at 174 0.05 MPa at its distal end with an aquarium air pump while the basal end was repeatedly shortened 175 by 2 cm under water until air bubbles emerged. The remaining sample length was assumed as 176 maximum vessel length. 177

An estimate of the amount of vessels longer than the centrifuge rotor diameter and longer than half 178 the rotor diameter (open to centre vessels) was assessed in four branches of H. dactyloides and five 179 branches of H. leucoptera by measuring the decrease in PLC after air injection (Cochard et al. 1994;180 Torres-Ruiz et al. 2014). Briefly, 35 cm long segments were flushed as described above to remove 181 embolism. Then, tubing was attached to the distal end of these segments and compressed air was 182 injected into the samples at 0.1 MPa for 10 min using a pressure chamber. This pressure was 183 sufficient to empty the open vessels but not high enough to move water through wet pit membranes 184 between adjacent vessels [START_REF] Ewers | Variation in vessel length and diameter in stems of six tropical and 629 subtropical lianas[END_REF]. PLC was determined in 3 cm long segments 185 across the sample as described for native embolism. At the injection point, PLC is close to 100% 186 because all the vessels are air filled and progressively decrease to 0 for a length longer than the 187 longest vessel in the sample. The PLC at each distance from the injection point corresponds to the 188 percentage of contribution to flow from vessels longer than this distance. If all the vessels were of 189 equal diameter, this percentage would correspond to the number of vessels longer than the distance 190 from the injection point. In this case of the two Hakea species used are diffuse porous and vessel 191 diameters within the same sample did not vary greatly. Thus the curves in Fig. 1 (wrapped with aluminium foil and a plastic bag at least 1 h before sampling). When the target Ψ X to 198 construct the VC was reached, branches were sealed into a plastic bag with moistened paper towels 199 for 1 h to equilibrate Ψ X . Water potential was measured again in two bagged leaves of the same 200 branchlet to confirm homogeneous Ψ X in the sample. The Ψ X of the sample was considered 201 equilibrated if the difference between the three Ψ X (one measured before sealing the branch and 202 two measured after equilibration) was not higher than 0.1 MPa. Afterwards tension was released for 203 30 minutes by cutting the branch proximal end under water and PLC was determined in one-year-old 204 segments as for native embolism. Vulnerability curves were generated by plotting PLC against Ψ X . 205 For H. leucoptera 7 branches were dehydrated and 4 different branchlets per branch were measured 206 at different Ψ X to construct the vulnerability curve and for H. dactyloides we used 12 branches and 207 two branchlets per branch. All branchlets were far apart (at least four branch orders) and after 208 collection the cutting surface was covered with parafilm to avoid air entry in the rest of the sample. 209

Centrifuge techniques 210

We compared two centrifuge techniques: i) the static centrifuge method described by Alder et al. 211 (1997) and ii) the in situ flow technique (Cavitron (Cochard 2002;Cochard et al. 2005)). In the static 212 centrifuge two different sizes of custom-built rotors, 14 cm and 27 cm, were used to test the effect 213 of segment length and fraction of open vessels. All hydraulic conductivity measurements were 214 performed using filtered, degassed 2 mmol KCl solution and a flow meter (see Native embolism 215 section). 216 trimmed under water and both ends were shaved to a final length of 14 or 27 cm. The initial 218 hydraulic conductivity was measured as described above (see Native embolism section) with a 219 pressure head of 7.5 kPa. Subsequently, 14-cm long branches were spun in the centrifuge (Sorvall RC 220 5C Plus) for 5 minutes at increasing pressure steps. Foam pads saturated with the solution used for 221 measurements were placed in the reservoirs of the rotor to maintain sample ends in contact with 222 the solution even when the rotor was stopped [START_REF] Tobin | Xylem vulnerability to cavitation can be 673 accurately characterised in species with long vessels using a centrifuge method[END_REF]). After each step, samples were 223 removed and K h was measured on the whole segment as described for native embolism. In the 224 27cm-long branches we modified the single spin method [START_REF] Hacke | The standard 631 centrifuge method accurately measures vulnerability curves of long-vesselled olive stems[END_REF]) so that two 225 measurements were made in each centrifuged segment. The initial K h was measured before spinning 226 in the 27-cm long sample. After spinning, K h was measured on the whole segment and the first PLC 227 was calculated. Subsequently, a 4 cm-long segment was cut from the middle section and its K h was 228 measured. The second PLC was determined in this 4 cm-long segment after flushing to obtained the 229 maximum K h (K max ) as described for native embolism. 230

In situ flow centrifuge measurements (Cavitron technique) were carried out on six branches per 231 species using a modified bench top centrifuge (H2100R, Cence Xiangyi, Hunan, China). For the static 232 centrifuge, samples were trimmed under water to a length of 27 cm to fit in the rotor. Initial 233 conductivity, K i , was determined at a xylem pressure of -0.5 MPa in H. dactyloides and 1.5 MPa in H. 234 leucoptera. The xylem pressure was then lowered stepwise by increasing the rotational velocity, and 235 K h was again determined while the sample was spinning. The PLC at each pressure step was 236 quantified as 237 (NSW, Australia). They were gradually dehydrated to five different xylem water potentials ranging 241 from -4.8 MPa to -9 MPa as for the bench dehydration method. After measuring Ψx, tension was 242 relaxed by cutting the proximal end of the branch under water leaving it submerged for 30 minutes. 243

PLC = 100 x (1-K h / K i ). ( equation
Then the branch was sequentially cut back under water and finally 10-mm-long segments were 244 excised under water from current-year shoots, wrapped in Parafilm, inserted into a plexiglass tube 245 and then placed in an X-ray microtomography system (GE-Phoenix V|tome|xs, GE Sensing & 246 Inspection Technologies, Wunstorf, Germany) to visualize embolized vessels. Another subset of 247 branches of H. leucoptera was centrifuged to five (-5, -6, -7, -8, -9 MPa) and three (-5, -6, -7 MPa) 248 different water potentials in the static centrifuge using 27 cm and 14 cm long segments, 249 respectively. They were immediately submerged in liquid paraffin wax and preserved at 4 °C for 250 three days until measured in the same facility (Cochard et al. 2015). Seven branches of H. dactyloides 251 were also centrifuged at four (-3, -4, -5, -6 MPa) and three (-3, -4, -5 MPa) water potentials with the 252 27 and 14 cm rotors, respectively, following the same protocol. One branch of H. leucoptera was 253 prepared as the centrifuged samples but was not spun in the centrifuge to detect any possible 254 artefact due to sample preparation. All samples were scanned at the middle of the sample. 255

Additionally, in three 27 cm long samples we scanned at 6 cm and 12 cm from the axis of rotation to 256 examine embolism profiles across a sample. 257 X-ray scan settings were 90 kV and 170 mA, and 1800 projections, 600 ms each, were acquired 258 during the 360° rotation of the sample. The resultant images covered the whole cross section of the 259 sample in 8.7 mm length with a spatial resolution of 8.7 μm per voxel. At the end of the scan, the 260 sample was cut back to 30 mm length, injected with air at >1 MPa pressure and rescanned at the 261 same location as before to visualize all empty vessels in the fully embolized cross section. After 262 three-dimensional reconstruction with Phoenix datos|x2 Reconstruction Version 2.2.1-RTM (GE 263 Sensing & Inspection Technologies, Wunstorf, Germany), volumes were imported into ImageJ 1.49k 264 [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. A median Z projection of c. 100 μm along the sample axis was extracted from 265

F o r P e e r R e v i e w
12 the middle of the scan volumes following the protocol in [START_REF] Nolf | Visualization of xylem 645 embolism by X-ray microtomography: a direct test against hydraulic measurements[END_REF]. PLC of each sample was 266 estimated calculating the theoretical hydraulic conductance based on the conduit dimensions of 267 embolized and functional vessels (Choat et al. 2016). To measure conduit dimensions, a radial sector 268 of the transverse section was selected in the same microCT scan and all their embolized vessels were 269 measured manually. The image of this sector was then binarized so the dimensions of the selected 270 embolized vessels matched with the manually drawn vessels. This threshold value was then used for 271 binarizing the image of the whole cross section and all the embolized vessels were measured using 272 the Analyse Particles function in Image J. Theoretical specific hydraulic conductivity (K sth ) was 273 calculated as: 274

ℎݐݏܭ = ∑൬ ವ ర ഏ భమఴಏ • ∆ ∆ೣ ൰ (equation 3) 275 276
Where D is the equivalent circular vessel diameter based on vessel area, η viscosity of water, Δp/Δx 277 pressure gradient per xylem length, A xylem cross-sectional area. 278

279

The current theoretical specific hydraulic conductivity (K sth ) for each sample was calculated by 280 subtracting the summed specific hydraulic conductivity of embolized vessels from the K sth(max) of that 281 sample, calculated as the K sth of the sample after air injection. The pressure gradient used for 282 calculations of K sth was similar to the pressure gradient used in the hydraulic measurements, 0.06 283 MPa m -1 . 284

Vulnerability curve fitting and statistical analysis 285

Vulnerability curves were fitted using a Weibull function [START_REF] Ogle | Hierarchical statistical modeling of xylem 648 vulnerability to cavitation[END_REF]) in R 3.2.0 (R Core Team, 286 2015) using the fitplc package [START_REF] Duursma | fitplc -an R package to fit hydraulic vulnerability curves[END_REF]. Confidence intervals of P 12 , P 50 and P 88 (Ψx 287 at 12, 50 and 88 % loss of conductivity, respectively) and the slope of the curve at 50% loss of 288 conductivity (S 50 ) were used to compare between methods. Confidence intervals (CI) for the bench 289 13 dehydration and the static centrifuge techniques were obtained using bootstrap resampling (999 290 replicates). Methods were considered to be statistically different if the 95% CIs did not overlap. 291 Differences in native embolism and specific initial conductivity between sampling dates were tested 292 with a one-way ANOVA. Means were compared using a Tukey test at 95% confidence. Vulnerability 293 curve parameters across methods were compared at the Ψx corresponding with three levels of loss 294 of conductivity: 12%, 50% and 88% (P 12 , P 50 and P 88 , respectively) and the slope of the VC at 50% loss 295 of conductivity (S 50 ). 296

CAVITOPEN-simulation of the effect of open vessels in a centrifuged sample 297

To disentangle the effects of centrifugation on 'true' vessel embolism at the centre of the samples, 298 where more vessels are closed at both ends and tension is maximum, from draining of open vessels 299 at both sample ends a new model, CAVITOPEN, was developed. In a centrifuged sample, the 300 variation of xylem pressure (P) with distance from the axis of rotation (r) is given by the following 301 equation (Alder et al. 1997): 302 dP/dr = ρω 2 r (equation 4) 303 where ρ is the density of water, and ω the angular velocity. 304

Integrating this equation from R (distance from the axis of rotation to the water reservoir) we can 305 obtain the pressure at r (P r ): 306

P r = 0.5 ρω 2 (R 2 -r 2 ) (equation 5) 307
The effect of vessel length on 'true' vessel embolism in a spun sample has already been modelled by 308 Cochard et al (2005). Briefly, if the vessels are infinitely long, the VC obtained by centrifugation 309 should yield the correct P 50 value. When the vessels are infinitely short the P 50 value is 310 underestimated due to the variation of xylem pressure inside the spun sample (eq. 4) and the 311 consequent gradient of embolism along the sample: xylem pressure is minimum in the middle of the 312 F o r P e e r R e v i e w 14 sample and null at the extremities (eq. 5). Since the loss of conductivity is measured on the whole 313 sample, an underestimation of the degree of embolism in the middle of the sample is predicted. This 314 effect of vessel length was further tested with the CAVITOPEN model and found marginal, i.e. the 315 shift in the VC was negligible, compared to the draining effect. For sake of simplicity, this effect was 316 no longer considered in the simulations. To simulate the draining effect at both sample ends, we first 317 hypothesized that vessel ends follow a logarithmic distribution following the vessel length 318 probability density function proposed by Cohen et al. (2003) and assuming vessel ends uniformly 319 distributed across the length of the sample: 320

N x = N 0 . exp(-x/L max ) (equation 6) 321
where N x is the number of open vessels at the distance x from sample ends, N 0 the total number of 322 vessels and L max the maximum vessel length. 323

The second assumption of the model is that open vessels drain when the minimum pressure in the 324 vessel exceeds a threshold value P open . Because of the quadratic distribution of the pressure in the 325 sample, vessels having their end wall located closer to the sample ends, i. e. further from the centre 326 of rotation, will drain at a higher rotational velocity. 327

The branch segment was discretised in 0.1 mm thick sections arranged in serial. The xylem pressure 328 in the middle of the segment was set to a pressure varying from 0 to -12 MPa in 1 MPa steps. The 329 model then computes the pressure at steady state in each 0.1 mm section and determines the PLC 330 caused by 'true' embolism (non-open vessels) both species across the sampling dates. We measured higher PLC in two-year-old branch segments 343 (< 13 %) than in current year growth (< 2 %) in H. leucoptera whereas in H. dactyloides native 344 embolism was lower than 2% in all samples. Maximum xylem specific conductivity (K smax ) was 0.87 ± 345 0.10 kg m -1 s -1 MPa -1 in H. leucoptera and 1.29 ± 0.09 kg m -1 s -1 MPa -1 in H. dactyloides (mean ± sd). No 346 significant differences in native PLC or K s (P > 0.05; Table S1) were detected between sampling dates. 347

Maximum vessel length and vessel length distribution 348

Maximum vessel length as determined by air injection was 25 cm (standard deviation, sd = 5) in H. 349 leucoptera and 10 cm (sd = 3) in H. dactyloides. Air injected branches of H. dactyloides showed 17% 350 PLC at 7 cm from the injection point, 5% at 14 cm and less than 1% at 28 cm, whereas in H. 351 leucoptera the PLC was always higher, 50%, 25%, and 5% at 7, 14 and 28 cm respectively (Fig. 1). 352

Thus the number of open vessels at both ends when using the centrifuge technique differed 353 between species. 354

Vulnerability curves 355

Vulnerability curves (VCs) obtained with the bench dehydration technique were s-shaped for both 356 species, with significant embolism only occurring once a threshold water potential had been 357 reached. This threshold was more negative in H. leucoptera

MPa) (Fig. 2). VCs obtained with bench dehydration had the most negative P 12 and the steepest 359 slopes of all methods (Table S2), meaning that embolism formation started at more negative Ψx and 360 conductivity was lost across a narrower range of Ψx compared with VCs generated by centrifugation. 361

When the centrifuge was used to induce embolism, results in the shorter-vesseled species, H. 362 dactyloides, were similar for the three techniques used to measure loss of conductivity, flowmeter, 363 Cavitron and microCT (average P 50 with the 27 cm rotor in the static centrifuge and the Cavitron -4.8 364 MPa), and the CI at 95% overlapped with bench dehydration (P 50 = -5.0 MPa). The VC generated with 365 the 14 cm rotor for H. dactyloides yielded slightly less negative values (P 50 = -4.3 MPa; Table S2; Fig. 366 2). In contrast, VCs for H. leucoptera differed considerably depending on the method and the sample 367 length. Vulnerability parameters (P 12 , P 50 , P 88 ) obtained with the Cavitron (-5.0, -7.1 and -9.0 MPa, 368 respectively) matched more closely with the bench dehydration VC . For 369 samples spun in the static centrifuge, we found a significant effect both of the rotor size and the 370 segment used to measure flow (whole, spun segment or excised middle section in the 27 cm rotor) 371 on apparent vulnerability to embolism: segments measured across their entire length exhibited 372 higher vulnerability to embolism compared to the bench-dehydration VC as shown by P 12 (-1.2 and -373 2.6 MPa for 14 and 27 cm rotors, respectively) and P 50 respectively), but seemed 374 less vulnerable towards the dry end of the curve (P 88 of -14.2 and -10.4 MPa, respectively; Table S2). 375 Both VCs were almost linear when flow was measured across the whole segment with a shift 376 towards more vulnerable values with the 14 cm rotor, but became s-shaped when only the middle 377 section of the 27 cm segment was measured (Fig. 2). Removing the segment ends resulted in a 378 steeper slope and significantly more negative values of P 12 and P 50 . The Cavitron and the middle 379 segment techniques yielded similar results and agreed well with the dehydration technique in P 50 380 and P 88 and with microCT image analysis (red triangles in Fig. 2 levels were consistently at their highest near the sample ends (at 12 cm from the axis of rotation) 384 when spun at equivalents of -5, -7 and -9 MPa in the static centrifuge (Fig. 3). At -5 and -7 MPa loss 385 of conductivity decreased from the basal end to the centre, contradicting theoretical expectations. 386 This trend was observed even at Ψx inducing less than 40% PLC based on the bench dehydration VC 387 (Fig. 3). Only at -9 MPa, that is, below P 88 on bench dehydration, did levels of embolism converge 388 along the length of the sample at 80-90%. 389

Influence of open vessels in the VC of a centrifuged sample 390

The simulations produced by the CAVITOPEN model confirmed that the shape of the VCs generated 391 by the centrifugation was largely dependent on vessel and sample lengths. As maximum vessel 392 length decreased, PLC of the whole sample decreased at a given Ψx, and the shape of the VC shifted 393 from exponential to sigmoidal (Fig. 4a). The same pattern was observed when the sample length 394 increased (Fig. 4b). For instance, with 14-cm-length centrifuged samples, P 50 ranged from -0.6 MPa 395 to -7.7 MPa varying the maximum vessel length of the sample from 50 cm to 5 cm. Likewise, the P 50 396 of a centrifuged sample with maximum vessel length of 15 cm ranged from -2.1 MPa in the 14-cm 397 rotor to -7.7 MPa using a 40-cm rotor. Embolism of vessels open from the cut surface (Fig. S1) 398 influenced values of PLC at high negative pressures, even in short-vesseled samples, resulting in 399 rapid loss of conductivity followed by a plateau. The more open vessels and the less negative the 400 threshold of embolism of open vessels (Fig. 4c), the higher is this plateau and stronger the impact on 401 the VC (Fig. 4). VCs can be corrected if the first inflection point of the curve is considered the starting 402 point for initial conductivity (K i ), i.e. 0% loss of conductivity. This is shown in Fig. 4d with actual 403 measurements of PLC obtained in 27-cm centrifuged samples of H. leucoptera. When the 404 CAVITOPEN model was fit (black circles and grey solid line, respectively) and we used the inflection 405 point as starting point for K i , the corrected curve matched the reference VC obtained with bench 406 dehydration (Fig. 4d the errors were significantly greater in the static centrifuge method. In a species with maximum 415 vessel length longer or similar to the centrifuge rotor diameter, the static centrifuge significantly 416 overestimated xylem vulnerability to embolism if the whole spun segment was used to measure 417 flow. Observations with microCT indicated that artefactual embolism caused by centrifugation of 418 samples occurred in the outer most portions of samples. However, we demonstrated that 419 artefactual embolism was largely eliminated from static centrifuge if flow was measured in an 420 excised central part of the segment. This altered protocol yielded VCs similar to those obtained on 421 the same species with bench dehydration thus allowing these centrifuge techniques to accurately 422 measure vulnerability to embolism in longer vesseled species. We also present a new model 423 (CAVITOPEN) that simulates the impact of vessel draining at the cut end on the whole VC curve and 424 showed that errors were largely dependent on vessel length and rotor diameter. This model allows 425 researchers to quantitative test and avoid errors associated with the artefactual embolism. The 426 bench dehydration technique indicated that significant embolism was only initiated in both species 427 after water potential dropped below a threshold value, -3.8 MPa in H. dactyloides and -6.3 MPa in H. 428 leucoptera. PLC then increased rapidly and hydraulic conductivity was lost almost completely within 429 a span of 1 MPa (Fig. 2). These vulnerability curves have been classified as sigmoidal or s-shaped as 430 opposed to exponential or r-shaped curves, characterized by rapid conductivity losses as soon as the 431 water potential declines below zero (Cochard et al. 2013;[START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF]. A third type of VC, 432

Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

F o r P e e r R e v i e w
19 intermediate between these two, exhibits a linear response, and is mainly found in diffuse porous 433 species when using centrifugation to induce embolism (Cochard et al. 2013). 434 Our results showed that VCs obtained with the static centrifuge technique and the Cavitron are 435 similar to bench dehydration in a short-vesseled species, i.e. a species with no through vessels (open 436 at both ends) in the segment and with few vessels open from the cut surface to the middle of the 437 segment. All centrifuge generated VCs for H. dactyloides were sigmoidal and similar to bench 438 dehydration VCs, with a slight shift towards more vulnerable values when using the 14 cm rotor (Fig. 439 2, Table S2) as recently found by Pengxian et al. (2018) in Acer mono when comparing in the static 440 centrifuge the 14 cm and 27 cm rotors. VCs of other short vesseled angiosperms such as Betula 441 pendula (Cochard et al. 2010), Fagus sylvatica (Aranda et al. 2014), Populus tremuloides (Schreiber et 442 al. 2011) or Acer negundo (Christman et al. 2009) were also sigmoidal when the static centrifuge or 443 the Cavitron were used. In contrast, the VC shape obtained for H. leucoptera samples differed 444 significantly depending on methodology resulting in a shift of P 50 of 2 MPa in samples from the same 445 population (Fig. 2, Table S2). This dramatic change was observed previously in peach (Prunus persica) 446 when the length of the centrifuged samples was varied in a Cavitron; shorter samples were more 447 vulnerable to embolism (P 50 shifted from -4.5 to -1 MPa) and VCs became r-shaped (Cochard et al. 448 2010). However, when using the static centrifuge to measure the same population, Sperry et al. 449 (Sperry et al. 2012) found that VCs were linear and relatively insensitive to the number of open 450 vessels with P 50 less negative than -2 MPa using 14 cm and 27 cm samples. This difference in 451 sensitivity to the proportion of open vessels in the centrifuged samples has led some to conclude 452 that the original centrifuge method and rotor design are not subject to the open vessel artefact 453 [START_REF] Hacke | The standard 631 centrifuge method accurately measures vulnerability curves of long-vesselled olive stems[END_REF][START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF]). However, Torres-Ruiz et al. (2017) results with both the static centrifuge and the Cavitron when compared with reference curves 459 generated by dehydration or non-invasive imaging (Choat et al. 2016;Choat et al. 2010;Pengxian et 460 al. ;[START_REF] Torres-Ruiz | Vulnerability to cavitation in Olea europaea current-year shoots: further 677 evidence of an open-vessel artifact associated with centrifuge and air-injection techniques[END_REF]. [START_REF] Li | Evaluation of centrifugal methods for measuring 640 xylem cavitation in conifers, diffuse-and ring-porous angiosperms[END_REF] and Pengxian et al. (2018) tested the two centrifuge 461 methods head to head and found close correspondence in VCs across species with different xylem 462

anatomy. An extended literature survey of methods to measure vulnerability to embolism showed 463 that when using the centrifuge, VCs were sigmoidal in conifers and in long vessel species 464 exponential, whereas in diffuse porous species VCs varied from sigmoidal to linear or exponential 465 (Cochard et al. 2013). Our measurements and simulations made with the CAVITOPEN model explain 466 the different shapes of VCs and some disagreements between the static centrifuge and the Cavitron. 467

In short-vesseled angiosperms, we have shown that VCs by centrifugation agreed with each other 468 and closely matched the curves based on bench dehydration and microCT (Choat et al. 2016;469 Cochard et al. 2010). In angiosperms with a proportion of vessels open to the middle but not the 470 whole way through, the standard protocol in the static centrifuge produces linear VCs (Sperry et al. 471 2012). Here the initial conductivity is measured before spinning, thus if the native embolism is low, 472 all the vessels are conductive, regardless of their length. As soon as the sample is spun, the 473 conductivity would be artificially reduced relative to the native state in proportion to the amount of 474 vessels open to centre. Sample with open vessels thus become artificially vulnerable to embolism at 475 the beginning of the VC (i.e. at less negative water potentials). For H. leucoptera, this translated into 476 less negative values of P 12 in all centrifuged samples compared with those measured with the bench 477 dehydration technique creating a linear response or a plateau at high water potentials. Higher 478 differences in P 12 were observed in H. leucoptera than in H. dactyloides according with a higher 479

proportion of vessels open to centre in the former species (Fig. 1). In the Cavitron, the initial 480 measurement was made while spinning at low tension and many open to centre vessels would 481 already be embolised in the initial measurement of conductivity, resulting in a lower artefactual loss 482 pushing them to more negative values but it did not appear to be significant effect here as the 484

Cavitron curves for H. leucoptera were similar to bench dehydration curves. 485

The simulations of PLC with the CAVITOPEN model confirmed that the impact of open vessels on the 486 VC was higher when vessels were long, samples short and when open vessels cavitated at less 487 negative pressures (Fig. 4). If the samples were much shorter than the maximum vessel length of the 488 branch (see the results in Fig. 4 for the 14 cm rotor with L max 50cm), the resulting VC was exponential 489 (r-shaped), as observed in long-vesseled angiosperms, and shifted to more linear or s-shaped when 490 L max was decreased or the sample length increased. One of the assumptions in the model is that 491 vessels open at the cut surface cavitate when they reach a threshold value; that is far less negative 492 than intact vessels whose two ends are included within the spun segment. This influences the shape 493 the VC at higher pressures creating a "bump" in the VC followed by a plateau. This effect can be 494 corrected to some extent if the first inflexion point of the VC is considered to be the 0% point for loss 495 of conductivity. In this case the initial conductivity (K i ) value is shifted to a lower value corresponding 496 to the hydraulic conductivity of the plateau (Fig. 4d). The estimated values of P 50 and S 50 when the 497 CAVITOPEN model was fit to actual measurements agreed quite well with those obtained with 498 reference techniques and confirmed that this model can be used to correct open vessel artefacts for 499 centrifuge based VCs. The estimated L max was however significantly shorter than L max measured with 500 the air injection technique. The air injection technique has shown to produce higher L max than the 501 rubber injection method [START_REF] Pan | A comparison of two methods for measuring vessel length in 650 woody plants[END_REF], thus our values could be overestimated. On the other 502 hand, the model assumed that vessel lengths in a sample follow the density function proposed by 503 Cohen et al. (2003) which can be sensitive to the clustering of vessel lengths (Cai and Tyree 2014) The physical mechanisms underlying this open-vessel artefact are yet to be fully elucidated. Some 508 studies suggest that microbubbles and particles can act as nucleation sites when they flow through 509 the sample as it spins in the Cavitron, causing premature embolism (Cochard et al. 2010;Sperry et al. 510 2012;[START_REF] Wang | Water relations of Robinia pseudoacacia L.: do 701 vessels cavitate and refill diurnally or are R-shaped curves invalid in Robinia?[END_REF]). In the static centrifuge, bubbles might be drawn into vessels while starting 511 the spin or while mounting or dismounting the stems to measure flow [START_REF] Wang | Water relations of Robinia pseudoacacia L.: do 701 vessels cavitate and refill diurnally or are R-shaped curves invalid in Robinia?[END_REF]. In both 512 centrifuge techniques bubbles in open vessels can move by buoyancy while spinning toward the 513 region of lowest pressure at the center of rotation (Rockwell et al., 2014). Draining from open 514 vessels as a consequence of artefactual embolism when the centrifuge starts spinning appears to be 515 a common phenomenon in both rotors. Our microCT images showed that after spinning in the 516 centrifuge, most of the vessels were empty near the ends even though tension ought to be zero 517 (Cochard et al. 2005). The use of water saturated foam pads to avoid desiccation did not prevent this 518 [START_REF] Hacke | The standard 631 centrifuge method accurately measures vulnerability curves of long-vesselled olive stems[END_REF][START_REF] Tobin | Xylem vulnerability to cavitation can be 673 accurately characterised in species with long vessels using a centrifuge method[END_REF]. We discarded the possibility that sample manipulation before 519 spinning or during wax embedding had triggered vessel draining because we scanned control 520 samples that were not spun. These samples showed no embolism (Fig. 3). Furthermore, patterns of 521 embolism did not follow theoretical expectations based on the distribution of tension within the 522 spun sample. The embolism levels decreased from the ends to the center in a fashion consistent 523 with the amount of vessels open to center, opposite to that expected from profile in tension and in 524 agreement with the assumption of the CAVITOPEN model than open vessels artificially cavitate 525 when they reach a threshold pressure that is much less negative than in intact vessels. This pattern 526 was observed at water potentials inducing less than 40% loss of conductivity based on the VC 527 obtained using the middle segment of the centrifuged sample (Fig. 3), even though the centre of the 528 sample experienced the highest tensions. Embolism levels converged within the sample at -9 MPa at 529 80-90%. These results confirm that centrifugation drains open vessels and only reliably measure the 530 vulnerability of intact xylem vessels within the sample (Fig. S1). This is consistent with observations 531 made previously by Cochard et al. (2010) The hydraulic continuity between vessels cut open at each end of the sample and vessels with their 539 terminal ends in this portion of the sample is probably re-established by refilling of vessels immersed 540 under water at both ends (Fig. 2 in Cochard et al. (2010)). This refilling would occur by capillarity 541 either while spinning in the Cavitron or while flow is measured gravimetrically (Fig. S2). Since the 542 middle of the centrifuged sample contains the majority of intact vessels, VCs constructed with the 543 static centrifuge technique of angiosperm species using only the central segment are more reliable 544 and in closer agreement with PLC generated by natural dehydration (Fig. 2). This modification is 545 technically easy to achieve and mitigates the open vessel artefact; however, it carries the 546 disadvantage that samples cannot be spun repeatedly to construct replicate curves for each sample 547 and thus more plant material is needed to construct each curve. 548

Conclusion 549

We confirmed the validity of vulnerability curves constructed with both centrifuge methods for short 550 conduit angiosperm species, those with most conduits shorter than half the length of the centrifuge 551 rotor. A new model, CAVITOPEN was developed to simulate the effect of vessel length, rotor size and 552 vulnerability of open vessels in loss of conductivity of centrifuged samples. In species with maximum 553 vessel length similar to the centrifuge rotor, we recommend constructing vulnerability curves with 554 the Cavitron or measuring flow exclusively in the central part of the spun segment when using the 555 static centrifuge. Alternatively, artefactual embolism at low xylem tensions can be corrected if the 556 first inflexion point of the VC is considered to be the starting point for K max ( Xylem water transport is dependent upon water held in a metastable state of water; evaporation of 45 water from the leaf cell walls generates tension, which is transmitted through the water column to 46 the roots. Water under tension is prone to cavitation, i.e. the abrupt transition from a metastable 47 liquid to a gas, resulting in the formation of gas emboli that block the xylem conduits and impairs 48 water transport [START_REF] Tyree | Do woody plants operate near the point of catastrophic xylem 691 dysfunction caused by dynamic water stress? : answers from a model[END_REF]. As tension in the xylem sap increases, for example during 49 drought, so does the probability of embolism formation. During severe or prolonged droughts, 50 hydraulic failure can result in the complete loss of hydraulic conductance in the xylem and subsequent 51 canopy dieback, or whole plant death (Brodribb and Cochard 2009;[START_REF] Nardini | Shoot desiccation and hydraulic failure in temperate woody 643 angiosperms during an extreme summer drought[END_REF]Rodríguez-52 Calcerrada et al. 2017;[START_REF] Urli | Xylem embolism threshold for 696 catastrophic hydraulic failure in angiosperm trees[END_REF][START_REF] Venturas | Chaparral shrub 698 hydraulic traits, size, and life history types relate to species mortality during California's 699 historic drought of 2014[END_REF]). Hydraulic failure is now considered a 53 principal cause of drought-induced plant mortality and forest die off (Choat et al. 2012;Sala et al. 54 2010). The projected rise in global mean temperature and frequency of extreme climate events over 55 the next century will impact forest ecosystems and shift species distribution ranges. In this sense, 56 resistance to embolism has emerged as a crucial parameter to understanding species ecology, 57 differences in water use strategies, and for predicting future mortality events (Brodribb 2017). 58

Xylem resistance to embolism is usually characterized with a vulnerability curve, showing the decrease 59 in hydraulic conductivity as a function of the xylem tension. Since the publication of the first 60 vulnerability curves for woody plants were published in 1985 [START_REF] Sperry | Xylem embolism in the palm Rhapis excelsa[END_REF] and 1986 (Tyree and Dixon 61 1986), a number of techniques that allow for more rapid measurement of vulnerability have been 62

introduced (see Cochard et al. (2013) for a detailed review). However, although the time required for 63 construction of a vulnerability curve has been dramatically reduced, recent work suggests that some 64 of these methods are prone to experimental artefact (Choat et al. 2010;Cochard et al. 2010;Sperry 65 et al. 2012;[START_REF] Torres-Ruiz | Vulnerability to cavitation in Olea europaea current-year shoots: further 677 evidence of an open-vessel artifact associated with centrifuge and air-injection techniques[END_REF]). This has led to re-examination of methodology used to measure 66 vulnerability to embolism [START_REF] Jansen | Current controversies and challenges in applying plant hydraulic 638 techniques[END_REF]. The most straightforward technique for inducing embolism is bench dehydration, wherein whole 68 plants or long branches are gradually dehydrated to various xylem tensions and hydraulic conductivity 69 of excised segments is measured gravimetrically before and after removing air from embolised 70 conduits [START_REF] Sperry | Mechanism of water stress-induced xylem embolism[END_REF][START_REF] Tyree | Hydraulic architecture of whole plants and plant performance 694 Xylem structure and the ascent of sap[END_REF]. Bench dehydration relies on natural 71 desiccation of plant tissues and is therefore considered as the best reference method with which to 72 validate other techniques (Cochard et al. 2013;[START_REF] Ennajeh | How reliable is the double-ended pressure sleeve 626 technique for assessing xylem vulnerability to cavitation in woody angiosperms?[END_REF][START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF]. This method 73 is not completely free of artefacts and issues associated with disequilibrium in water potential within 74 a stem, blockage of flow by resin/mucilage (Cobb et al. 2007), and excision of samples under tension 75 can all alter the vulnerability curve significantly [START_REF] Wheeler | Cutting xylem under tension or 704 supersaturated with gas can generate PLC and the appearance of rapid recovery from 705 embolism[END_REF]. Although most of these issues 76 can be minimised by adoption of suitable protocols (eg. [START_REF] Torres-Ruiz | Direct X-ray microtomography observation confirms the induction 684 of embolism upon xylem cutting under tension[END_REF], the bench 77 dehydration technique requires several days and a substantial amount of plant material to obtain a 78 vulnerability curve for one species. As such, [START_REF] Holbrook | Negative xylem pressures in plants: a test of the balancing 634 pressure technique[END_REF] and [START_REF] Pockman | Sustained and significant negative water pressure in xylem[END_REF] proposed 79 the use of a centrifugal force to create a defined negative pressure in the xylem sap of excised plant 80 stems, allowing for rapid and consistent generation of vulnerability curves. [START_REF] Pockman | Sustained and significant negative water pressure in xylem[END_REF] 81 constructed vulnerability curves for several species by comparing the hydraulic conductivity before 82 and after spinning branches with their ends exposed to air, removing segments at both ends before 83 measuring conductivity in the remaining, middle section of the sample. Alder et al. (1997) modified 84 this technique with a centrifuge rotor designed to keep the segment ends immersed in water during 85 spinning, allowing the conductivity of a single segment to be remeasured at different tensions to 86 create an entire vulnerability curve for a single sample. This important innovation allowed repeated 87 measurements to be made on the same plant material, reducing the number of samples required for 88 construction of a curve and strengthening the results statistically. Finally, Cochard (2002) patterns of embolism spread through the sample may differ from a naturally dehydrated sample (Cai 95 et al. 2010). The tension profile in the centrifuged segment is highest in the axis of rotation (i.e. in the 96 middle section of the segment) and declines towards the segment ends (Cochard et al. 2005), while 97 during natural dehydration the tension profile across the segment is expected to remain 98 approximately constant (Cai et al. 2010). Nevertheless, the vulnerability curves generated by 99 centrifugation agree well with the bench-top method in conifers and short-vesseled angiosperm 100 species (Alder et al. 1997;Cochard et al. 2005;Cochard et al. 2010;[START_REF] Li | Evaluation of centrifugal methods for measuring 640 xylem cavitation in conifers, diffuse-and ring-porous angiosperms[END_REF]. In contrast, 101 inconsistent results have been obtained for species with long vessels, specifically those in which a 102 significant number of vessels in the sample are longer than the centrifuge rotor (Choat et al. 2010;103 Jacobsen and Pratt 2012;[START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF][START_REF] Torres-Ruiz | Vulnerability to cavitation in Olea europaea current-year shoots: further 677 evidence of an open-vessel artifact associated with centrifuge and air-injection techniques[END_REF]. 104

Since 2005 the number of vulnerability curves constructed by centrifugation has increased 105 exponentially (see Fig. 3 in Cochard et al. (2013)). Accordingly, considerable effort has been devoted 106 to testing and validation of centrifuge techniques, whether measuring the flow gravimetrically after 107 spinning (static centrifuge method), or while centrifuging (Cavitron method). However, we are yet to 108 reach a consensus on the nature and extent of artefactual embolism observed with centrifuge 109 techniques. In particular, there is disagreement over whether these artefacts influence both spin 110 (Cavitron rotor) and static versions of the centrifuge technique equally [START_REF] Hacke | The standard 631 centrifuge method accurately measures vulnerability curves of long-vesselled olive stems[END_REF]Sperry et 111 al. 2012). In recent years, the application of x-ray computed microtomography (microCT) to the study 112 of plant hydraulics has emerged as a potentially powerful tool to validate hydraulic techniques. In 113 addition to providing a non-invasive assay of xylem function, it allows for analyses of spatial and 114 temporal patterns of embolism formation (Brodersen et al. 2013;Choat et al. 2016;Dalla-Salda et al. 115 2014;Torres-Ruiz et al. 2016). 116 In this study we evaluated the performance of both centrifuge techniques against bench dehydration 117 in order to examine possible discrepancies associated with each technique. 6 methods for inducing embolism: bench dehydration and centrifugation. We then tested three ways of 119 measuring the resulting loss of conductivity: gravimetric flow measured in bench-dehydrated and 120 centrifuged samples (static centrifuge), in situ flow measured under tension during spinning in the 121 centrifuge (Cavitron), and direct imaging using X-ray microCT observation. All experiments were 122 carried out with two species of the genus Hakea that differ in vessel length. H. dactyloides is a short 123 vesseled species with maximum vessel length shorter than 14 cm, whereas H. leucoptera has longer 124 vessels and maximum vessel length is ca. 25 cm. Additionally, we compared results obtained using 125 two rotor diameters (14 and 27 cm) to assess the effect of sample length, and measured hydraulic 126 flow both in the whole, spun segments and excised middle sections. Spatial patterns of embolism 127 within samples were visualized with X-ray microCT after centrifugation in order to provide further 128 insight into potential discrepancies. Finally, a new model, CAVITOPEN was developed to simulate the 129 effect of vessel and sample lengths on centrifuge estimates of embolism resistance. We hypothesized 130 that i) both centrifuge techniques, the static centrifuge and the cavitron, are prone to similar artefacts 131 when constructing vulnerability curves of long-vesseled species; ii) the shape of the vulnerability curve 132 Native embolism was determined in current-year, one-year and two-year old segments of 5 branches 151 per species to ensure that the effects of previous natural water stress were minimised. due toNote 152 that segments containing 1-year and 2-year-old growth were necessary to fit in the 27 cm rotor of the 153 centrifuge. Measuring native embolism we also wanted and to control for sample collection date 154 because branches were cut at different times during late autumn-winter 2016 to avoid long storage. 155 Branch proximal end was cut underwater to release tension for 30 min [START_REF] Torres-Ruiz | Direct X-ray microtomography observation confirms the induction 684 of embolism upon xylem cutting under tension[END_REF]156 Wheeler et al. 2013) and then the branch was progressively recut under water to segments 50 mm 157 long. Note that at least twice the maximum vessel length was removed from the cut end after tension 158 relaxation. Thereafter, the edges of these segments were trimmed using a razor blade. Initial 159 conductivity (Kh) was measured in 50 mm long segments with filtered, degassed 2 mmol KCl solution 160 at low pressure (≤ 4 kPa) with a liquid flowmeter (LiquiFlow L13-AAD-11-K-10S; Bronkhorst High-Tech 161 B.V., Ruurlo, the Netherlands). The segments were then flushed with the same solution at a minimum 162 of 0.20 MPa for 15 min to remove embolism and subsequently determine maximum hydraulic 163 conductivity (Kmax). The native percentage loss of conductivity (PLC) was calculated for each segment 164

Maximum vessel length and vessel length distribution 169

Ten branches per species were sampled from the same plants as used for hydraulic measurements to 170 determine maximum vessel length with the air perfusion technique [START_REF] Ewers | Variation in vessel length and diameter in stems of six tropical and 629 subtropical lianas[END_REF]. Once in 171 the lab, 60 cm long segments were flushed for 1 h with degassed, filtered 2 mmol KCl solution at 0.18-172 0.20 MPa to remove any embolism. Then each segment was infiltrated with compressed air at 0.05 173 MPa at its distal end with an aquarium air pump while the basal end was repeatedly shortened by 2 174 cm under water until air bubbles emerged. The remaining sample length was assumed as maximum 175 vessel length. 176

An estimate of the amount of vessels longer than the centrifuge rotor diameter and longer than half 177 the rotor diameter (open to centre vessels) was assessed in four branches of H. dactyloides and five 178 branches of H. leucoptera by measuring the decrease in PLC after air injection (Cochard et al. 1994;179 Torres-Ruiz et al. 2014). Briefly, 35 cm long segments were flushed as described above to remove 180 embolism. Then, tubing was attached to the distal end of these segments and compressed air was 181 injected into the samples at 0.1 MPa for 10 min using a pressure chamber. This pressure was sufficient 182

to empty the open vessels but not high enough to move water through wet pit membranes between 183 adjacent vessels [START_REF] Ewers | Variation in vessel length and diameter in stems of six tropical and 629 subtropical lianas[END_REF]. PLC was determined in 3 cm long segments across the 184 sample as described for native embolism. At the injection point, PLC is close to 100% because all the 185 vessels are air filled and progressively decrease to 0 for a length longer than the longest vessel in the 186 sample. The PLC at each distance from the injection point corresponds to the percentage of 187 contribution to flow from vessels longer than this distance. If all the vessels were of equal diameter, 188 this percentage would correspond to the number of vessels longer than the distance from the injection 189 point. In this case of the two Hakea species used are diffuse porous and vessel diameters within the 190 same sample did not vary greatly. Thus the curves in Fig. 1 (wrapped 196 with aluminium foil and a plastic bag at least 1 h before sampling). When the target ΨX to construct 197 the VC was reached, branches were sealed into a plastic bag with moistened paper towels for 1 h to 198 equilibrate ΨX. Water potential was measured again in two bagged leaves of the same branchlet to 199 confirm homogeneous ΨX in the sample. The ΨX of the sample was considered equilibrated if the 200 difference between the three ΨX (one measured before sealing the branch and two measured after 201 equilibration) was not higher than 0.1 MPa. Afterwards tension was released for 30 minutes by cutting 202 the branch proximal end under water and PLC was determined in one-year-old segments as for native 203 embolism. Vulnerability curves were generated by plotting PLC against ΨX. For H. leucoptera 7 204 branches were dehydrated and 4 different branchlets per branch were measured at different ΨX to 205 construct the vulnerability curve and for H. dactyloides we used 12 branches and two branchlets per 206 branch. All branchlets were far apart (at least four branch orders) and after collection the cutting 207 surface was covered with parafilm to avoid air entry in the rest of the sample. 208

Centrifuge techniques 209

We compared two centrifuge techniques: i) the static centrifuge method described by Alder et al. 210 (1997) and ii) the in situ flow technique (Cavitron (Cochard 2002;Cochard et al. 2005)). In the static 211 centrifuge two different sizes of custom-built rotors, 14 cm and 27 cm, were used to test the effect of 212 under water and both ends were shaved to a final length of 14 or 27 cm. The initial hydraulic 217 conductivity was measured as described above (see Native embolism section) with a pressure head of 218 7.5 kPa. Subsequently, 14-cm long branches were spun in the centrifuge (Sorvall RC 5C Plus) for 5 219 minutes at increasing pressure steps. Foam pads saturated with the solution used for measurements 220 were placed in the reservoirs of the rotor to maintain sample ends in contact with the solution even 221 when the rotor was stopped [START_REF] Tobin | Xylem vulnerability to cavitation can be 673 accurately characterised in species with long vessels using a centrifuge method[END_REF]). After each step, samples were removed and Kh was 222 measured on the whole segment as described for native embolism. In the 27cm-long branches we 223 modified the single spin method [START_REF] Hacke | The standard 631 centrifuge method accurately measures vulnerability curves of long-vesselled olive stems[END_REF]) so that two measurements were made in each 224 centrifuged segment. The initial Kh was measured before spinning in the 27-cm long sample. After 225 spinning, Kh was measured on the whole segment and the first PLC was calculated. Subsequently, a 4 226 cm-long segment was cut from the middle section and its Kh was measured. The second PLC was 227 determined in this 4 cm-long segment after flushing to obtained the maximum Kh (Kmax) as described 228 for native embolism. 229

In situ flow centrifuge measurements (Cavitron technique) were carried out on six branches per 230 species using a modified bench top centrifuge (H2100R, Cence Xiangyi, Hunan, China). For the static 231 centrifuge, samples were trimmed under water to a length of 27 cm to fit in the rotor. Initial 232 conductivity, Ki, was determined at a xylem pressure of -0.5 MPa in H. dactyloides and 1.5 MPa in H. 233 leucoptera. The xylem pressure was then lowered stepwise by increasing the rotational velocity, and 234

Kh was again determined while the sample was spinning. The PLC at each pressure step was quantified 235 as 236 Technologies, Wunstorf, Germany) to visualize embolized vessels. Another subset of branches of H. 246 leucoptera was centrifuged to five (-5, -6, -7, -8, -9 MPa) and three (-5, -6, -7 MPa) different water 247 potentials in the static centrifuge using 27 cm and 14 cm long segments, respectively. They were 248 immediately submerged in liquid paraffin wax and preserved at 4 °C for three days until measured in 249 the same facility (Cochard et al. 2015). Seven branches of H. dactyloides were also centrifuged at four 250 (-3, -4, -5, -6 MPa) and three (-3, -4, -5 MPa) water potentials with the 27 and 14 cm rotors, 251 respectively, following the same protocol. One branch of H. leucoptera was prepared as the 252 centrifuged samples but was not spun in the centrifuge to detect any possible artefact due to sample 253 preparation. All samples were scanned at the middle of the sample. Additionally, in three 27 cm long 254 samples we scanned at 6 cm and 12 cm from the axis of rotation to examine embolism profiles across 255 a sample. 256 X-ray scan settings were 90 kV and 170 mA, and 1800 projections, 600 ms each, were acquired during 257 the 360° rotation of the sample. The resultant images covered the whole cross section of the sample 258 in 8.7 mm length with a spatial resolution of 8.7 μm per voxel. At the end of the scan, the sample was 259 cut back to 30 mm length, injected with air at >1 MPa pressure and rescanned at the same location as 260 before to visualize all empty vessels in the fully embolized cross section. After three-dimensional 261 reconstruction with Phoenix datos|x2 Reconstruction Version 2.2.1-RTM (GE Sensing & Inspection 262 Technologies, Wunstorf, Germany), volumes were imported into ImageJ 1.49k [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. 12 volumes following the protocol in [START_REF] Nolf | Visualization of xylem 645 embolism by X-ray microtomography: a direct test against hydraulic measurements[END_REF]. PLC of each sample was estimated calculating the 265 theoretical hydraulic conductance based on the conduit dimensions of embolized and functional 266 vessels (Choat et al. 2016). To measure conduit dimensions, a radial sector of the transverse section 267 was selected in the same microCT scan and all their embolized vessels were measured manually. The 268 image of this sector was then binarized so the dimensions of the selected embolized vessels matched 269 with the manually drawn vessels. This threshold value was then used for binarizing the image of the 270 whole cross section and all the embolized vessels were measured using the Analyse Particles function 271 in Image J. Theoretical specific hydraulic conductivity (Ksth) was calculated as: 272 13 dehydration and the static centrifuge techniques were obtained using bootstrap resampling (999 289 replicates). Methods were considered to be statistically different if the 95% CIs did not overlap. 290 Differences in native embolism and specific initial conductivity between sampling dates were tested 291 with a one-way ANOVA. Means were compared using a Tukey test at 95% confidence. Vulnerability 292 curve parameters across methods were compared at the Ψx corresponding with three levels of loss 293 of conductivity: 12%, 50% and 88% (P12, P50 and P88, respectively) and the slope of the VC at 50% loss 294 of conductivity (S50). 295

PLC = 100 x (1-Kh / Ki). ( equation
𝐾𝑠𝑡ℎ = ∑ • ∆ ∆ ( equation

CAVITOPEN-simulation of the effect of open vessels in a centrifuged sample 296

To disentangle the combined effects of centrifugation on 'true' vessel embolism at the centre of the 297 samples, where more vessels are closed at both ends and tension is maximum, from draining of open 298 vessels at both sample ends a new model, CAVITOPEN, was developed. In a centrifuged sample, the 299 variation of xylem pressure (P) with distance from the axis of rotation (r) is given by the following 300 equation (Alder et al. 1997): 301 dP/dr = ρω 2 r (equation 4) 302 where ρ is the density of water, and ω the angular velocity. 303

Integrating this equation from R (distance from the axis of rotation to the water reservoir) we can 304 obtain the pressure at r (Pr): 305

Pr = 0.5 ρω 2 (R 2 -r 2 ) (equation 5) 306
The effect of vessel length on 'true' vessel embolism in a spun sample has already been modelled by 307 Cochard et al (2005). Briefly, if the vessels are infinitely long, the VC obtained by centrifugation should 308 yield the correct P50 value. When the vessels are infinitely short the P50 value is underestimated due 309 to the variation of xylem pressure inside the spun sample (eq. 4) and the consequent gradient of 310 14 extremities (eq. 5). Since the loss of conductivity is measured on the whole sample, an 312 underestimation of the degree of embolism in the middle of the sample is predicted. This effect of 313 vessel length was further tested with the CAVITOPEN model and found marginal, i.e. the shift in the 314 VC was negligible, compared to the draining effect. For sake of simplicity, this effect was no longer 315 considered in the simulations. To simulate the draining effect at both sample ends, we first 316 hypothesized that vessel ends follow a logarithmic distribution following the vessel length probability 317 density function proposed by Cohen et al. (2003) and assuming vessel ends uniformly distributed 318 across the length of the sample: 319

Nx = N0 . exp(-x/Lmax) (equation 6) 320
where Nx is the number of open vessels at the distance x from sample ends, N0 the total number of 321 vessels and Lmax the maximum vessel length. 322

The second assumption of the model is that open vessels drain when the minimum pressure in the 323 vessel exceeds a threshold value Popen. Because of the quadratic distribution of the pressure in the 324 sample, vessels having their end wall located closer to the sample ends, i. e. further from the centre 325 of rotation, will drain at a higher rotational velocity. 326

The branch segment was discretised in 0.1 mm thick sections arranged in serial. The xylem pressure 327 in the middle of the segment was set to a pressure varying from 0 to -12 MPa in 1 MPa steps. The 328 model then computes the pressure at steady state in each 0.1 mm section and determines the PLC 329 both species across the sampling dates. We measured higher PLC in two-year-old branch segments (< 342 13 %) than in current year growth (< 2 %) in H. leucoptera whereas in H. dactyloides native embolism 343 was lower than 2% in all samples. Maximum xylem specific conductivity (Ksmax) was 0.87 ± 0.10 kg m -1 344 s -1 MPa -1 in H. leucoptera and 1.29 ± 0.09 kg m -1 s -1 MPa -1 in H. dactyloides (mean ± sd). No significant 345 differences in native PLC or Ks (P > 0.05; Table S1) were detected between sampling dates. 346

Maximum vessel length and vessel length distribution 347

Maximum vessel length as determined by air injection was 25 cm (standard deviation, sd = 5) in H. 348 leucoptera and 10 cm (sd = 3) in H. dactyloides. Air injected branches of H. dactyloides showed 17% 349 PLC at 7 cm from the injection point, 5% at 14 cm and less than 1% at 28 cm, whereas in H. leucoptera 350 the PLC was always higher, 50%, 25%, and 5% at 7, 14 and 28 cm respectively (Fig. 1). Thus the number 351 of open vessels at both ends when using the centrifuge technique differed between species. 352

Vulnerability curves 353

Vulnerability curves (VCs) obtained with the bench dehydration technique were s-shaped for both 354 species, with significant embolism only occurring once a threshold water potential had been reached. 355

This threshold was more negative in H. leucoptera (-6.3 MPa) than in H. dactyloides (-3.8 MPa) (Fig. 356 2). VCs obtained with bench dehydration had the most negative P12 and the steepest slopes of all 357 S2), meaning that embolism formation started at more negative Ψx and conductivity 358 was lost across a narrower range of Ψx compared with VCs generated by centrifugation. 359

When the centrifuge was used to induce embolism, results in the shorter-vesseled species, H. 360 dactyloides, were similar for the three techniques used to measure loss of conductivity, flowmeter, 361

Cavitron and microCT (average P50 with the 27 cm rotor in the static centrifuge and the Cavitron -4.8 362 MPa), and the CI at 95% overlapped with bench dehydration (P50 = -5.0 MPa). The VC generated with 363 the 14 cm rotor for H. dactyloides yielded slightly less negative values (P50 = -4.3 MPa; Table S2; Fig. 364 2). In contrast, VCs for H. leucoptera differed considerably depending on the method and the sample 365 length. Vulnerability parameters (P12, P50, P88) obtained with the Cavitron (-5.0, -7.1 and -9.0 MPa, 366 respectively) matched more closely with the bench dehydration VC . For 367 samples spun in the static centrifuge, we found a significant effect both of the rotor size and the 368 segment used to measure flow (whole, spun segment or excised middle section in the 27 cm rotor) on 369 apparent vulnerability to embolism: segments measured across their entire length exhibited higher 370 vulnerability to embolism compared to the bench-dehydration VC as shown by P12 (-1.2 and -2.6 MPa 371 for 14 and 27 cm rotors, respectively) and P50 (-5.3 and -6.0 MPa, respectively), but seemed less 372 vulnerable towards the dry end of the curve (P88 of -14.2 and -10.4 MPa, respectively; Table S2). Both 373 VCs were almost linear when flow was measured across the whole segment with a shift towards more 374 vulnerable values with the 14 cm rotor, but became s-shaped when only the middle section of the 27 375 cm segment was measured (Fig. 2). Removing the segment ends resulted in a steeper slope and 376 significantly more negative values of P12 and P50. The Cavitron and the middle segment techniques 377 yielded similar results and agreed well with the dehydration technique in P50 and P88 and with microCT 378 image analysis (red triangles in Fig. 2). 379

Patterns of embolism across a centrifuged sample 380

Within 27-cm-length centrifuged samples of H. leucoptera, microCT scans revealed that embolism 381 levels were consistently at their highest near the sample ends (at 12 cm from the axis of rotation 17 when spun at equivalents of -5, -7 and -9 MPa in the static centrifuge (Fig. 3). At -5 and -7 MPa loss of 383 conductivity decreased from the basal end to the centre, contradicting theoretical expectations. This 384 trend was observed even at Ψx inducing less than 40% PLC based on the bench dehydration VC (Fig. 385 3). Only at -9 MPa, that is, below P88 on bench dehydration, did levels of embolism converge along the 386 length of the sample at 80-90%. 387Influence of open vessels in the VC of a centrifuged sample 388

The simulations produced by the CAVITOPEN model confirmed that the shape of the VCs generated 389 by the centrifugation was largely dependent on vessel and sample lengths. As maximum vessel length 390 decreased, PLC of the whole sample decreased at a given Ψx, and the shape of the VC shifted from 391 exponential to sigmoidal (Fig. 4a). The same pattern was observed when the sample length increased 392 (Fig. 4b). For instance, with 14-cm-length centrifuged samples, P50 ranged from -0.6 MPa to -7.7 MPa 393 varying the maximum vessel length of the sample from 50 cm to 5 cm. Likewise, the P50 of a centrifuged 394 sample with maximum vessel length of 15 cm ranged from -2.1 MPa in the 14-cm rotor to -7.7 MPa 395 using a 40-cm rotor. Embolism of vessels open from the cut surface (Fig. S1) influenced values of PLC 396 at high negative pressures, even in short-vesseled samples, resulting in rapid loss of conductivity 397 followed by a plateau. The more open vessels and the less negative the threshold of embolism of open 398 vessels (Fig. 4c), the higher is this plateau and stronger the impact on the VC (Fig. 4). VCs can be 399 corrected if the first inflection point of the curve is considered the starting point for initial conductivity 400 (Ki), i.e. 0% loss of conductivity. This is shown in Fig. 4d with actual measurements of PLC obtained in 401 27-cm centrifuged samples of H. leucoptera. When the CAVITOPEN model was fit (black circles and 402 grey solid line, respectively) and we used the inflection point as starting point for Ki, the corrected 403 curve matched the reference VC obtained with bench dehydration (Fig. 4d the errors were significantly greater in the static centrifuge method. In a species with maximum vessel 412 length longer or similar to the centrifuge rotor diameter, the static centrifuge significantly 413 overestimated xylem vulnerability to embolism if the whole spun segment was used to measure flow. 414

Observations with microCT indicated that artefactual embolism caused by centrifugation of samples 415 occurred in the outer most portions of samples. However, we demonstrated that artefactual 416 embolism was largely eliminated from static centrifuge if flow was measured in an excised central part 417 of the segment. This altered protocol yielded VCs similar to those obtained on the same species with 418 bench dehydration thus allowing these centrifuge techniques to accurately measure vulnerability to 419 embolism in longer vesseled species. We also present a new model (CAVITOPEN) that simulates the 420 impact of vessel draining at the cut end on the whole VC curve and showed that errors were largely 421 dependent on vessel length and rotor diameter. This model allows researchers to quantitative test 422 and avoid errors associated with the artefactual embolism. The bench dehydration technique 423 indicated that significant embolism was only initiated in both species after water potential dropped 424 below a threshold value, -3.8 MPa in H. dactyloides and -6.3 MPa in H. leucoptera. PLC then increased 425 rapidly and hydraulic conductivity was lost almost completely within a span of 1 MPa (Fig. 2). These 426 vulnerability curves have been classified as sigmoidal or s-shaped as opposed to exponential or r-427 shaped curves, characterized by rapid conductivity losses as soon as the water potential declines 428 below zero (Cochard et al. 2013;[START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF]. A third type of VC, intermediate between these 429 two, exhibits a linear response, and is mainly found in diffuse porous species when using 430 centrifugation to induce embolism (Cochard et al. 2013). 431 Our results showed that VCs obtained with the static centrifuge technique and the Cavitron are similar 432 to bench dehydration in a short-vesseled species, i. All centrifuge generated VCs for H. dactyloides were sigmoidal and similar to bench dehydration VCs, 435 with a slight shift towards more vulnerable values when using the 14 cm rotor (Fig. 2, Table S2) as 436 recently found by Pengxian et al. (2018) in Acer mono when comparing in the static centrifuge the 14 437 cm and 27 cm rotors. VCs of other short vesseled angiosperms such as Betula pendula (Cochard et al. 438 2010), Fagus sylvatica (Aranda et al. 2014), Populus tremuloides [START_REF] Schreiber | Genetic variation of hydraulic and wood 665 anatomical traits in hybrid poplar and trembling aspen[END_REF] or Acer 439 negundo (Christman et al. 2009) were also sigmoidal when the static centrifuge or the Cavitron were 440 used. In contrast, the VC shape obtained for H. leucoptera samples differed significantly depending on 441 methodology resulting in a shift of P50 of 2 MPa in samples from the same population (Fig. 2, Table 442 S2). This dramatic change was observed previously in peach (Prunus persica) when the length of the 443 centrifuged samples was varied in a Cavitron; shorter samples were more vulnerable to embolism (P50 444 shifted from -4.5 to -1 MPa) and VCs became r-shaped (Cochard et al. 2010). However, when using 445 the static centrifuge to measure the same population, Sperry et al. [START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF] found that VCs 446 were linear and relatively insensitive to the number of open vessels with P50 less negative than -2 MPa 447 using 14 cm and 27 cm samples. This difference in sensitivity to the proportion of open vessels in the 448 centrifuged samples has led some to conclude that the original centrifuge method and rotor design 449 are not subject to the open vessel artefact [START_REF] Hacke | The standard 631 centrifuge method accurately measures vulnerability curves of long-vesselled olive stems[END_REF][START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF]). However, Torres-450 Ruiz et al. (2017) demonstrated that if the amount of open vessels is relatively high in both rotors, 14 451 and 27 cm, VCs could be equally biased and would appear statistically indistinguishable. 452

Recent publications have addressed this controversy, showing that long-vesseled species such as 453 grape vine, oaks, robinia or olive, with a high proportion of open vessels, produce similarly biased 454 results with both the static centrifuge and the Cavitron when compared with reference curves 455 generated by dehydration or non-invasive imaging (Choat et al. 2016;Choat et al. 2010;Pengxian et 456 al. ;[START_REF] Torres-Ruiz | Vulnerability to cavitation in Olea europaea current-year shoots: further 677 evidence of an open-vessel artifact associated with centrifuge and air-injection techniques[END_REF]. [START_REF] Li | Evaluation of centrifugal methods for measuring 640 xylem cavitation in conifers, diffuse-and ring-porous angiosperms[END_REF] and Pengxian et al. (2018) tested the two centrifuge 457 methods head to head and found close correspondence in VCs across species with different xylem 458

anatomy. An extended literature survey of methods to measure vulnerability to embolism showed 459 F o r P e e r R e v i e w 20 that when using the centrifuge, VCs were sigmoidal in conifers and in long vessel species exponential, 460 whereas in diffuse porous species VCs varied from sigmoidal to linear or exponential (Cochard et al. 461 2013). Our measurements and simulations made with the CAVITOPEN model explain the different 462 shapes of VCs and some disagreements between the static centrifuge and the Cavitron. In short-463 vesseled angiosperms, we have shown that VCs by centrifugation agreed with each other and closely 464 matched the curves based on bench dehydration and microCT (Choat et al. 2016;Cochard et al. 2010). 465

In angiosperms with a proportion of vessels open to the middle but not the whole way through, the 466 standard protocol in the static centrifuge produces linear VCs [START_REF] Sperry | Vulnerability curves by 668 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 669[END_REF]. Here the initial 467 conductivity is measured before spinning, thus if the native embolism is low, all the vessels are 468 conductive, regardless of their length. As soon as the sample is spun, the conductivity would be 469 artificially reduced relative to the native state in proportion to the amount of vessels open to centre. 470Sample with open vessels thus become artificially vulnerable to embolism at the beginning of the VC 471 (i.e. at less negative water potentials). For H. leucoptera, this translated into less negative values of 472 P12 in all centrifuged samples compared with those measured with the bench dehydration technique 473 creating a linear response or a plateau at high water potentials. Higher differences in P12 were 474 observed in H. leucoptera than in H. dactyloides according with a higher proportion of vessels open to 475 centre in the former species (Fig. 1). In the Cavitron, the initial measurement was made while spinning 476 at low tension and many open to centre vessels would already be embolised in the initial 477 measurement of conductivity, resulting in a lower artefactual loss of conductivity in the subsequent 478 water potentials of the VC. This may bias the curves slightly pushing them to more negative values but 479 it did not appear to be significant effect here as the Cavitron curves for H. leucoptera were similar to 480 bench dehydration curves. 481

The simulations of PLC with the CAVITOPEN model confirmed that the impact of open vessels on the 482 VC was higher when vessels were long, samples short and when open vessels cavitated at less negative 483 pressures (Fig. 4). If the samples were much shorter than the maximum vessel length of the branch 484 (see the results in Fig. 4 for the 14 cm rotor with Lmax 50cm), the resulting VC was exponential (r-485 open at the cut surface cavitate when they reach a threshold value; that is far less negative than intact 488 vessels whose two ends are included within the spun segment. This influences the shape the VC at 489 higher pressures creating a "bump" in the VC followed by a plateau. This effect can be corrected to 490 some extent if the first inflexion point of the VC is considered to be the 0% point for loss of 491 conductivity. In this case the initial conductivity (Ki) value is shifted to a lower value corresponding to 492 the hydraulic conductivity of the plateau (Fig. 4d). The estimated values of P50 and S50 when the 493 CAVITOPEN model was fit to actual measurements agreed quite well with those obtained with 494 reference techniques and confirmed that this model can be used to correct open vessel artefacts for 495 centrifuge based VCs. The estimated Lmax was however significantly shorter than Lmax measured with 496 the air injection technique. The air injection technique has shown to produce higher Lmax than the 497 rubber injection method [START_REF] Pan | A comparison of two methods for measuring vessel length in 650 woody plants[END_REF], thus our values could be overestimated. On the other hand, 498 the model assumed that vessel lengths in a sample follow the density function proposed by Cohen et 499 al. (2003) which can be sensitive to the clustering of vessel lengths (Cai and Tyree 2014). It is clear that 500 the actual distribution of vessel lengths, network topology and connectivity are crucial for the 501

sensitivity to an open vessel artefact. 502

Origin of the open-vessel artefact 503

The physical mechanisms underlying this open-vessel artefact are yet to be fully elucidated. Some 504 studies suggest that microbubbles and particles can act as nucleation sites when they flow through 505 the sample as it spins in the Cavitron, causing premature embolism (Cochard et al. 2010;Sperry et al. 506 2012;[START_REF] Wang | Water relations of Robinia pseudoacacia L.: do 701 vessels cavitate and refill diurnally or are R-shaped curves invalid in Robinia?[END_REF]). In the static centrifuge, bubbles might be drawn into vessels while starting 507 the spin or while mounting or dismounting the stems to measure flow [START_REF] Wang | Water relations of Robinia pseudoacacia L.: do 701 vessels cavitate and refill diurnally or are R-shaped curves invalid in Robinia?[END_REF]. In both 508 centrifuge techniques bubbles in open vessels can move by buoyancy while spinning toward the region 509 of lowest pressure at the center of rotation (Rockwell et al., 2014). Draining from open vessels as a 510 F o r P e e r R e v i e w 22 consequence of artefactual embolism when the centrifuge starts spinning appears to be a common 511 phenomenon in both rotors. Our microCT images showed that after spinning in the centrifuge, most 512 of the vessels were empty near the ends even though tension ought to be zero (Cochard et al. 2005). 513

The use of water saturated foam pads to avoid desiccation did not prevent this [START_REF] Hacke | The standard 631 centrifuge method accurately measures vulnerability curves of long-vesselled olive stems[END_REF]514 Tobin et al. 2013). We discarded the possibility that sample manipulation before spinning or during 515 wax embedding had triggered vessel draining because we scanned control samples that were not 516 spun. These samples showed no embolism (Fig. 3). Furthermore, patterns of embolism did not follow 517 theoretical expectations based on the distribution of tension within the spun sample. The embolism 518 levels decreased from the ends to the center in a fashion consistent with the amount of vessels open 519 to center, opposite to that expected from profile in tension and in agreement with the assumption of 520 the CAVITOPEN model than open vessels artificially cavitate when they reach a threshold pressure 521 that is much less negative than in intact vessels. This pattern was observed at water potentials 522 inducing less than 40% loss of conductivity based on the VC obtained using the middle segment of the 523 centrifuged sample (Fig. 3), even though the centre of the sample experienced the highest tensions. 524 Embolism levels converged within the sample at -9 MPa at 80-90%. These results confirm that 525 centrifugation drains open vessels and only reliably measure the vulnerability of intact xylem vessels 526 within the sample (Fig. S1). This is consistent with observations made previously by Cochard et al. 527 (2010) using the Cavitron; they reported that embolism was higher in the basal and upstream ends 528 relative to the centre of samples from species with vessels that are predominately at least half as long 529 as the spun segment. Cai et al. (2010) and Pengxian et al. (2018) also reported higher PLC values than 530 predicted by theory at both ends after spinning samples in a Cavitron. Given that our results were 531 obtained with the static centrifuge it is clear that the overestimation of vulnerability for open to centre 532 vessels occurs in both versions of the centrifuge technique. 533

The hydraulic continuity between vessels cut open at each end of the sample and vessels with their 534 terminal ends in this portion of the sample is probably re-established by refilling of vessels immersed 535 under water at both ends (Fig. 2 in Cochard et al. (2010)). This refilling would occur by capillarity either 536

  techniques induce embolism by increasing tension in sample xylem, the 95

  black plastic bags with moistened paper towels to prevent transpiration with 145 their cut ends covered with Parafilm. In the laboratory they were kept at 4 °C until measured. 146 Midday xylem water potential in the field and Native embolism 147 Midday xylem water potential was measured in the field in November 2015, February 2016 and June 148

  (K S ) was calculated dividing K max by the xylem cross-sectional area 168 (average distal and proximal xylem area measured with a calliper). 169

  were carried out on 20 branches per species. Branches were 217

  branches of H. leucoptera was transported to the University of New England in Armidale 240

  Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H.,Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 the subsequent water potentials of the VC. This may bias the curves slightly 483

  . It 504 is clear that the actual distribution of vessel lengths, network topology and connectivity are crucial 505 for the sensitivity to an open vessel artefact. 506Origin of the open-vessel artefact 507Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H.,Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

Figure legends 715 Figure 1 .Figure 2 .Figure 3 .

 715123 Figure legends 715

Figure 4 .

 4 Figure 4. Simulations with the CAVITOPEN model of the effect of threshold of embolism formation 734

  67 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

  , Cochard et 89 al. (2005) and[START_REF] Li | Evaluation of centrifugal methods for measuring 640 xylem cavitation in conifers, diffuse-and ring-porous angiosperms[END_REF] further modified the centrifuge method and designed new rotors which 90 allowed measuring the conductivity of the segment while it is spinning and under tension. This further 91 increased the efficiency of measurement and allowed for flow measurements to be made under 92 tension. 93 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 techniques induce embolism by increasing tension in sample xylem, the 94

  First, we tested two 118 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

  of centrifuged samples will depend on the amount of cut open vessels; iii) image techniques and 133 standard flow measurements will produce similar vulnerability curves. out on branch material of two diffuse-porous species of the same genus 138 exhibiting different vessel lengths, Hakea dactyloides (Gaertn.) Cav. and Hakea leucoptera R. Br. 139Branches were sampled from natural populations of H. dactyloides at Mount Banks(33° 34' 46'' S, 140 150° 21' 56'' E; NSW, Australia) and H. leucoptera at Binya State Forest (34° 11' 16'' S, 146° 16' 13'' E; 141 NSW, Australia) from May to September 2016 (late autumn-winter in the South Hemisphere). Sun 142 Page 35 of 67 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 length were collected in the field in the early morning and immediately 143 placed in black plastic bags with moistened paper towels to prevent transpiration with their cut ends 144 covered with Parafilm. In the laboratory they were kept at 4 °C until measured. 145 Midday xylem water potential in the field and Native embolism 146 Midday xylem water potential was measured in the field in November 2015, February 2016 and June 147 2016. Two leaves of five plants per species were covered with aluminium foil and sealed with a plastic 148 bag 1 hour before excision and measurement with a pressure chamber (PMS Instrument Co., Albany, 149 OR, USA). 150

  mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 (KS) was calculated dividing Kmax by the xylem cross-sectional area 167 (average distal and proximal xylem area measured with a calliper). 168

  segment length and fraction of open vessels. All hydraulic conductivity measurements were 213 performed using filtered, degassed 2 mmol KCl solution and a flow meter (see Native embolism 214 section). 215 Page 38 of 67 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 were carried out on 20 branches per species. Branches were trimmed 216

  mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 branches of H. leucoptera was transported to the University of New England in Armidale 239 (NSW, Australia). They were gradually dehydrated to five different xylem water potentials ranging 240 from -4.8 MPa to -9 MPa as for the bench dehydration method. After measuring Ψx, tension was 241 relaxed by cutting the proximal end of the branch under water leaving it submerged for 30 minutes. 242 Then the branch was sequentially cut back under water and finally 10-mm-long segments were excised 243 under water from current-year shoots, wrapped in Parafilm, inserted into a plexiglass tube and then 244 placed in an X-ray microtomography system (GE-Phoenix V|tome|xs, GE Sensing & Inspection 245

  263 A median Z projection of c. 100 μm along the sample axis was extracted from the middle of the scan 264 Page 40 of 67 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

  the equivalent circular vessel diameter based on vessel area, η viscosity of water, Δp/Δx 275 pressure gradient per xylem length, A xylem cross-sectional area. 276 277 The current theoretical specific hydraulic conductivity (Ksth) for each sample was calculated by 278 subtracting the summed specific hydraulic conductivity of embolized vessels from the Ksth(max) of that 279 sample, calculated as the Ksth of the sample after air injection. The pressure gradient used for 280 calculations of Ksth was similar to the pressure gradient used in the hydraulic measurements, 0.06 MPa 281 m -1 . 282 283 Vulnerability curve fitting and statistical analysis 284 Vulnerability curves were fitted using a Weibull function (Ogle et al. 2009) in R 3.2.0 (R Core Team, 285 2015) using the fitplc package (Duursma and Choat 2017). Confidence intervals of P12, P50 and P88 (Ψx 286 at 12, 50 and 88 % loss of conductivity, respectively) and the slope of the curve at 50% loss of 287 conductivity (S50) were used to compare between methods. Confidence intervals (CI) for the bench 288 Page 41 of 67 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

  embolism along the sample: xylem pressure is minimum in the middle of the sample and null at the 311 Page 42 of 67 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

  caused by 'true' embolism (non-open vessels) and by draining (open vessels). Finally, the PLC of the 330 whole segment is computed which enables the construction of the vulnerability curve. We tested the 331 model for different theoretical Lmax values and the 4 rotors sizes used in our experiments. To validate 332 the model we used the values of PLC obtained for H. leucoptera in the static centrifuge with the 27 cm 333 rotor. The CAVITOPEN model was fit to the measurements using constrained numerical optimization 334 Page 43 of 67 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 parameters: P50, S50, Lmax and Popen. All routines were implemented as an R package 335(available from[START_REF] Duursma | An R implementation of the CAVITOPEN model 624[END_REF]. 336 Results 337 Native embolism and minimum xylem water potential in the field. 338 Midday xylem water potential decreased from -1.02 to -1.51 MPa in H. dactyloides and from -1.35 to 339 -2.62 MPa in H. leucoptera from November 2015 to February 2016. In June 2016, the water potential 340 was -1.16 MPa in H. dactyloides and -1.42 MPa in H. leucoptera. Native embolism remained low in 341

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  ) 382 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

  black solid line and orange 404 dashed line, respectively). Alternatively, by fitting the model using numerical optimization we 405 estimated values of P50 = -6.9 MPa, S50 = 49.7, Lmax = 15.21 and Popen = -0.75. 406 Discussion 407 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 reliability of two centrifuge based techniques commonly used to measure 408 vulnerability to embolism in angiosperm species and present a protocol that mitigates experimental 409 artefacts associated with open xylem vessels. Both the static centrifuge method and the in-situ flow 410 centrifuge method (Cavitron) were prone to artefactual embolism caused by open vessels, although 411

  e. a species with no through vessels (open at both 433 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 the segment and with few vessels open from the cut surface to the middle of the segment. 434

  as observed in long-vesseled angiosperms, and shifted to more linear or s-shaped when Lmax 486 was decreased or the sample length increased. One of the assumptions in the model is that vessels 487

Figure 1 .

 1 Figure legends 709

Figure 2 .Figure 3 .

 23 Figure 2. Xylem vulnerability to embolism curves and 95% confidence intervals (grey shaded areas) 714 of Hakea dactyloides (left panels) and Hakea leucoptera (right panels) obtained with two methods to 715 induce cavitation in the xylem, bench dehydration and centrifuge force and three methods to 716 measure the loss of conductivity, flowmeter (close circles), in situ flow method (open circles) and X-717 ray microCT visualisation (red triangles). Vertical solid lines indicate P50 and vertical dashed lines 718 indicate the 95% confidence interval for P50. Horizontal dashed lines indicated native xylem 719 embolism measured in the field. Two rotor sizes, 14 cm and 27 cm, were used in the static 720 centrifuge, and water flow in the whole segment or only in the central part was measured (see 721 methods for details). 722

Figure 4 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 1 .

 412341 Figure 4. Simulations with the CAVITOPEN model of the effect of threshold of embolism formation 728 (MPa) of cut open vessels (A), maximum vessel length (cm) (B), and rotor size (cm) (C) on xylem 729 vulnerability to embolism curves generated with centrifugation. In red, vulnerability curve of close 730 vessels at both ends. (D) The CAVITOPEN model was fit to measurements in H. leucoptera using 731 numerical optimization to estimate all four parameters: water potential at 50% loss of conductivity 732

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Xylem vulnerability to embolism curves and 95% confidence intervals (grey shaded areas) of Hakea dactyloides (left panels) and Hakea leucoptera (right panels) obtained with two methods to induce cavitation in the xylem, bench dehydration and centrifuge force and three methods to measure the loss of conductivity, flowmeter (close circles), in situ flow method (open circles) and X-ray microCT visualisation (red triangles). Vertical solid lines indicate P50 and vertical dashed lines indicate the 95% confidence interval for P50. Horizontal dashed lines indicated native xylem embolism measured in the field. Two rotor sizes, 14 cm and 27 cm, were used in the static centrifuge, and water flow in the whole segment or only in the central part was measured (see methods for details).254x338mm (300 x 300 DPI)

  the two species, although not as accurate as anatomy, and allow to estimate 193 the amount of open vessels from a certain cut point. 194

	195	vessel distribution of Bench dehydration technique
	196	Branches were dehydrated gradually in the laboratory at ca. 23 °C. Xylem water potential (Ψ X ) was
	197	measured with a pressure chamber (PMS Instrument Co., Albany, OR, USA) in bagged leaves
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  demonstrated that if the 454 amount of open vessels is relatively high in both rotors, 14 and 27 cm, VCs could be equally biased 455 and would appear statistically indistinguishable. 456 Recent publications have addressed this controversy, showing that long-vesseled species such as 457 grape vine, oaks, robinia or olive, with a high proportion of open vessels, produce similarly biased 458

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  using the Cavitron; they reported that embolism was 532 higher in the basal and upstream ends relative to the centre of samples from species with vessels 533 at least half as long as the spun segment.Cai et al. (2010) andPengxian et al. 534 (2018) also reported higher PLC values than predicted by theory at both ends after spinning samples 535 in a Cavitron. Given that our results were obtained with the static centrifuge it is clear that the 536 overestimation of vulnerability for open to centre vessels occurs in both versions of the centrifuge 537

	538	that are predominately technique.
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  0 % loss of conductivity) 557 or by fitting the CAVITOPEN model to the measurements to estimate P 50 and S 50 . When samples 558 proportion of open to centre vessels, the centrifuge technique is prone to error and 559 overestimates vulnerability to embolism. Determining the proportion of open to centre vessels or 560 performing the simple test recently proposed by Torres-Ruiz et al. (2017), which compares changes 561 in K s before and after spinning in the centrifuge at low tensions, are highly advisable before using 562 any of the centrifuge techniques. 563 The shape of the vulnerability curves obtained with bench dehydration were always sigmoidal while 564 in centrifuged samples the shape was determined by the presence of open vessels. While previous 565 studies have demonstrated that species with the longest vessel classes (eg. lianas, ring porous trees) 566 open vessels tend to exhibit exponential curves when measured in the centrifuge. Here we showed 567 that VCs with a linear shape are symptomatic of species with intermediate vessel lengths in which a 568 higher proportion of vessels open to centre of the test segment. The occurrence of this incipient 569 open vessel artefact can be mitigated by measurement of the excised central portion of the 570

		Manuscripts submitted to Tree Physiology
	627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 571 643 644 645 646 647 652 653 693 694 695 696 697 698 699 700 701 702 703	F o r P e e r Dalla-Salda G, Fernández ME, Sergent A-S, Rozenberg P, Badel E, Martinez-Meier A (2014) Dynamics of cavitation in a Douglas-fir tree-ring: transition-wood, the lord of the ring? Journal of Plant Hydraulics. 1:005. Duursma R (2017) An R implementation of the CAVITOPEN model Duursma R, Choat B (2017) fitplc -an R package to fit hydraulic vulnerability curves. 2017. 4 Ennajeh M, Simoes F, Khemira H, Cochard H (2011) How reliable is the double-ended pressure sleeve technique for assessing xylem vulnerability to cavitation in woody angiosperms? Physiologia Plantarum. 142:205-10. Ewers FW, Fisher JB (1989) Variation in vessel length and diameter in stems of six tropical and subtropical lianas. American Journal of Botany:1452-1459. Hacke UG, Venturas MD, MacKinnon ED, Jacobsen AL, Sperry JS, Pratt RB (2015) The standard centrifuge method accurately measures vulnerability curves of long-vesselled olive stems. New Phytologist. 205:116-27. Holbrook NM, Burns MJ, Field CB (1995) Negative xylem pressures in plants: a test of the balancing pressure technique. Science. 270:1193-1195. Jacobsen AL, Pratt RB (2012) No evidence for an open vessel effect in centrifuge-based vulnerability Sperry JS, Tyree MT (1988) Mechanism of water stress-induced xylem embolism. Plant Physiology. 88:581-7. Tobin MF, Pratt RB, Jacobsen AL, De Guzman ME (2013) Xylem vulnerability to cavitation can be accurately characterised in species with long vessels using a centrifuge method. Plant Biology. 15:496-504. Torres-Ruiz JM, Cochard H, Mayr S, Beikircher B, Diaz-Espejo A, Rodriguez-Dominguez CM, Badel E, Fernandez JE (2014) Vulnerability to cavitation in Olea europaea current-year shoots: further evidence of an open-vessel artifact associated with centrifuge and air-injection techniques. Physiologia Plantarum. 152:465-74. Torres-Ruiz JM, Cochard H, Mencuccini M, Delzon S, Badel E (2016) Direct observation and modelling of embolism spread between xylem conduits: a case study in Scots pine. Plant, Cell & Environment. 39:2774-2785. Torres-Ruiz JM, Jansen S, Choat B, McElrone AJ, Cochard H, Brodribb TJ, Badel E, Burlett R, Bouche PS, Brodersen CR (2015) Direct X-ray microtomography observation confirms the induction of embolism upon xylem cutting under tension. Plant Physiology. 167:40-43. Torres-Ruiz JM, Cochard H, Choat B, Jansen S, López R, Tomášková I, Padilla-Díaz CM, Badel E, Burlett contained a high segment. F o r P e e r F o r P e e r curves of a long-vesselled liana (Vitis vinifera). New Phytologist. 194:982-990. Jansen S, Schuldt B, Choat B (2015) Current controversies and challenges in applying plant hydraulic techniques. New Phytologist. 205:961-964. Li Y, Sperry JS, Taneda H, Bush SE, Hacke UG (2008) Evaluation of centrifugal methods for measuring xylem cavitation in conifers, diffuse-and ring-porous angiosperms. New Phytologist. 177:558-68. New Phytologist. 214:890-898. F o r P e e r R, King A (2017) Xylem resistance to embolism: presenting a simple diagnostic test for the open vessel artefact. New Phytologist. 215:489-499. Tyree MT, Dixon MA (1986) Water stress induced cavitation and embolism in some woody plants. Physiologia Plantarum. 66:397-405. Tyree MT, Sperry JS (1988) Do woody plants operate near the point of catastrophic xylem F o r dysfunction caused by dynamic water stress? : answers from a model. Plant Physiology. 88:574-80. Tyree MT, Zimmermann MH (2002) Hydraulic architecture of whole plants and plant performance P e Xylem structure and the ascent of sap. Springer, pp 175-214. Urli M, Porte AJ, Cochard H, Guengant Y, Burlett R, Delzon S (2013) Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees. Tree Physiology. 33:672-83. e r
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	657 658 659 660 661 662 707 708 709 710 711 712	v i e w v i e w v i e w woody plants. Plant, Cell & Environment. 38:2519-2526. Pengxian Y, Feng M, Qing L, Rui A, Jing C, Guangyuan D A comparison of two centrifuge techniques for constructing vulnerability curves: insight into the 'open-vessel' artifact. Physiologia Plantarum. 0 Pockman WT, Sperry JS, Leary JW (1995) Sustained and significant negative water pressure in xylem. Nature. 378:715. v i e w Wang R, Zhang L, Zhang S, Cai J, Tyree MT (2014) Water relations of Robinia pseudoacacia L.: do vessels cavitate and refill diurnally or are R-shaped curves invalid in Robinia? Plant, cell & v environment. 37:2667-2678. Wheeler JK, Huggett BA, Tofte AN, Rockwell FE, Holbrook NM (2013) Cutting xylem under tension or i e supersaturated with gas can generate PLC and the appearance of rapid recovery from embolism. Plant, Cell & Environment. 36:1938-1949. w
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http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 648 Nardini A, Battistuzzo M, Savi T (2013) Shoot desiccation and hydraulic failure in temperate woody 649 angiosperms during an extreme summer drought. New Phytologist. 200:322-329. 650 Nolf M, Lopez R, Peters JM, Flavel RJ, Koloadin LS, Young IM, Choat B (2017) Visualization of xylem 651 embolism by X-ray microtomography: a direct test against hydraulic measurements. 673 Sperry JS, Christman MA, Torres-Ruiz JM, Taneda H, Smith DD (2012) Vulnerability curves by 674 centrifugation: is there an open vessel artefact, and are 'r' shaped curves necessarily invalid? 675 Plant, Cell & Environment. 35:601-10. 676 http://mc.manuscriptcentral.com/tp Manuscripts submitted to Tree Physiology Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083 Comment citer ce document : Lopez Rodriguez, R. A., Nolf, M., Duursma, R. A., Badel, E., Flavel, R. J., Cochard, H., Choat, B. (2018). Mitigating the open vessel artefact in centrifuge-based measurement of embolism resistance. Tree Physiology, 13 p. , DOI : 10.1093/treephys/tpy083

  represent a good proxy of vessel distribution 191 Branches were dehydrated gradually in the laboratory at ca. 23 °C. Xylem water potential (ΨX) was 195 measured with a pressure chamber (PMS Instrument Co., Albany, OR, USA) in bagged leaves

	192	of the two species, although not as accurate as anatomy, and allow to estimate the amount of open
	193	vessels from a certain cut point.
	194	Bench dehydration technique
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Acknowledgments 572 This research was supported by a Marie Curie Fellowship to R.L. (FP7PEOPLE-2013-IOF-624473) and 573 an ARC Future Fellowship to B.C. (FT130101115). We thank Dr. Javier Cano, Adrián Cano, Teresa Acknowledgments 566