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. 2022 Feb 10;129(5):555–566. doi: 10.1093/aob/mcac016

Foliar water uptake does not contribute to embolism repair in beech (Fagus sylvatica L.)

Jeroen D M Schreel 1,2,✉, Craig Brodersen 3, Thomas De Schryver 4,2, Manuel Dierick 4,2, Adriana Rubinstein 3, Koen Dewettinck 5, Matthieu N Boone 4, Luc Van Hoorebeke 4, Kathy Steppe 1
PMCID: PMC9007097  PMID: 35141741

Abstract

Background and Aims

Foliar water uptake has recently been suggested as a possible mechanism for the restoration of hydraulically dysfunctional xylem vessels. In this paper we used a combination of ecophysiological measurements, X-ray microcomputed tomography and cryo-scanning electron microscopy during a drought treatment to fully evaluate this hypothesis.

Key Results

Based on an assessment of these methods in beech (Fagus sylvatica L.) seedlings we were able to (1) confirm an increase in the amount of hydraulically redistributed water absorbed by leaves when the soil water potential decreased, and (2) locate this redistributed water in hydraulically active vessels in the stem. However, (3) no embolism repair was observed irrespective of the organ under investigation (i.e. stem, petiole or leaf) or the intensity of drought.

Conclusions

Our data provide evidence for a hydraulic pathway from the leaf surface to the stem xylem following a water potential gradient, but this pathway exists only in functional vessels and does not play a role in embolism repair for beech.

Keywords: Climate change, computed tomography, cryo-scanning electron microscopy, drought, embolism formation, embolism repair, foliar absorption, foliar water uptake, microCT

INTRODUCTION

Recent drought events (e.g. Ciais et al., 2005; Moore et al., 2016; Hauser et al., 2017; Goulden and Bales, 2019) have demonstrated how predicted shifts in global precipitation patterns and longer and more intense droughts as a result of climate change will impact the water cycle and tree survival (IPCC, 2018). One of the primary problems in trees during drought is tissue dehydration and the concomitant dwindling of internal water reserves (Körner, 2019). This reduction in water availability translates into a decreased xylem water potential. If the xylem water potential drops below a critical threshold, xylem conduits may embolize and become blocked by air bubbles, which disrupt the flow of water to the foliage (Zimmermann, 1983). Air seeding, the spread of air from embolized conduits to adjacent conduits, mostly likely via pits (Zimmermann, 1983), further diminishes the hydraulic conductivity of trees, thus impairing their hydraulic functionality and eventually resulting in tree dieback (Brodribb and Cochard, 2009; Choat et al., 2018a).

It has been suggested (e.g. Hacke et al., 2001; Baert et al., 2014) and heavily debated (e.g. Cochard and Delzon, 2013; Rockwell et al., 2014) that embolism and embolism repair can occur on a daily basis, making xylem hydraulic conductivity dependent on the interplay between these two phenomena. During the recent International Xylem Meeting (XIM4), however, a consensus has been reached identifying embolism repair when xylem is under a negative tension (e.g. during transpiration) as improbable (Cochard et al., 2019). Notwithstanding, the underlying mechanism of embolism repair during relaxation of the water column is still unclear (Zwieniecki and Holbrook, 2009), can be affected by embolism fatigue (Hacke et al., 2001) and is unlikely to be a universal trait of woody plants (Choat et al., 2015, 2018b; Brodersen et al., 2018).

Several mechanisms for embolism repair have been suggested, such as water influx from surrounding tissue (Zwieniecki and Holbrook, 2009), with foliar water uptake (FWU) being one of the more recently proposed contributing plant mechanisms (Burgess and Dawson, 2004; Laur and Hacke, 2014; Schreel and Steppe, 2020). It is hypothesized that FWU might result in embolism repair (Brodersen and McElrone, 2013; Laur and Hacke, 2014; Mayr et al., 2014) by generating reduced tension in the xylem sap, and associated reduced water demand (Burgess and Dawson, 2004; Steppe et al., 2018). Moreover, during FWU, the minimum requirements for embolism repair, being (1) a water source, and (2) a beneficial water potential gradient (Earles et al., 2016), are met.

Foliar water uptake has been reported for ~93 % of >180 tested plant species belonging to >70 different families worldwide (Dawson and Goldsmith, 2018; Berry et al., 2019; Schreel and Steppe, 2020). This research has led to the understanding that FWU results in hydraulic redistribution and rehydration of dehydrated plant tissues, thus reducing a plant’s soil water dependency (Nadezhdina et al., 2010; Eller et al., 2013; Earles et al., 2016). This allows plants to benefit from water condensation on the foliage and small precipitation events that wet the foliage but are unavailable to the root system due to crown interception or runoff. The amount of water taken up by FWU is generally small, but could be sufficient to prevent permanent drought-induced damage to root structures (Nadezhdina et al., 2010) and increase survival rates (Boucher et al., 1995; Emery, 2016). Thus, FWU and embolism repair should be taken into account when assessing future effects of climate change on plant hydraulics (Mayr et al., 2014).

Multiple techniques have been used to investigate embolism formation and/or repair, such as the hydraulic method (De Baerdemaeker et al., 2019a, b), acoustic emissions (Mayr et al., 2007; Vergeynst et al., 2015), magnetic resonance imaging (MRI) (Holbrook et al., 2001; Scheenen et al., 2007), cryo-scanning electron microscopy (cryo-SEM) (Cochard et al., 2000; Mayr et al., 2007) and microcomputed tomography (microCT) (Brodersen et al., 2010, 2018; De Baerdemaeker et al., 2019b). In the category of destructive methods, cryo-SEM can provide a thorough assessment of the number of embolized vessels, while the hydraulic method and acoustic emissions cannot. Among the non-invasive imaging techniques, microCT has a higher resolution compared with MRI, which makes it more ideal for a detailed in vivo analysis of embolism formation and repair (Choat et al., 2016).

It has been speculated that FWU could result in embolism repair (Burgess and Dawson, 2004; Laur and Hacke, 2014; Schreel and Steppe, 2020), but so far no protocol to assess this hypothesis has been suggested. To meet this need, we developed a methodology based on a novel synthesis of existing techniques (microCT and cryo-SEM). This method allows a three-step build-up to assess the contribution of FWU to embolism repair: (1) an assessment of the amount of water absorbed by FWU in an intact tree seedling, (2) localization of water absorbed by leaves in a stem segment by using a contrast agent (sodium iodide), and (3) a statistical assessment showing whether FWU results in embolism repair during varying intensities of drought (Fig. 1). Beech (Fagus sylvatica L.) seedlings were selected for this study, based on their high potential relative importance and average actual relative importance for FWU during drought (Schreel et al., 2019a).

Fig. 1.

Fig. 1.

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Schematic of the suggested workflow to assess the contribution of FWU to embolism repair, starting with the identification of FWU in intact plants (left), followed by localization of water absorbed by leaves in the stem (middle) and ending with the actual assessment of embolism repair and the addition of statistical rigour during a range of environmental conditions (right).

MATERIALS AND METHODS

Assessment of the magnitude of FWU in intact seedlings: ecophysiological measurements

One-year-old beech (Fagus sylvatica L.) seedlings were planted into pots (diameter 30 cm, height 27 cm) containing seed soil for tree nurseries (VP308, Peltracom, Belgium) on 23 March 2016 and placed in a greenhouse at the Faculty of Bioscience Engineering, Ghent University, Belgium. A total of six Duo-Sticks (Substral, Scotts Miracle-Gro, USA) containing nutrition and pesticides were added to the soil of each pot, one on 11, one on 20 and four on 23 May 2016. Seedlings had an initial stem diameter of ~6 mm (measured 10 cm above the soil) and an average height of ~40 cm. Plants were randomly divided into two groups and subjected to different treatments: (1) well-watered control treatment (n = 4) and (2) drought treatment (n = 5). All seedlings were initially well-watered. To investigate the effect of a drought treatment on FWU, irrigation in drought-treated seedlings was stopped on 14 June 2016 (DOY 165). Artificial rain events (AREs) were imposed by spraying the seedlings with tap water for 1 h between 1100 and 1300 h, while the soil was sealed off with plastic. During the well-watered period (12 d; DOY 15–166) and the first 15 d of drought (DOY 166–181), AREs were imposed daily. In the subsequent drought period (77 d, DOY 181–258) biweekly AREs were carried out, i.e. spraying on two consecutive days per week. After an ARE, the plastic covering the soil was removed to avoid anaerobic conditions.

Microclimatic conditions were monitored with a relative humidity (RH) (HIH-4000, Honeywell, USA), temperature (Thermistor 10k, Epcos, Germany) and photosynthetically active radiation (PAR) (JYP 1000, SDEC, France) sensor. An average RH, temperature and daily light integral of 71.0 ± 1.0 %, 20.8 ± 0.3 °C and 25.4 ± 0.8 mol photons PAR m−2 d−1 (mean ± standard error for the period DOY 155–259) were obtained, respectively.

Sap flow sensors (below lowest branch), point dendrometers (above the sap flow sensor) and tensiometers were installed on all seedlings. Sap flow was measured with mini-HFD (heat field deformation) sensors. These sensors are able to measure a continuous bidirectional sap flow (Hanssens et al., 2013; Schreel and Steppe, 2018). The HFD ratio (−) was calculated [eqn (1)] as a proxy of the actual sap flow (Vandegehuchte and Steppe, 2012) and will henceforth be referred to as sap flow:

HFD ratio=K+dTs−adTas (1)

with dTs–a the temperature difference between upper axial and tangential thermocouple (°C), K the absolute value of dTs–a or dTas during zero flow (°C) and dTas the temperature difference between tangential and lower axial thermocouple (°C). The K value was calculated as the absolute value of the intersection with the dTs–a axis by linear extrapolation of dTs–a versus dTsym/dTas (Nadezhdina et al., 2012). Following sensor installation, each mini-HFD was covered with bubble wrap for thermal insulation purposes, and with aluminium foil to reflect incident radiation.

Stem diameter variations were measured with point dendrometers (DF 5.0, Solartron, UK). Soil water potential was measured with tensiometers (CV5 U, Tensio-Technik, Germany); it can be converted to soil water content based on Fig. S2 in Schreel et al. (2019a).

All sensor signals were recorded at 10-s intervals, averaged every 60 s with a Campbell data logger (CR6, Campbell Scientific Inc., Logan, USA) and stored using the PhytoSense cloud service (Phyto-IT, Gent, Belgium).

Locating water absorbed by leaves in the stem: laboratory-based microCT imaging

While the previous experiment can demonstrate FWU, it does not locate hydraulically redistributed water absorbed by FWU. A contrast agent (NaI or sodium iodide) was applied to the leaves to identify which conduits are moving foliar-absorbed water (Pratt and Jacobsen, 2018). By subsequently imaging the stem with X-ray microCT, water absorbed by FWU can be located in the stem, allowing us to assess whether this water is used to refill previously embolized vessels and if some parts of the xylem are preferentially used or not.

For the contrast agent experiment (21–22 September 2016), a beech seedling (n = 1) from the same batch as described above (i.e. stem diameter of ±6 mm measured at 10 cm above the soil; height ±40 cm) was grown under the same microclimatic conditions and treated in the same way; however, this seedling was planted in different a pot (diameter 11 cm, height 30 cm) to fit the scanner gantry. During the start of the experiment the seedling had a leaf water potential of −2.0 MPa. This corresponds to an approximate soil water potential of −8 kPa, based on a leaf and soil water potential correlation constructed with soil water potential measurements collected with a tensiometer and discrete leaf water potential measurements collected with a thermocouple-psychrometer (HR-33T, Wescor, Logan, USA) (n = 3; Fig. 2). Top leaves were glued in dishes (Fig. 3) that were filled with 55 g L−1 NaI (contrast agent), giving the solution a water potential of −1.9 MPa. After 7 h the contrast agent was diluted to about half of the original concentration to increase the difference in water potential between the leaves and the contrast agent.

Fig. 2.

Fig. 2.

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Soil water potential (Ψsoil) as a function of leaf water potential (Ψleaf; n = 3) of beech seedlings. Error bars indicate standard error.

Fig. 3.

Fig. 3.

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Top view (left) and schematic (right) of the set-up for the laboratory-based microCT imaging experiment with contrast agent.

The seedling was scanned in the laboratory-based environmental microCT scanner [EMCT (Dierick et al., 2014)] located at the Centre for X-ray Tomography of Ghent University [UGCT, www.ugct.ugent.be (Masschaele et al., 2007)]. This scanner was designed and built in-house in collaboration with the UGent spin-off company XRE (www.xre.be, today part of Tescan Orsay Holding a.s.). The system consists of a directional microfocus Hamamatsu L9181-02 X-ray tube with integrated high-voltage power supply. The detector is a Xineos CMOS flat-panel detector (1316 × 1312 pixels with a 100-µm pitch) with a thick, structured CsI scintillator.

Scanning was performed by a single rotation, with 2000 2-D projections. The scan parameters used were a tube voltage of 60 kV, a target power of 5 W and a magnification yielding 3-D images with 7.4 voxel size. The CT reconstructions were performed using Octopus Reconstruction software (Vlassenbroeck et al., 2007) (currently distributed by Tescan-XRE, formerly known as XRE, spin-off company of UGCT). Volume rendering was done in Drishti (Limaye, 2012).

Contribution of FWU to embolism repair in the stem: laboratory-based microCT imaging

MicroCT imaging with a contrast agent provided information on locality, clearly identifying where water absorbed by FWU is redistributed. However, the set-up with contrast agent has drawbacks as it is not easily replicated, sacrificing statistical rigor for spatial localization. To compensate for this shortcoming, FWU can be assessed by microCT imaging with pure water used for leaf wetting. This set-up does not allow the observer to make a distinction between water absorbed by leaves or roots, but it can provide statistical rigour as it is more easily replicated and allows comparison between the number of embolized vessels found in the stems of seedlings before and after a leaf wetting event.

Two unused beech seedlings, from the same batch as described above for the experiment with contrast agent, were scanned (EMCT scanner) at different levels of soil drought before and after a 1-h ARE. Seedling 1 was scanned on 9 and 27 June 2016 at a soil water potential of −4 and −7 kPa, respectively, indicating well-watered conditions. After initial scanning, drought was induced by stopping irrigation. Seedling 2 was used for subsequent scans as seedling 1 died during the drought treatment. Seedling 2 was scanned on 11 July and 19 August 2016 at a soil water potential of −28 and −57 kPa, respectively.

Seedlings were scanned by three rotations, with 2000 2-D projections per rotation. The scan parameters used were a tube voltage of 60 kV, a target power of 5 W and a magnification yielding 3-D images with 6.7 μm voxel size. The CT reconstructions were performed using Octopus Reconstruction software (Vlassenbroeck et al., 2007) (currently distributed by TESCAN-XRE, formerly known as XRE, spin-off company of UGCT). Volume rendering was done in Drishti (Limaye, 2012). The number of embolized vessels was determined with Fiji (Schindelin et al., 2012) in three slices of every scan, using the same vertical slice in both scans (before and after ARE). The influence of image noise on the counted number was minimized by only measuring embolized vessels with an area ≥900 μm2, based on minimum beech vessel areas in the literature [250 500 μm2 (Sass and Eckstein, 1995) and 1500 μm2 (Diaconu et al., 2016)]. The number of embolized vessels was determined by using the ‘auto threshold’ function, separating clusters by ‘watershed’ and ‘analyze particles’ with an area of 900 μm2 – infinite. Paired, one-sided (after < before) t-tests were performed on three pseudoreplications per scan-couple (one tree, before and after ARE at a given soil water potential) to check for a significant decrease in the number of embolized vessels after 1-h AREs. Statistical analyses were done in RStudio (R Core Team, 2018).

Contribution of FWU to embolism repair in petioles: cryo-SEM imaging

To check for differences regarding the possible link between embolism repair and FWU in different organs, we move from the stem towards the petioles (i.e. closer to the leaves, the point of uptake). This point can be assessed by microCT imaging; however, it is easier to obtain a larger number of replicates and concomitant statistical rigour with cryo-SEM.

Petioles were sampled from a beech seedling subjected to the same drought treatment as seedlings used for the ecophysiological measurements (pot diameter 30 cm and height 27 cm), on 7 July and 11 and 25 August 2016 at a soil water potential of −11, −44 and −51 kPa, respectively. Sampling was performed with deep-freezing pliers in liquid nitrogen, after which the pliers were used to rapidly freeze water in the petioles, break the petioles from the plant and put them in liquid nitrogen. Petiole samples were vertically fixed by applying carbon glue [a mixture of an embedding medium for frozen tissue samples (Scigen Scientific, Gardena, USA) and colloidal graphite (Agae Scientific Ltd, Sussex, UK)] on an aluminium stub. Samples were kept cool in nitrogen slush and transferred to a cryo-preparation chamber (PP3010 Cryo-SEM Preparation System, Quorum Technologies, UK) under vacuum conditions at −140 °C. Subsequently, samples were sublimated for 15 min at −70 °C to remove frost artefacts, sputter-coated with platina by using argon gas, transferred to the SEM stage at −140 °C and visualized by using a 3-keV electron beam on a Jeol JSM 7100F scanning electron microscope (JEOL Ltd, Tokyo, Japan).

Five sub-images of 100 × 100 μm were taken per sample. The number of embolisms per sub-image was counted and averaged to one value per sample. Subsequently, the average of all samples was calculated from the averaged values of individual samples.

Contribution of FWU to embolism repair in leaves: synchrotron-based microCT imaging

When checking for differences concerning the link between embolism repair and FWU in different organs, the largest possible disparities are expected to be observed between leaves and other organs. Assessing embolism repair in leaves as a result of FWU can be done by both cryo-SEM and microCT imaging. An advantage of microCT imaging is its potential to provide a 3-D view, which cannot be obtained with cryo-SEM. While a 3-D view is not necessary at this stage, it can provide important insights regarding vessel connectivity, making microCT the preferred tool for this assessment. To reduce sample scanning time, we opted to use synchrotron microCT imaging.

Beech seedlings in square pots (side 7.5 cm, height 23 cm) were separately ordered for synchrotron-based microCT imaging (June 2019). Seedlings were initially well-watered until 3 d before microCT imaging. At this point they were transported from Oakland, CA (Rolling River Nursery) to the X-ray microtomography facility at the Lawrence Berkeley National Laboratory Advanced Light Source (Beamline 8.3.2).

Four leaves of four different beech seedlings were glued to the inside of the lid of a plastic cup [mesophyll had to be partly cut to allow gluing; see Fig. S3 in Schreel et al., (2020)] and were allowed to air-dry. After 3.5 h of air-drying, leaves were scanned to evaluate leaf dehydration. After scanning, cups were partially filled with water to allow leaf rehydration by submergence, while avoiding contact between the water and the petiole or the cut mesophyll edge, and rescanned 4 h after rehydration. Leaves were rotated in the 24-keV synchrotron X-ray beam in 1/12° increments over 180°, yielding 2160 2-D projection images with a 0.65-μm pixel pitch resolution captured on a 4008 × 2672-pixel CCD camera (PCO.4000, Cooke Corporation). Approximately 1.3 mm2 of leaf tissue was scanned from a region surrounding the mid-vein located below the water level. Two-dimensional projection images were reconstructed into a 3-D data set using TomoPy [a Python-based framework for reconstructing tomographic data (Gürsoy et al., 2014)].

Only one sample (n = 1) did not move during scanning after rehydration and was used to assess embolism repair. Three random slices per scan (one scan after air drying and one scan after rehydration) were used to count the number of embolized vessels with ImageJ/Fiji image processing software (Schindelin et al., 2012). ‘Noise’ was excluded by only measuring embolized vessels of the midvein with an area ≥10 μm2 as vessel area was assumed to be smaller in leaf veins compared with stems and less noise was present in synchrotron-based microCT images compared with laboratory-based microCT images. The number of embolized vessels was determined by using the ‘auto threshold’ function, separating clusters by ‘watershed’ and ‘analyze particle’ with an area of 10 μm2 – infinite.

RESULTS

Assessing the magnitude of FWU in intact seedlings: ecophysiological measurements

During the AREs we detected reverse sap flow in beech seedlings exposed to both the well-watered and the drought treatment (Fig. 4A, B). Initially, reverse flow in both groups had the same order of magnitude (Fig. 4A). As the soil water potential declined, daily sap flow decreased relative to the control group while reverse flow during subsequent AREs increased compared with the control treatment (Fig. 4B).

Fig. 4.

Fig. 4.

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(A, B) HFD ratio and (C, D) stem diameter variation (set to zero at the start of the 2-d measurement campaigns; control, n = 4; drought, n = 5) measured in beech seedlings. Corresponding soil water potential (A, C) 20 d after stopping irrigation for the drought treatment (DOY 185–186), control, −4 ± 0 kPa; drought, −9 ± 1 kPa; and (B, D) 70 d after stopping irrigation for the drought treatment (DOY 235–236), control, −4 ± 0 kPa; drought, −49 ± 9 kPa (mean ± standard error). Coloured bands indicate the mean ± standard error. Artificial rainfall events are indicated by areas shaded in grey.

Artificial rain events resulted in small transient increases in stem diameter (Fig. 4C, D), which were negated by stem diameter decreases in the hour following the AREs. Although negative sap flow during severe drought increased, the increase in diameter became less pronounced during AREs in the drought treatment (Fig. 4D).

Locating water absorbed by leaves throughout the stem and assessing the contribution of FWU to embolism repair in different organs during drought: microCT and cryo-SEM observations

To locate water absorbed across the leaf surface and transported into the stem xylem, we used microCT imaging with a contrast agent applied to the foliage (Fig. 3). When the water potential of the contrast agent (−1.9 MPa) nearly matched the leaf water potential (−2.0 MPa) of the beech seedling, no uptake of contrast agent was observed in the stem xylem (data not shown). After the contrast agent had been applied to the foliage for 7 h, it was diluted to about half the concentration in order to increase the difference in water potential between leaves and contrast agent. Ten hours after the initial start and ~3 h after diluting the contrast agent, no uptake was observed (Fig. 5B). Twenty-four hours after the initial start, contrast agent was visible in functional vessels of the stem and kept accumulating over time (Fig. 5C, D). Contrast agent was first observed in xylem near the central pith, and then spread outward radially, primarily in early wood of both growth rings (Fig. 5). The contrast agent was clearly localized in the xylem conduit lumen, while ray parenchyma showed no contrast agent (Figs 5 and 6A). The number of embolized vessels remained constant throughout the entire measurement campaign (Fig. 5).

Fig. 5.

Fig. 5.

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Laboratory-based microCT images of cross-sections of a beech seedling stem at (A) 0, (B) 10, (C) 24 and (D) 34 h after applying contrast agent to the leaves. White vessels are embolized. Dark grey vessels are functional and turn light grey when containing contrast agent. Scale bars = 1 mm. CP, central pith; Y1, first-year growth ring; Y2, second-year growth ring; EV, embolized vessel; FV, functional water-filled vessel; VCA, vessel filled with contrast agent; RP, ray parenchyma.

Fig. 6.

Fig. 6.

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Laboratory-based microCT 3-D reconstruction of the anatomy of a beech seedling stem 34 h after applying a contrast agent to the leaves (see Fig. 5D). (A) Transverse section with indication of the location of intersections shown in panels (B), (C) and (D). (B) Radial cross-section. (C) Tangential cross-section, halfway between the centre and the surface. (D) Tangential cross-section through the outer xylem ring. CP, central pith; Y1, first-year growth ring; Y2, second-year growth ring; LW, late wood; EW, early wood; RP, ray parenchyma. Black vessels are embolized. White vessels are functional and contain contrast agent. Scale bars = 1 mm.

The number of embolized vessels in both the stem (Fig. 7, Table 1) and the petioles (Fig. 8, Table 2) increased as soil water potential (kPa) became more negative, which was confirmed with microCT and SEM, respectively. Extensive embolism spread occurred in the stem at a soil water potential between −28 and −57 kPa. In petioles, the number of embolized vessels per mm2 increased as soil water potential decreased but did not result in complete hydraulic failure at a soil water potential of −51 ± 1 kPa as seen in the stem xylem. Both experiments showed no difference between the number of embolized vessels before and after an ARE, irrespective of the investigated plant organ (stem or petiole), the degree of embolism and the soil water potential.

Fig. 7.

Fig. 7.

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Laboratory-based microCT images of the cross-sections of beech seedling stems before and after an ARE with the average associated soil water potential (Ψsoil; kPa) measured from 0900 to 1700 h ± standard error (see Table 1). Two seedlings are shown, with seedling 1 at a Ψsoil of −7 kPa and seedling 2 at a Ψsoil of −28 and −57 kPa. White vessels are embolized. Scale bars = 1 mm.

Table 1.

Number of embolized vessels (mean ± standard error based on three pseudoreplications) in a beech stem (n = 1), before and after an ARE with the average associated soil water potential (Ψsoil; kPa) measured from 0900 to 1700 h  ± standard error (see Fig. 7). P-values are based on one-sided (after < before) paired t-tests

Ψ soil (kPa) Before After P-value
−7 ± 0 77 ± 3 81 ± 4 0.83
−28 ± 0 132 ± 5 131 ± 4 0.36
−57 ± 0 640 ± 4 633 ± 6 0.17
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Fig. 8.

Fig. 8.

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Cryo-SEM images of the cross-section of beech petioles during a drought treatment (soil water potential −51 ± 1 kPa; see Table 2). Scale bars, left to right = 200, 100, 20 and 5 μm, respectively.

Table 2.

Average number (×10−2) of embolized vessels in beech (F. sylvatica) petioles per mm2 ± standard error (see Fig. 8), before and after an artificial rainfall event with the average associated soil water potential (Ψsoil; kPa) measured from 0900 to 1700 h ± standard error

Ψ soil (kPa) Before (n*) After (n*)
−11 ± 0 0 ± 0 (3) 0 ± 0 (2)
−44 ± 1 0.3 ± 0.1 (2) 0.4 ± 0.5 (3)
−51 ± 1 30.2 ± 9.2 (2) 26.6 ± 4.2 (2)
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*Number of samples.

MicroCT images of the leaves indicated rehydration of subepidermal tissues surrounding the midvein and an increase in lamina thickness of 28 % (from 77 ± 1 to 98 ± 3 μm) following AREs (Fig. 9). No difference in the number of embolized vessels was observed in the midvein of a leaf (Fig. 9), with 280 ± 7 embolized vessels after 3.5 h of air-drying and 280 ± 7 embolized vessels after rehydrating the leaf surface.

Fig. 9.

Fig. 9.

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Synchrotron-based microCT cross-sections of a beech leaf (A) after 3.5 h of air-drying and (B) after 4 h of leaf surface rehydration. Scale bars = 200 μm.

DISCUSSION

To assess the magnitude of FWU in intact beech seedlings (Fig. 1), ecophysiological measurements were conducted during 1-h AREs. When exposed to AREs, a negative sap flow and a transient increase in stem diameter were observed (Fig. 4). These results indicate that enough water was absorbed by the leaves to be hydraulically redistributed towards the stem (similar to FWU by mangrove trees; Steppe et al., 2018; Schreel et al., 2019b). More water was taken up by FWU and transported towards lower plant organs as soil water potential decreased (Fig. 4B), contrasting with the findings of FWU for beech in detached leaves (Schreel et al., 2019a) and signifying the importance of water sinks external to the leaves themselves. As non-significant transient stem diameter increments in drought-treated seedlings (Fig. 4D) were smaller compared with the significant transient stem diameter increments in well-watered ones (Fig. 4D), we hypothesize that the capacitance of the roots must have been larger than that of the stem, thus allowing roots to serve as the principle sink for water absorbed by leaves. This reasoning might also explain why no turgor-driven growth was observed in beech seedlings, contrary to observations in the mangrove species Avicennia marina (Steppe et al., 2018; Schreel et al., 2019b).

Showing that water absorbed by leaves can be found in intact stem xylem and could contribute to embolism repair was demonstrated by supplying a contrast agent to the leaves (Fig. 1), which was transported downward and observed at stem level (Figs 5 and 6). As this set-up is challenging and time-consuming (Fig. 3), it is only used to locate foliar absorbed water in the stem and not to add significant statistical rigour to the observed findings. Contrast agent was first detected in water-filled xylem vessels near the pith and over time spread outwards in the direction of the phloem (Fig. 5). Surprisingly, less contrast agent was observed in late wood of the first growth ring and no contrast agent was transported by or accumulated in late wood of the second-year growth ring (Fig. 6). This observation might be explained by a predominant connection of leaf traces to early wood formed when leaves are developing. Leaf traces could thus result in an improved vertical alignment of early wood in the first-year growth ring and a more direct connection between leaves and stem for early wood of the second growth ring. Alternatively, if the xylem vessels of the second-year late wood were still developing, it is possible that no contrast agent was observed because these vessels were not yet fully functional.

While microCT imaging with a contrast agent provided the information on locality, clearly identifying where water absorbed by FWU is redistributed to, statistical rigour needs to be added to confirm or reject the envisaged hypothesis of FWU resulting in embolism repair. As the set-up with contrast agent is not easily replicated, more replicable assessments need to be added. This can be done by microCT or cryo-SEM imaging with spraying of pure water for leaf wetting. As some species are expected to absorb more water by FWU during drought (Schreel et al., 2019a), this experiment should be repeated during different degrees of drought to assess a potential selective occurrence of FWU and/or embolism repair during different degrees of drought. Furthermore, to check for differences regarding the possible link between embolism repair and FWU in different organs, this assessment should be repeated on several plant organs, such as the stem, petioles and leaves.

As the number of embolized vessels remained constant before and after leaf wetting (ARE) (Fig. 7, Table 1), no FWU-induced embolism repair was observed at the stem level. Because no embolism repair was observed during both aforementioned experiments (Figs 5–7, Table 1), a lack of embolism repair due to the low osmotic water potential of contrast agent inhibiting transport to embolized vessels seems unlikely. Embolism repair might only occur within a specific range of stem water potential; however, contrary to observation in Vitis vinifera (Brodersen et al., 2013), no embolism repair was observed in the stem of beech seedlings, irrespective of soil water potential (−7.4, −27.9 or −57.1 kPa) or degree of embolism (Fig. 7). As soil water potential and stem water potential are coupled and experiments were performed up to full runaway embolism (Fig. 7), these results indicate that embolism repair at the stem level might not occur as a result of FWU in beech seedlings.

When embolized vessels are not hydraulically isolated, embolism repair could be inhibited by drainage of water from refilling vessels towards adjacent vessels under tension (Brodersen et al., 2010). Additionally, as long as some vessels are filled with water, water taken up by FWU could be hydraulically redistributed within the plant and possibly towards the roots, before inducing embolism repair. This redistribution would increase the water potential of tissues and reduce the chance of permanent root damage. When all xylem vessels are embolized, rehydration would be much slower and could be preceded by embolism repair, emphasizing the need to repeat this experiment during varying degrees of drought.

If FWU continues for a long enough time, the stem water potential will approximate zero, which could potentially result in embolism repair, governed by the interplay between recharge of capacitive tissues and embolism repair (Knipfer et al., 2017). However, as FWU in our study resulted in a rapid shift from a positive to a negative sap flow (Fig. 4A, B) and the microCT scans with contrast agent did not reveal embolism repair after 36 h (Figs 5 and 6), it seems less likely that longer AREs would induce embolism repair, even though repair can be a lengthy process, ranging from <1 h in Vitis riparia (Knipfer et al., 2016) up to 5–14 h in Cucumis sativus ‘Hurona’ (Scheenen et al., 2007), illustrating the need to repeat this experiment with varying lengths of leaf-wetting events.

Even though the minimum requirements for embolism repair indicated for coniferous species by Earles et al. (2016) (i.e. water source and supporting water potential gradient) were met and rehydration of subepidermal cells surrounding the main leaf vein was observed, no notable difference in the number of embolized vessels at petiole (Table 2) or leaf (Fig. 9) level were observed before and after AREs. As no embolisms were found in petioles during higher soil water potential conditions (−11 ± 0 kPa), possible artificial embolisms inherent to this technique (Cochard et al., 2000) are assumed to be absent in our dataset.

Embolism repair has been shown to be an energy-demanding process that requires an adequate supply of non-structural carbohydrates (Nardini et al., 2011; Trifilò et al., 2019). Even though non-structural carbohydrates are not always essential for embolism repair (Rolland et al., 2015), their limited occurrence in the stem during drought due to stomatal closure could explain why plants preferably rehydrate tissues in leaves (Fig. 9) and possibly the roots (Fig. 4) rather than invest in embolism repair. In this scenario, surrounding tissue would attract water because of its low osmotic water potential, resulting from an increased intercellular concentration of compounds due to dehydration. This led to the observed rehydration of subepidermal cells and increased thickness of the mesophyll by up to 28 % due to water absorption (Fig. 9).

Conclusions

Our set-up allowed us to indicate that beech seedlings are able to absorb water with their leaves and redistribute this water towards lower plant parts (Fig. 4). We were able to observe a predominant use of hydraulically active vessels from early xylem for this hydraulic redistribution and an apparent absence of embolism repair (Figs 5 and 6). When adding additional assessments, we found no degree of embolism repair at stem (Fig. 7, Table 1), petiole (Fig. 8, Table 2) or leaf (Fig. 9) level, irrespective of the degree of drought at the soil level. Thus, the suggested set-up succeeded in providing a confirmation of FWU, localizing hydraulically redistributed water absorbed by FWU and identifying a potential absence of embolism repair as a result of FWU. Thus, the proposed set-up allows a full assessment of the FWU–embolism repair hypothesis.

ACKNOWLEDGEMENTS

We thank Geert Favyts, Philip Deman, Erik Moerman and Benny Lewille for technical support. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract number DE-AC02-05CH11231. J.D.M.S. and K.S. conceived the research plans and designed the experiments; K.S. and C.B. supervised the experiments; J.D.M.S. performed the experiments; C.B., T.D.S., M.D. and A.R. provided technical assistance; J.D.M.S. analysed the data; J.D.M.S. wrote the article with contributions from all authors; K.S. and C.B. supervised and complemented the writing. The authors declare that there are no competing interests.

FUNDING

This work was supported by the Fund for Scientific Research–Flanders (FWO) through the PhD grant to J.D.M.S. (SB PhD fellow at FWO) and a travel grant allowing the research stay of J.D.M.S. at Yale University (V423619N). This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. The Hercules Foundation is acknowledged for its financial support in the acquisition of the scanning electron microscope JEOL JSM-7100F equipped with the cryo-transfer system Quorum PP3010T (grant number AUGE-09-029) used in this research.

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