Plant Material

To assess the role of root pressure in the refilling of embolized conduits we selected one species with known root pressure (B. vulgaris) and one species with minimal root pressure (B. blakeana). Root pressure in B. vulgaris has previously been assessed (Cao et al. 2012), and measurements of B. blakeana root pressure associated with this study showed minimal root pressure compared to similar sized plants of B. vulgaris (31.4  1.8 kPa compared to ~120 kPa, data not shown). Plants selected for sampling were located adjacent to two small man-made lakes on the Guangxi University campus, Nanning, Guangxi Zhuang-Nationality Autonomous Region, China (22.841°, 108.286°; 84 m asl). Details on the collection of leaf material are included in the appropriate sections below.

Pressure-Volume curves

Pressure volume curves were measured on leaves collected from five different individuals of B. blakeana and B. vulgaris to estimate turgor loss point (Ψ_TLP), osmotic potential at full turgor (Ψ_osm), modulus of elasticity (ε), and leaf capacitance (C_leaf). Branches of each species were collected from sun-lit upper canopy positions (sampling height ranged from c. 3-5 m for B. vulgaris and 2-3 m for B. Blakeana) prior to sunrise on the morning of measurements. The total height of plants sampled ranged from c. 6-8 m among B. vulgaris individuals and c. 4-6 m among B. Blakeana individuals. For B. blakeana, branches were cut at least 100 cm long, with at least 6-10 leaves attached along the branch, immediately sealed in a plastic bag and transported back to the laboratory. For B. vulgaris, branches were cut with 3-5 branchlets attached, with each branchlet consisting of ~7-9 leaf blades, immediately sealed in a plastic bag and transported back to the laboratory. In the lab, each branch was recut underwater, placed in a reservoir of water and allowed to rehydrate for >2 hours. One leaf was removed at a time and placed in either a plastic bag (B. blakeana) or wrapped in paraffin (B. vulgaris) to slow dehydration. Leaves were kept in plastic bag/paraffin-wrap during the measurement of leaf water potential to prevent dehydration while in the chamber. Successive measurements of leaf water potential (Ψ_leaf, Model 1505D, PMS Instruments, Albany, OR, USA) and leaf mass (0.0001 g, ML204T, Mettler Toledo) were made to generate pressure volume curves. Following the completion of the PV curve, leaf area was measured with a leaf area meter (LI-3000C, Li-Cor Biosciences, Lincoln, NE, USA) and then leaves were dried for 72 h and weighed.

Leaf vulnerability to hydraulic failure using the Rehydration Kinetics Method (RKM)

The vulnerability to hydraulic failure of leaves was estimated by developing vulnerability curves for B. blakeana and B. vulgaris using the Rehydration Kinetics Method (RKM; Brodribb and Holbrook, 2003). Branches were sampled during pre-dawn hours and allowed to rehydrate as described above (height, branch length, and number of branches). Thirty-nine individual leaves were used to build a vulnerability curve for B. blakeana and 27 small branchlets (ca 10-15 cm), each with 7-9 leaves, were used to build a vulnerability curve for B. vulgaris. The response of leaf hydraulic conductance to decreasing Ψ_leaf was measured for both species to develop vulnerability curves. Leaf hydraulic conductance (K_leaf) was determined by measuring the change in Ψ_leaf that occurred during rehydration and calculated after Brodribb and Holbrook (2003):

Equation 1. $K_{leaf}=\frac{C_{leaf}ln\left[\frac{{\Psi }_0}{{\Psi }_f}\right]}{t}$

where Ψ₀ and Ψ_f are the Ψ_leaf before and after rehydration, respectively. ‘t’ is the duration of rehydration, and C_leaf is the initial capacitance of the leaf estimated from the pressure volume curves described above. Prior to K_leaf measurements the branchlets were set on the bench under an LED light source with a PAR of ~1800 μmol m^-2 m^-1 (SP501-power, Senpro Mechanical & Electrical Co. LTD, Shanghai), and leaves were kept under this light during measurements. K_leaf was measured at a range of initial Ψ_leaf values generated by allowing leaves to dehydrate for different lengths of time on the lab bench. The relationship between Ψ_leaf and K_leaf was fit with sigmoidal, logistic, exponential, and linear functions (Table 1) and then AIC values were used to select the function that best represented the decline in K_leaf (Scoffoni et al., 2012; Ocheltree et al., 2016). For both the RKM method and the optical method, a single model was fit to the pooled data for each species, i.e. individual trees/branches/leaves were not fit with separate models. Using the selected function, we then calculated the maximum K_leaf when leaves were fully hydrated (K_sat) and the Ψ_leaf when leaves lost 12%, 50%, and 88% of K_sat (P₁₂, P₅₀, P₈₈, respectively).

Recovery of K_leaf using RKM

Branches were collected from the field and allowed to rehydrate as previously described. Branches were then allowed to dehydrate on the lab bench to achieve a range of Ψ_leaf values. Two branchlets (ca 30 cm long) were cut from a single dehydrating branch. Ψ_leaf and K_leaf were then measured on two adjacent leaves taken from one of the branchlets, while the other branchlet (assumed to be at the same Ψ_leaf) was recut under water and allowed to rehydrate for 8-12 hours in a reservoir of DI H₂0. After the branchlet had rehydrated (Ψ_leaf had returned to near zero), it was taken from the reservoir and allowed to dehydrate once again for ~5 minutes underneath an LED light source and then K_leaf was determined again as described above. This short dehydration time was required to generate a water potential gradient between Ψ_leaf and the reservoir of water.

Visualization of leaf embolism formation and repair using the optical method

Leaves on rehydrated branches, collected from the field as described above (height, branch length, number of branches), were measured using the leaf ‘optical’ method (Brodribb et al., 2016b). Eight individual leaves were used to build an optical vulnerability curve for B. blakeana and seven individual leaves were used to build a vulnerability curve for B. vulgaris. Leaves were left attached to branches during both the dehydration and rehydration portions of the experiment. Branches were all >100 cm in length. The cut end of rehydrated branches were transferred from a large DI water reservoir to 15 mL centrifuge tubes that were filled with water and sealed with paraffin to ensure the cut end of the branch remained under water during transfer to a high-resolution flatbed scanner (Epson Perfection V800, Epson Inc., Suwa, Japan). One fully-expanded leaf per branch, located towards the apex of the branch, was selected and placed on the scanner bed, sandwiched between two 4-mm thick plates of glass. This placement ensured that the leaf was at the proper focal length from the scanner bed, and also minimized transpiration from the leaf surface, such that Ψ_leaf could be closely approximated by Ψ_stem. Four leaves were placed on the scanner bed at the same time with the full length of the branch (~1.5 m) attached and laid carefully alongside the scanner. For the dicot, B. blakeana, Ψ_stem was measured continuously at 10-min intervals using a stem psychrometer (model PSY1, ICT International, Armidale, NSW, Australia) placed on a stem that was closely adjacent to the leaves in the scanner and verified periodically (ca every two hours) by measuring Ψ_leaf using a pressure chamber. The unique stem morphology of B. vulgaris prevented the use of the stem psychrometer, and so, more frequent Ψ_leaf measurements (ca ~30 min) were taken with the pressure chamber. Dehydration times varied markedly between the species. B. blakeana branches required c. 12-18 h to progress from fully hydrated to fully embolized, whereas B. vulgaris required c. 9-12 h. The time required to progress from the moment of first embolism occurrence to full embolization, ranged from 6-11 hours for B. blakeana and 5-7 h for B. vulgaris.

    Dehydrated branches (K_leaf ~50% of maximal) were rehydrated under two treatment conditions: 1) positive pressure (~120 kPa), and 2) atmospheric pressure whilst still on the scanner to assess their capacity to reverse embolism. 120 kPa was chosen to approximate the recorded root pressure in B. vulgaris (Cao et al., 2012a). For the atmospheric pressure treatment, whilst still on the scanner branches were re-cut underwater and sealed with paraffin in a 15 mL centrifuge tube filled with DI H₂0 to allow rehydration. The elevation of the vial was kept at, or slightly below, the level of the scanned leaf during this process to minimize the chance of any positive pressure generated by the placement of the reservoir of water, although the capillary pressure due to vessel diameter could not be controlled. For positive pressure treatment, branches were re-cut underwater and sealed into water-filled tubing connected to a small pressure chamber. Pressure was maintained between 117 and 124 kPa throughout the rehydration treatment. Scanning of the leaves continued during the rehydration treatment at 10 min intervals, as described above.

    Leaves were scanned every 10 min using VueScan software (Hamrick Software, Sunny Isles Beach, Florida, USA). AutoIt freeware (Bennett, 1999) was used to automate the scanning. The VueScan settings were: mode = ‘transparency’, media = ‘slide film’, bits = ‘8-bit gray’, scan resolution = ‘3200 dpi’. The scanned area (ie. the full area of the scanner bed) was divided into four quadrants so that each leaf could be assigned to an individual quadrant and scanned separately. For B. vulgaris, a ~4-cm² area of each leaf was scanned at high resolution (3200 dpi), whereas the larger leaves of B. blakeana allowed for a ~16-cm² area to be scanned at high resolution. As the xylem became more embolized in both species, individual veins could be observed to darken, presumably from the liquid-to-gas phase change occurring in the vessels (Brodribb et al., 2016a,b). As such, the transmission of light through the leaf decreased during xylem embolization, and therefore, the fraction of dark-colored pixels increased in the scanned images. Changes in the fraction of dark-colored pixels were analyzed using ‘ImageJ’ analysis software (Schneider et al., 2012). For each leaf dry-down sequence, the threshold of the first image was adjusted such that the individual veins were represented by white pixels when converted to a binary image (an image consisting of only white or black pixels). The fraction of black pixels was recorded and this value was assumed equal to 100% xylem functioning. This threshold was then kept constant for each subsequent image in the sequence (all images taken from a single leaf as it dried down). As the xylem became more embolized, the images became darker and the recorded fraction of black pixels increased. Images were analyzed in this way until the xylem was completely embolized, which was taken as the point when no further meaningful change in either light transmission or the fraction of dark-colored pixels occurred between two subsequent images. The fraction of black pixels in the final image was assumed equal to 0% xylem functioning. The percent loss of functioning (as estimated via the fraction of black pixels present in each image) was then plotted against Ψ_leaf to generate percent loss of ‘conductance’ curves. Although shrinkage and subsequent movement of the leaves on the scanner prevented automated analysis of our images, our fraction of black pixels should, in principle, be the same measurement of “cumulative area of cavitated xylem” described elsewhere (Brodribb et al. 2017). For B. vulgaris, Ψ_leaf values were estimated for each scanned image (every 10 min) by plotting measured Ψ_leaf values against time and then solving this equation for the time each image was taken. For B. blakeana, the recorded psychrometer data were used because these were taken on the same time interval as the images (10 min).

Pressure-volume analyses

Parameters from the pressure volume curves (Table 2) provided several drought tolerant metrics and capacitance values used to calculate K_leaf. The turgor loss point (Ψ_TLP) of the monocotyledon B. vulgaris was -3.07 MPa, which was ~1.94 MPa more negative than the woody dicotyledon B. blakeana. The difference in Ψ_TLP was mirrored by differences in the osmotic potential (Ψ_osm) between the two species; Ψ_osm of B. vulgaris was 1.16 MPa more negative than B. blakeana. B. vulgaris also had a larger modulus of elasticity (ε) than B. blakeana. Conversely, the leaf capacitance of B. blakeana was ~4 times greater than for B. vulgaris.

Loss and recovery of leaf conductance using RKM

Sigmoidal functions provided the best fit to the RKM data for both species (Figure 1, grey symbols and dashed lines). B. vulgaris lost 12, 50, and 88% of its maximum leaf hydraulic conductance at leaf water potentials of -0.6, -2.5 and -4.3 MPa, respectively. B. blakeana lost the same percentage of hydraulic conductance at Ψ_leaf values of -2.4, -3.3 and -4.6 MPa, respectively, but did maintain consistently positive K_leaf down to -4.0 MPa.

Figure 1: Loss and recovery of Kleaf for B. blakeana (a) and B. vulgaris (b) following dehydration and rehydration as measured by RKM. Pairs of red and blue symbols (connected by solid lines) represent pairs of leaves on the same branch. Red symbols denote Kleaf of dehydrated leaves and the blue symbols denote Kleaf of leaves that had been rehydrated for 8-12 hours. Note that for B. blakeana leaves that were dehydrated to Ψleaf <-3.5 MPa did not recover, whereas, B. vulgaris did not exhibit any recovery of Kleaf.

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    The recovery of K_leaf following a dehydration/rehydration cycle differed between the two species. In Figure 1, the red symbols represent K_leaf of dehydrated leaves, and the blue symbols represent K_leaf of leaves from the same branches that were rehydrated for 8-12 hours. The arrows connecting the red and blue symbols are intended to help visualize the trajectory of recovery (or lack there-of) of leaves from the same branch. K_leaf of the dicotyledon leaves investigated, B. blakeana, dehydrated down to -4 MPa recovered along the same fitted line (ie. no hysteresis) as the decline in K_leaf (Figure 1a). However, once leaves were dehydrated < -4 MPa, K_leaf did not recover but, rather, continued to decline during the rehydration treatment (Figure 1a) despite the leaf rehydrating (i.e., Ψ_leaf became less negative). None of the leaves of B. vulgaris showed a recovery of K_leaf following rehydration (Figure 1b).

Figure 2: Leaf embolization curves during dehydration of B. blakeana and B. vulgaris using the optical method (Kleaf*opt). Changes in Kleaf*opt were approximated from changes in the light transmission through leaves. Note that embolism in both species was not detected until Ψleaf decline below the P88 values (estimated using RKM; Figure 1) for both species.

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Estimated loss and recovery of leaf conductance using the optical method

Vulnerability curves constructed using data from the optical method were fit with sigmoidal models similar to the RKM results. B. blakeana, the dicotyledon species, exhibited less negative P12, P50, and P88 values than B. vulgaris when compared using the optical method (Figure 2). However, in marked contrast to RKM, the optical method resulted in much lower (more negative) PX values for both species. Using the optical method, B. blakeana leaves exhibited very little embolism until Ψ_leaf values fell below ca -3.4 MPa (Figure 2a, Figure 3a), which is the point when this species lost 50% of its K_leaf (-3.3 MPa). Similarly, markedly little embolism was observed in B. vulgaris until Ψ_leaf declined below -4.9 MPa (Figure 2b, Figure, 3b); K_leaf was reduced by 88% of its maximum at -4.3 MPa in this species. Both species showed clear declines in K_leaf by -3 MPa when analyzed using the RKM (Figure 1), but no embolized vessels were detected at this water potential, and so the optical method resulted in PX values that were more negative than RKM (Figs 1 & 2).

Figure 3: Example of an embolization sequences for B. blakeana (a – d) and B. vulgaris (e – h) using the optical method. The transition of veins from light colored pixels to dark colored pixels are interpreted as the liquid-to-gas phase change (i.e., embolism) in vessels. Ψleaf (MPa) is given in the upper-left corner of each panel. Videos of the entire sequence have been supplied online (Ocheltree, 2017) as well as in the supplemental materials.

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Embolism in B. blakeana began first in the higher vein orders and progressed to lower vein orders as water potential declined (Figure 3 a, b, c, d). Similarly, embolism in B. vulgaris began in vascular bundles closer to the leaf margins and progressed closer to the midrib as water potential declined (Figure 3 e, f, g, h). Neither species exhibited refilling of embolized vessels (i.e., increased light transmission) when stems were re-cut and allowed to rehydrate under atmospheric pressure. Additionally, applying positive pressure to the stems of B. blakeana during rehydration did not result in refilling of embolized vessels, but did result in the discoloration (brown color development) of the lamina directly adjacent to the veins (Figure 4a-d, video link: https://www.youtube.com/watch?v=0xiCk_0rUaU). We speculate that this may indicate that the mesophyll cell membranes had ruptured, either during the dry-down or when positive pressure was applied. In the case of B. vulgaris, applying 120 kPa of positive pressure did result in partial recovery (Figure 4e-h, video link: https://www.youtube.com/watch?v=pZbw4NVcoI0), particularly evident near the midrib, whereas veins closer to the leaf margins continued to embolize even after pressurizing the stem (Figure 3e-h).

Figure 4: Example of a rehydration (120 kPa) sequence for B. blakeana (a – d) and B. vulgaris (e – h). Rehydration time is denoted in the upper-left corner of each image. Panel ‘a’ shows the leaf with darkened veins and lamina that developed during rehydration; panels ‘c’-‘d’ show the continual darkening of the veins and lamina while water was forced through the vasculature without refilling. The blue arrow in panel ‘d’ indicates where cytoplasm has been pushed through damaged membranes in B. blakeana leaves. The red arrows in panel ‘e’ denote the section of the leaf undergoing refilling, and thus becoming lighter. Note that the dark areas away from these arrows, and closer to leaf edges, continued to embolize during rehydration. Videos of the entire sequence have been supplied online (Ocheltree, 2017) as well as in the supplemental materials.

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Figure 5: Section of a Bauhinia blakeana leaf that had been dehydrated followed by a positive-pressure treatment. After this treatment combination, leaves exhibited leaf lamina that appeared completely saturated and quickly turned brown in color. That saturation stopped at the point that embolisms had formed, such that it appeared that water could travel through xylem until it hit the point of embolisms and then the water ‘leaked’ from the vasculature. At the locations where water leaked out, the lamina and vein tissue turned brown in color, which we suspect is from the leakage of cell-contents due to membrane damage.

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Possible mechanisms for the decline and recovery of Kleaf using RKM

We found that leaf hydraulic conductance (K_leaf) of both species declined by >50% (Figure 1) prior to the observation of any embolism formation (Figure 2). These results suggest that the initial decline in K_leaf does not result from embolism formation in either Bauhinia blakeana or Bambusa vulgaris (Figs 1 & 2), but is likely a result of declining conductance in extra-xylary tissues (Scoffoni et al., 2014; Trifiló et al., 2016) or vessel deformation/collapse (Cochard et al., 2004; Blackman et al., 2010; Zhang et al., 2016). These two mechanisms could potentially reduce K_leaf independently of any gas obstruction in the conducting elements and would explain the large reduction in K_leaf we observed prior to any embolism formation in the veins of these species. Although this data represents only two species, the species selected have very different anatomical traits and vastly different evolutionary histories.

Although the results from these species highlight the potential role of vessel collapse and extra-xylary conductance on declining K_leaf, the recovery of K_leaf show markedly different results. If vessel collapse/deformation caused the initial decline in K_leaf, we might expect a relatively rapid recovery of K_leaf because it has been shown that the collapsed vessels in Quercus spp. recover their original shape within seconds to minutes following collapse (Zhang et al., 2016). Similarly, if the initial decline in K_leaf was associated with aquaporin down-regulation in extra-xylary tissues, we might expect upregulation to occur within minutes to hours following rehydration (Cochard et al., 2007; Kim & Steudle, 2009), although the signal to upregulate aquaporins during rehydration has not been studied in leaves. In this study, leaves were provided a recovery period of 8-12 h, which would likely have allowed the recovery of K_leaf via both of these proposed mechanisms. When Ψ_leaf stayed above -3.5 MPa, we did observe recovery of K_leaf in B. blakeana leaves, although there were only 4 replicate leaves to which this condition applied. However, we observed no recovery in B. vulgaris for the 4 leaves we measured, highlighting the inability of K_leaf to recover in this species in the absence of root-pressure. This suggests that either the mechanism of conductance loss and/or the mechanism or signal to initiate conductance recovery differ between these two species, highlighting the need for additional research focused on the recovery of K_leaf during leaf re-hydration. Although intriguing, we acknowledge the low replicates of K_leaf recovery leave much uncertainty in the degree and thresholds of leaf recovery and encourage the measurement of this response in a much larger set of species.

To visualize the timing of K_leaf decline, embolism formation, and the point of stomatal closure, we combined the vulnerability curves from the RHK and optical method along with the turgor loss point of each species (Figure 6), which is typically correlated with the point of stomatal closure (Farrell et al., 2017). The stomata of B. blakeana leaves closed before any decline in K_leaf and well before any embolism formation. B. vulgaris, on the other hand, maintained open stomates until Ψ_leaf reached the same point of ~50% loss in K_leaf, but stomates closed well before any embolism formation. These results suggest stomata close in order to prevent embolism formation in both species, which agrees with other recent research on tree species (Creek et al., 2020). However, B. vulgaris operates with a more ‘aggressive’ strategy in that it continues water-use despite reductions in K_leaf. B. vulgaris may be able to maintain positive stomatal conductance despite losses of leaf hydraulic conductance because it can recover from these losses if positive pressure can be supplied, presumably from root tissue (Cao et al., 2012; Yang et al., 2012). B. blakeana, however, did not exhibit an ability to recover with positive pressure, and had much lower root pressure, and so may be more conservative in closing stomates to prevent permanent hydraulic damage. Interestingly, B. blakaeana did show some ability to regain lost leaf hydraulic conductance, as long as it did not result from embolism formation. Therefore, the conservative stomatal control in this species may be an adaptation to prevent embolism formation.

Figure 6. Fitted relationships of the decline in Kleaf (solid black line), change in optical properties suggesting embolism formation (dotted grey line), and leaf turgor loss point (ΨTLP) of B. blakeana (‘a’) and B. vulgaris (‘b’). ΨTLP of both species occurs prior to any embolism formation and, for B. blakeana, ΨTLP also occurs prior to any loss of Kleaf. However, B. blakeana maintains turgor until ~50% loss of Kleaf.

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Possible mechanisms for the decline and recovery of Kleaf using the optical method

Decline in light transmission through individual leaf veins could be clearly observed in both species, though occurring at different values of Ψ_leaf. Assuming that this decline in light transmission is indeed related to the sudden gas-filling of vessels, we can then conclude that no appreciable embolism occurs in either species above ca -3.5 MPa, and therefore cannot be responsible for the initial loss of K_leaf that was measured using RKM. Recovery of embolism (ie. refilling) in the monocotyledon B. vulgaris was observed under positive pressure (red arrows in Figure 4e-h, video link: https://www.youtube.com/watch?v=pZbw4NVcoI0), but recovery was confined to veins near the leaf midrib, which were also the last veins to embolize during dry-down (Figure 3e-h). It is possible that because these veins had experienced embolism only shortly before positive-pressure was applied, they were more prone to embolism reversal than veins located further from the midrib. It is unlikely that veins located further from the midrib would have also recovered from embolism if given a longer rehydration time because embolism in these veins continued to accumulate even during rehydration (Figure 4e-h). Considering both the lack of K_leaf recovery as measured with the RKM (in which no positive pressure was applied during rehydration), as well as the observed recovery of embolism following positive-pressure rehydration, it is possible that B. vulgaris requires positive root pressure to regain conductance and also to reverse embolization. Although the mechanism driving declines in K_leaf of Sinarundinaria nitida, another bamboo, was not identified, recovery of mid-day depressions in K_leaf and stomatal conductance were attributed to the generation of positive root pressure (c. 60 kPa) at night in this species (Yang et al., 2012). In contrast, out results suggest that the physiology of B. blakeana may be coordinated to facilitate the recovery of leaf conductance in the absence of root pressure based on the RKM results, with the important caveat that embolization is likely irreversible in this species, even in the presence of root pressure.

    Although we did not observe recovery from embolism formation in the dicotyledon B. blakeana under atmospheric or positive pressure, the positive pressure rehydration treatment appeared to have caused the leakage of cytoplasm/water from either the vessels or mesophyll cells, presumably into either the apoplast or air spaces within the leaf (blue arrow in Figure 4d, video link: https://www.youtube.com/watch?v=0xiCk_0rUaU). The apparent leakage was confined to areas where embolism was not observed during the dry-down phase (Figure 3a-d), which is important for several reasons. First, the absence of leakage adjacent to embolized cells suggests that no water was flowing through these areas and refilling could not occur. But, because embolisms formed in higher order veins first, water could move through the basal portion of the vein, but was then forced out of the vasculature at the point it encountered an embolism. Second, these results support the validity of the optical method of detecting embolisms. The theory of this method is based on the idea that changes in the light-transmittance is caused by a change from liquid to air filled vessels, and since the only places we observed the leakage (which would require a supply of water) were the areas where embolisms were not identified by this method. And third, the leakage suggests that the dry-down resulted in significant membrane damage at water potentials above that required for embolism to occur. Therefore, the point of embolism formation in this species likely occurred after the cells were already damaged beyond recovery. It is also possible that this membrane damage was, at least partially, responsible for the initial decline in K_leaf in this species.

The apparent leakage of cell contents that was observed in the dicotyledon B. blakeana (Figure 5) was not observed in the monocotyledon B. vulgaris, even after Ψ_leaf had declined well below the turgor loss point (Figure 4e-h). Bamboo possesses stiff cell walls with a high modulus of elasticity (Table 2) and, therefore, we would expect the cells of this species to be more resistant to expansion and contraction. It is possible that this is why B. vulgaris resisted damage during dehydration, and therefore, that the hydraulic pathway was not compromised during dry-down or during positive pressure rehydration. The elasticity of the cell wall is dynamic and can change in response to drought through the production of expansins (Tenhaken, 2015; Houston et al., 2016) and membrane properties affecting leakage, such as lipid content, can also change in response to drought stress (Zhang et al., 2005). If, however, cell wall elasticity does play a role in membrane stability during periods of cell dehydration, then trade-offs between cell wall function related to stiffness may be important to consider in the recovery of leaves from dehydration stress. For example, if it is true that cell wall flexibility is required for high capacitance, but that cell wall rigidity is required to reduce the risk of cell wall deformation and membrane damage during severe dehydration, there must exist a tradeoff between these two traits (cells cannot be both flexible and rigid at the same time). Given this possible tradeoff, we might therefore expect species with high capacitance (flexible cell walls) to be unable to resist membrane damage at very low xylem water potentials (Lechthaler et al., 2016).

Initial sites of embolism

Our results suggest that embolism formation in the palmate-veined dicotyledon occurred in 2^rd/3^rd order veins first, followed quickly by the 2^nd and 1^st order veins. Although this species did not have a single midrib, but had a palmate venation pattern, the primary veins were the last to experience embolism formation. The imaging techniques currently being employed have shown some species to experience embolism in the midrib or petiole first (Brodribb et al., 2016a; Scoffoni et al., 2017), but some species of palms exhibit greater vulnerability to embolism in higher order veins (Emilio et al., 2019), and an earlier study using dye on Cercis siliquastrum reported embolism in the higher order veins first (Nardini et al., 2003). Similar to the dicotyledonous plant, the monocot leaves exhibited cavitation in the veins near the leaf margins, while the veins in the midrib and central region of the leaves remained water-filled. Unfortunately, we are unaware of any other data on the order of embolism formation among the veins of monocot leaves with which to compare our results.

Cavitation in the leaf, petiole, or midrib has been suggested as a mechanism to provide segmentation of the plant; isolating embolism to the more distal organs and thus reducing whole-plant water loss (Zimmermann, 1983; Pivovaroff et al., 2014; Nolf et al., 2015; Charrier et al., 2016; Johnson et al., 2016). Whereas cavitation in the petiole is likely to lead to loss of the entire leaf, embolism in the higher order veins could leave part of the venation system of leaves intact and functioning. Alternatively, embolism in the distal veins, which are most closely linked with the supply of water to stomates, might be detrimental for a species that cannot refill these conduits. There appears to be no convincing argument for why we should expect embolization to initiate in the same location within the leaf vasculature across all species and habitats. Rather, it is likely that as we search across a wider set of species, we may find variation in the ranking of vein orders to embolism vulnerability. Therefore, investigating the potential ecological correlates with vein order vulnerability may provide a more satisfying answer to this question than assuming a general pattern across species.