TY - JOUR
AU - McElrone, Andrew J
AB - Abstract Drought decreases water transport capacity of leaves and limits gas exchange, which involves reduced leaf leaf hydraulic conductance (Kleaf) in both the xylem and outside-xylem pathways. Some literature suggests that grapevines are hyper-susceptible to drought-induced xylem embolism. We combined Kleaf and gas exchange measurements, micro-computed tomography of intact leaves, and spatially explicit modeling of the outside-xylem pathways to evaluate the role of vein embolism and Kleaf in the responses of two different grapevine cultivars to drought. Cabernet Sauvignon and Chardonnay exhibited similar vulnerabilities of Kleaf and gs to dehydration, decreasing substantially prior to leaf xylem embolism. Kleaf and gs decreased by 80% for both cultivars by Ψ leaf approximately –0.7 MPa and –1.2 MPa, respectively, while leaf xylem embolism initiated around Ψ leaf = –1.25 MPa in the midribs and little to no embolism was detected in minor veins even under severe dehydration for both cultivars. Modeling results indicated that reduced membrane permeability associated with a Casparian-like band in the leaf vein bundle sheath would explain declines in Kleaf of both cultivars. We conclude that during moderate water stress, changes in the outside-xylem pathways, rather than xylem embolism, are responsible for reduced Kleaf and gs. Understanding this mechanism could help to ensure adequate carbon capture and crop performance under drought. Anatomy, drought, ecophysiology, gas exchange, leaf hydraulics, micro-CT, modeling, Vitis Introduction Grapes are an economically important and widely cultivated crop worldwide, but vineyards that produce premium wine are most commonly located in regions characterized by dry and hot summers. Climate change is predicted to have a major impact on wine grape production due in part to increased crop water demands associated with higher evapotranspiration and increased water scarcity in arid growing regions (Diffenbaugh and Scherer, 2013; Hannah et al., 2013). Deficit irrigation strategies that purposefully induce water stress are also commonly used to control grapevine vigor and improve fruit quality. Wine grape cultivars exhibit contrasting responses to drought, which influences cultivar selection that will be a key factor for wine grape growing in a changing climate (Morales-Castilla et al., 2020). Cabernet Sauvignon (red wine grape) and Chardonnay (white wine grape) are the two most widely cultivated wine grapes worldwide, and are grown in contrasting environmental conditions. A better understanding of how water stress-induced changes in hydraulic traits impact grapevine physiology could help to predict tolerance of given cultivars in future climatic conditions. Leaf-level mechanisms are hypothesized to play an important role in the differential responses of grapevine cultivars to drought (Schultz, 2003). Grapevines exhibit long and wide xylem conduits that can extend from stems to leaf tips (Chatelet et al., 2006; Thorne et al., 2006), exacerbating the threat of hydraulic dysfunction due to water stress. Once water leaves the petiole, it flows through the veins from the lower to higher orders, finally reaching the free-ending veins in the leaf areole while also leaking radially across all veins along the way (Sack and Holbrook, 2006). To enter the outside-xylem pathways, water crosses the bundle sheath (BS) and flows across the mesophyll cells, evaporating into the intercellular airspace, and finally diffusing out of the stomata to the atmosphere (Rockwell et al., 2014; Buckley et al., 2017; Scoffoni et al., 2017b). Declining leaf hydraulic conductance (Kleaf) under drought stress can be driven not only by reduced xylem hydraulic conductance (Kx) but also by reduced outside-xylem hydraulic conductance (Kox). Declines in Kleaf and stomatal conductance (gs) during drought have been largely attributed to xylem embolism formation in several species including grapevines (Salleo et al., 2000; Nardini et al., 2001, 2003; Bucci et al., 2003; Trifilò et al., 2003a, b; Brodribb and Holbrook, 2004; Zufferey et al., 2011; Johnson et al., 2009, 2012). Similarly, a strong link between petiole xylem embolism and drought-induced leaf death was recently found in avocado trees (Cardoso et al., 2020), and Lovisolo et al. (2008) found that grapevine petioles lost ~80% of their hydraulic conductance due to embolism at leaf water potentials (Ψ leaf) of –1.4 MPa, but these results were obtained with hydraulic methods on excised leaves that have recently been questioned (e.g. McElrone et al., 2012; Torres-Ruiz et al., 2015). Hochberg et al. (2017a, 2019) used an optical method to examine embolism spread in grapevine leaves under drought, and found xylem to be more resistant to embolism than previous studies. Recent work in several other species from different ecological backgrounds showed that leaf veins are extremely resistant to drought-induced embolism formation and pointed towards the outside-xylem pathways as the driver of declines in Kleaf and gs during initial stages of water stress (Scoffoni et al., 2017a, b, 2018). Trifilò et al. (2016) found that the relative contribution of xylem versus outside-xylem pathways to declining Kleaf is species-specific across several studied species, but found also that both pathways contribute equally in a grapevine species. Direct measures of Kleaf combined with high-resolution visualization of embolism in intact leaves would help to further resolve their respective roles in hydraulic decline and their role in stomatal closure under drought stress. Work is needed to resolve incongruences in previous findings on grapevine in particular, given its central role in ongoing debates within the plant hydraulics community. Here, we compared leaf-level responses of Vitis vinifera L. cvs ‘Cabernet Sauvignon’ and ‘Chardonnay’ wine grapes to drought stress; the former is largely grown in warmer and drier regions, whereas the latter is more commonly cultivated in cooler, humid climates. In 2019, these two cultivars alone accounted for >39% of the cultivated wine grape acreage in California, which leads US production at ~85% of the country’s total. We used a combination of high-resolution X-ray micro-computed tomography (microCT) of intact leaves on potted plants subjected to different water potentials, light microscopy for anatomical comparisons, and physiological measurements such as Kleaf, gs, and turgor loss point to clarify the role of xylem embolism in grape leaves during dehydration. We also used a spatially explicit model of water transport outside-xylem (MOFLO 2.0; Buckley et al., 2017; Scoffoni et al., 2018) to elucidate the anatomical drivers of Kleaf during dehydration. Materials and methods Plant material Own-rooted V. vinifera L. cvs ‘Cabernet Sauvignon’ and ‘Chardonnay’ propagated from woody cuttings were obtained from the Foundation Plant Services (FPS, Davis, CA, USA) and grown in a greenhouse at the University of California, Davis. Greenhouse temperature was maintained between 16 °C and 32 °C, with a photoperiod of 15/9 h using supplemental light to maintain a stable photoperiod and a relative humidity of 40–80%. Plants were grown in 1.5 liter pots with a soil composed of 40% washed sand, 20% sphagnum peat moss, 20% redwood compost, and 20% pumice rock. Plants were irrigated daily with a diluted fertilized solution to maintain well-watered status, thus ensuring that they were grown in the same conditions. X-ray micro-computed tomography Leaves still attached to intact plants were scanned with high-resolution X-ray microCT imaging at the Lawrence Berkeley National Laboratory Advanced Light Source (ALS; Beamline 8.3.2) in July and August 2015. Each sample was scanned in the midrib, targeting the first branching of secondary veins after the petiole–lamina junction (see Supplementary Fig. S1 at JXB online). This location was chosen due to the importance of major veins for leaf hydraulics and function (Sack et al., 2003), especially in the basal portions of the leaf, so all midrib xylem conduits would be scanned before branching of conduits to secondary veins. Plants were allowed to naturally dehydrate in the greenhouse by discontinuing irrigation before being taken to the ALS, to achieve a range of water potentials. At the ALS, a leaf located a maximum of three nodes below the scanned leaf was sealed in a plastic bag lined with aluminum foil for at least 20 min to minimize transpiration and equilibrate the leaf with stem water potential, which was then measured with a pressure chamber (Plant Moisture Stress, Model 1000, Albany, OR, USA). The range of water potentials used in this study was between Ψ w= –0.3 MPa and Ψ w= –2 MPa, which is similar to values grapevines usually experience between Ψ w= –0.3 and Ψ w= –1.8 MPa in the field (Choat et al., 2010; Charrier et al., 2018). Levels of water stress have been previously defined by Smith and Prichard (2002) for several field-grown winegrape cultivars as: no stress Ψ w> –1 MPa; mild –1 MPa<Ψ w< –1.2 MPa; moderate –1.2 MPa<Ψ w< –1.4 MPa; and severe stress Ψ w< –1.6 MPa. A total of 22 samples of Cabernet Sauvignon ranging from Ψ w= –0.1 MPa to Ψ w= –2.8 MPa and 19 of Chardonnay spanning from Ψ w= –0.2 MPa to Ψ w= –2.9 MPa were taken. A subset of samples were used to evaluate embolism in all veins and tissues dimensions within the scanned field of view (FOV), which was on average 3.89 mm2 (± 0.15 SE, n=23). The pot was then bagged to maintain the plant at this water status to prepare the plants to be scanned. We then fixed a 2 mm copper wire to the leaf midrib with a piece of Kapton tape (DuPont, Wilmington, DE, USA). This wire served as an X-ray-dense reference point during the initial image preparation process to center the midrib in the middle of the FOV (Suppplementary Fig. S1). The leaf still attached to the intact plant was then placed between two half-cylinder pieces of styrofoam to be fixed in the center of the stage and the whole plant was placed inside a 9 cm diameter custom-made plexiglass cylinder. The plant and cylinder were then attached to the scanning stage. After centering the sample in the stage using the copper wire reference, the scan was performed between 10 min and 15 min depending on the area scanned. One single scan was performed per plant to eliminate potential effects associated with repeated X-ray scans on this soft tissue. The energy used was between 19 keV and 23 keV, and 1024 projection images were taken in continuous tomography mode, resulting in images with 1.27 µm3 voxel resolution. Image analysis for embolism quantification The resulting 2D projection images were then reconstructed in 3D in Fiji (FIJI-ImageJ version 1.46r; National Institutes of Health) using a custom plugin that utilizes Octopus 8.3 (Institute for Nuclear Sciences, University of Ghent, Belgium) to create a stack of images. The resulting image stacks were then evaluated in the transverse plane using Fiji to determine the presence of air-filled vessels in the midrib. Two datasets were analyzed, one from July 2015 and another from August 2015. The first dataset had 8 samples of Cabernet Sauvignon and 10 of Chardonnay, and only midrib embolisms were analyzed. In August, 14 samples of Cabernet Sauvignon and 9 of Chardonnay were obtained and their excellent resolution allowed an extensive 3D analysis of embolism in all veins of each scan and the measurement of cell dimensions to be used in our model. Each sample from August was extensively analyzed in 3D using visualization software Avizo 8.1.1 (VSG, Inc., Burlington, MA, USA). Analyses and leaf vein embolisms were quantified within the FOV in 2D and 3D using both software packages (i.e. Fiji and Avizo). The high resolution of the scans allowed us to determine whether xylem conduits were filled with water or air in each individual vein of all vein orders within the FOV. Each individual vein was examined in transverse cross-section, paradermal, and longitudinal views for the presence of embolisms. Measuring leaf hydraulic conductance (K leaf) K leaf was measured using the EFM protocol (Sack et al., 2002; Sack and Scoffoni, 2012; Scoffoni et al., 2012) with some adaptations. The night prior to performing measurements, potted plants grown in a greenhouse were watered until the soil was saturated, and placed inside a closed dark plastic bag filled with wet paper towels to maintain high humidity and avoid transpiration. The following morning, shoots were excised under water and submerged for 30–60 s. Shoots were recut under water from the end toward the tip until only four healthy mature leaves remained, which were bench-dried to achieve a range of water potentials. Leaves were then placed in plastic bags (Whirl-Pak, Nasco) that have been previously humidified (i.e. exhaled into) and the whole shoot was then placed inside a large bag with wet paper towels to avoid transpiration. After being allowed to equilibrate for at least 30 min, the water potential of both proximal and distal leaves was measured using a digital pressure chamber (Model 1000D, PMS Instruments) and, if the difference between water potential of both leaves was >0.2 MPa (or 0.3 MPa for very dehydrated shoots), then samples were discarded. One of the two leaves in the middle of the shoot was chosen for the flow measurements. The bagged leaf still attached to the shoot was excised in the stem–petiole junction with a fresh razor blade under ultrapure degassed water in a new sterilized Petri dish. Distilled water had been degassed with a vacuum pump (Gast, Bent Harbor, MI, USA) overnight on the day preceding measurements and then refiltered (0.22 µm, Syringe Filter, EMD Millipore, Billerica, MA, USA). This water was used for cutting the leaves under water in the Petri dishes and it was supplied to the leaves during the measurements. Parafilm was then wrapped around the base of the petiole and it was recut under water, where it was immediately inserted in tubing connected to a cylinder filled with ultrapure degassed water inside a ±10 µg sensitivity analytical balance (models MS205DU and AB135-S, Mettler Toledo, Columbus, OH, USA). Flow was measured as the rate of water loss in the cylinder. The sample leaf was then placed over a fan and under a light source (500 W Halogen Work Light, Ace Hardware, Oak Brook, IL, USA) providing photosynthetically active radiation (PAR) of 1100 µmol m–2 s–1 (±200 µmol m–2 s–1) as measured with a quantum sensor (190R, LiCor Biosciences, Lincoln, NE, USA). A water bath was placed between the leaf and the light to avoid excessive heat since leaf temperature should range between 23 °C and 28 °C to account for variations in water viscosity. The balance was connected to a data logger (CR1000, Campbell Scientific, Logan, UT, USA) and the weight of the water in the cylinder was logged every 30 s. When flow reached steady state (<5% coefficient of variation of the last 10 measurements and of the last five measurements for very low flow rates), leaf temperature was measured with a type E thermocouple placed on the abaxial side of the leaf. Steady-state flow rates had to be stable with no upwards or downwards trends, as shown in Supplementary Fig. S2, otherwise the sample was discarded. After stable steady-state flow was reached and temperature measured, the leaf was then quickly disconnected from the tubing, placed inside a humidified plastic bag, and the petiole tip was dabbed dry before placing it inside a larger bag with wet paper towels. After at least 30 min of equilibration, the final Ψ w was measured. Leaf area was determined as the average of five measurements taken using a leaf area meter (LI-3000C and LI-3050C, LiCor Biosciences). Kleaf was calculated as the average of the last 10 flow rate measurements and the last five samples with very low flow divided by the most negative Ψ w (i.e. between the average of two initials and the final), and normalized by leaf area and water viscosity at 25 °C. A vulnerability curve was generated for each cultivar, with 24 samples for Cabernet Sauvignon ranging from Ψ leaf= –0.31 MPa to Ψ leaf= –1.96 MPa and 23 samples for Chardonnay from Ψ leaf= –0.35 MPa to Ψ leaf= –2.07 MPa. Measuring stomatal conductance Stomatal conductance (gs) was measured using a portable gas exchange system with a red/blue light source and a CO2 mixing system (LI-6400XT; LiCor Biosciences). Measurements were taken in the morning between 09.00 h and 12.00 h, and in the afternoon between 15.00 h and 18.00 h in the greenhouse in 12 individuals of each cultivar. Plants were well watered on the first day of measurements and irrigation was discontinued during the experiment so plants dehydrated naturally until gs approached zero, when the experiment was terminated. Two plants of each cultivar were kept as controls and hydrated daily until soil saturation. A young fully expanded leaf per plant was chosen on the first day and measured repeatedly throughout the experiment. Cuvette CO2 was set at 400 ppm, PAR of 1100 µmol m–2 s–1, similar to what was used to measure Kleaf, block relative humidity between 30% and 50%, and block temperature set at 25 °C. After cuvette conditions stabilized and gs reached <1% of the coefficient of variation, data were logged five times between 30 s and 60 s, and their average was the final value. Ψ stem was taken immediately after each measurement using a pressure chamber (Model 1000D, PMS Instruments) in a leaf distal to the sampled leaf and that had been previously bagged in a plastic bag lined with aluminum foil for at least 20 min prior to being measured. Measuring turgor loss point Pressure/volume curves were obtained from one mature leaf of five different plants of each cultivar following the protocol detailed in Sack et al. (2011). The ‘squeeze’ method was used with some modifications. Intact potted plants were rehydrated the night before measurements as for Kleaf measurements (see above). In the morning, a mature healthy leaf was chosen, excised from the stem with a razor blade under water, and allowed to rehydrate for 2 min. The petiole was then carefully dried with paper tissue to avoid overestimation of water content and the leaf was equilibrated in a sealed Whirl-Pak bag previously exhaled into within a larger sealed bag with wet paper towels, and allowed to equilibrate for at least 30 min. Parafilm was wrapped around the petiole to prevent damage and prevent gas through the pressure chamber (Model 1000D, PMS Instruments) seal ring leaking during water potential measurements. The first water potential was measured and the pressure inside the chamber was held constant for 15 s after water exudation from the petiole ceased, after which the petiole tip was carefully dried with paper tissue and the pressure in the chamber was slowly released, not exceeding a rate of 0.005 MPa s–1. The bagged leaf was weighed on an analytical balance (±0.1 mg; model LA230S, Sartorius, Goettingen, Germany) and then replaced in the pressure chamber. The first water potential measurement was determined as the fresh weight measurement. This procedure was repeated approximately every 0.2 MPa interval until no water was coming out of the petiole for 20 s. Finally, the leaf was placed in an oven for 48 h at 80 °C for measurement of dry weight. Modeling the effect of anatomical changes during dehydration on water transport outside the xylem We used a spatially explicit model of outside-xylem water transport (MOFLO 2.0; Buckley et al., 2017), driven by anatomical measurements obtained from light microscopy and microCT scans, to elucidate the drivers of declining leaf hydraulic conductance during dehydration. For fully hydrated leaves, we assumed that membrane osmotic permeability to water (Pm) was equal to the default value used previously in this model (40 µm s–1), whereas, for dehydrated leaves, we compared simulations with and without an 80% reduction in Pm hypothetically caused by dehydration (i.e. Pm of either 40 µm s–1 or 8 µm s–1). For all leaves, we compared simulations with and without apoplastic water transport across the BS blocked by a hydrophobic Casparian-like barrier in the BS cell walls. Light microscopy of cells and tissues within leaves Measurements of cells and tissues of leaves (Supplementary Table S1) were taken from 2D leaf cross-section light microscopy images obtained following a previously used protocol (John et al., 2013). Three different leaves from each cultivar were used to make the measurements. Samples were then sectioned using glass knives at 0.5 μm thickness in a rotary microtome. Sections were stained in 0.01% toluidine blue in 1% sodium borate. Images observed in the microscope (Leica M205C, Wetzlar, Germany) were acquired with a digital microscope camera (Leica DFC 425) attached to it using ×10, ×20, and ×40 magnifications, and captured with the Leica Application Suite Software (Leica Microsystems CMS GmbH). Measurements were taken from images at ×20 magnification using Fiji (FIJI-ImageJ version 1.46r; National Institutes of Health) with a 2.7767 pixel µm–1 resolution and were obtained from three randomly chosen locations within the mesophyll in both sides of the midrib. MicroCT scans during dehydration Measurements were obtained from 2D cross-sectional images of slices of the same scans used for embolism quantification. Using Fiji-ImageJ across each scan analyzed, cell thicknesses of the epidermis (upper and lower), and palisade and spongy mesophylls were randomly measured in five cells from three slices (i.e. cross-sectional images) along the stack of images on both sides of the midrib (i.e. the midrib was in the center of scans as shown in Fig. 1). Leaf porosity in both palisade and spongy mesophylls was measured in five slices across the same stack of images and on both sides of the midrib. Fig. 1. Open in new tabDownload slide Leaf midrib of Vitis vinifera L. cv. Cabernet Sauvignon. (A) Light microscopy showing vascular bundles (VB) and a portion of mesophyll. (B) MicroCT slice of midrib 2D cross-section with embolized conduits (darker) and a portion of the mesophyll. Note that water-filled conduits in the vascular bundle can be seen by their brighter outline. (C) 3D reconstruction of the same leaf in (B) showing how embolism was analyzed. Samples were analyzed in 3D in all planes (x,y,z), allowing us to fully visualize the leaf xylem network, including each vein individually and its connectedness. Only embolized conduits are represented. The arrow in (B) and (C) is pointing to the same embolized conduit. Xylem branching and embolism spread to the secondary vein can be visualized in this conduit as it bends towards the left side while others in the same vascular bundle continue in a straight direction in the midrib. Scale bar=200 µm. (This figure is available in color at JXB online.) Fig. 1. Open in new tabDownload slide Leaf midrib of Vitis vinifera L. cv. Cabernet Sauvignon. (A) Light microscopy showing vascular bundles (VB) and a portion of mesophyll. (B) MicroCT slice of midrib 2D cross-section with embolized conduits (darker) and a portion of the mesophyll. Note that water-filled conduits in the vascular bundle can be seen by their brighter outline. (C) 3D reconstruction of the same leaf in (B) showing how embolism was analyzed. Samples were analyzed in 3D in all planes (x,y,z), allowing us to fully visualize the leaf xylem network, including each vein individually and its connectedness. Only embolized conduits are represented. The arrow in (B) and (C) is pointing to the same embolized conduit. Xylem branching and embolism spread to the secondary vein can be visualized in this conduit as it bends towards the left side while others in the same vascular bundle continue in a straight direction in the midrib. Scale bar=200 µm. (This figure is available in color at JXB online.) Vein length per leaf area Vein length per leaf area (VLA) was measured on minor veins of both cultivars from the microCT scans using the digital tracing tool in the software Dragonfly [Object Research Software (ORS) Inc., Montreal, Canada]. VLA was measured in a subregion of the scanned FOV (0.48 mm2, ±0.04 SE, n=23), which permitted an accurate measurement of minor veins on a small region of the leaf lamina. A 3D analysis was performed on the sampled region to identify all the veins as well as its connections and diameters along the axes (x,y,z) to determine vein orders. When all veins were identified, their lengths were measured using digital tracing. Statistics Both Kleaf and gs curves were constructed after being tested for the best fit with models previously used in the literature (Scoffoni et al., 2017b). Four functions were tested: a linear function; a three-parameter sigmoidal function; a logistic function; and an exponential function. The y in the equations represents Kleaf or gs, and Ψ w the water potential. The function choice was based on the lowest Akaike information criterion (AIC) corrected for low n and, if the difference was <2, the function with higher r2 was selected as the maximum likelihood. Plots were constructed using SigmaPlot v.13 (Systat Software, San Jose, CA, USA). A best fit curve was generated for embolism of midrib veins for each cultivar, with 24 samples for Cabernet Sauvignon ranging from Ψ leaf= –0.31 MPa to Ψ leaf= –1.96 MPa and 23 samples for Chardonnay from Ψ leaf= –0.35 MPa to Ψ leaf= –2.07 MPa. Results Drought-induced embolism in grapevine leaves was primarily restricted to the midrib and occurred after the turgor loss point when Kleaf and gs declined close to zero. MicroCT scans of intact leaves still attached to drying plants showed little to no embolism at tensions considered as severe water stress for wine grapes (Figs 2, 3) according to a previously established classification for field-grown V. vinifera (see the Materials and methods). Fig. 2. Open in new tabDownload slide Cabernet Sauvignon midrib (left column) and mesophyll (right column) microCT cross-sections of intact plants showing low vulnerability to embolism formation during drought at different stem water potentials. Each panel is a different leaf as each of them was scanned once only. Each panel show a microCT slice of a midrib cross-section with darker embolized conduits (left) and a portion of the mesophyll with a minor vein (right). Water-filled conduits can be seen by their brighter outline. (A) Control showing no embolism. (B) No embolism in the midrib but with reduced leaf thickness and cells beginning to shrink. (C) As dehydration progresses, midrib embolism initiates and mesophyll continues shrinking. (D) Midrib severely embolized, no minor vein embolism. Midrib scale=200 µm. Mesophyll scale=150 µm. Fig. 2. Open in new tabDownload slide Cabernet Sauvignon midrib (left column) and mesophyll (right column) microCT cross-sections of intact plants showing low vulnerability to embolism formation during drought at different stem water potentials. Each panel is a different leaf as each of them was scanned once only. Each panel show a microCT slice of a midrib cross-section with darker embolized conduits (left) and a portion of the mesophyll with a minor vein (right). Water-filled conduits can be seen by their brighter outline. (A) Control showing no embolism. (B) No embolism in the midrib but with reduced leaf thickness and cells beginning to shrink. (C) As dehydration progresses, midrib embolism initiates and mesophyll continues shrinking. (D) Midrib severely embolized, no minor vein embolism. Midrib scale=200 µm. Mesophyll scale=150 µm. Fig. 3. Open in new tabDownload slide Chardonnay midrib (left column) and mesophyll (right column) microCT cross-sections of intact plants showing low vulnerability to embolism formation during drought at different stem water potentials. Each panel is a different leaf as each one was scanned once only. Each panel show a microCT slice of a midrib cross-section with darker embolized conduits (left) and a portion of the mesophyll with a minor vein (right). Water-filled conduits can be seen by their brighter outline. (A) Control showing no embolism. (B) No embolism in the midrib but with reduced leaf thickness and cells beginning to shrink. (C) As dehydration progresses, midrib embolism initiates and more conduits embolize. (D) Midrib severely embolized, no minor vein embolism. Midrib scale=200 µm. Mesophyll scale=150 µm. Fig. 3. Open in new tabDownload slide Chardonnay midrib (left column) and mesophyll (right column) microCT cross-sections of intact plants showing low vulnerability to embolism formation during drought at different stem water potentials. Each panel is a different leaf as each one was scanned once only. Each panel show a microCT slice of a midrib cross-section with darker embolized conduits (left) and a portion of the mesophyll with a minor vein (right). Water-filled conduits can be seen by their brighter outline. (A) Control showing no embolism. (B) No embolism in the midrib but with reduced leaf thickness and cells beginning to shrink. (C) As dehydration progresses, midrib embolism initiates and more conduits embolize. (D) Midrib severely embolized, no minor vein embolism. Midrib scale=200 µm. Mesophyll scale=150 µm. Both cultivars exhibited similar sigmoidal vulnerability curves, with a sharp increase in midrib embolized conduits below Ψ w= –1.5 MPa, with 25/41 samples exhibiting midrib embolisms (Fig. 5). Cabernet Sauvignon showed slightly greater vulnerability than Chardonnay at embolism inception, having five midrib conduits embolized at Ψ w= –1.35 MPa, while Chardonnay reached the same number at Ψ w= –1.65 MPa. As tensions increased, both cultivars showed less difference, with Chardonnay exhibiting a steeper slope, reaching 20 midrib embolized conduits at Ψ w= –1.91 MPa, while Cabernet Sauvignon had 20 midrib embolized conduits at Ψ w= –2 MPa. Even though this increase occurred primarily in the midrib, in both cultivars some additional conduits embolized in the secondary veins at more negative Ψ w; in 8/23 samples embolisms in the secondary veins were found and in all cases they were connected to embolisms in the midrib, showing clear connections between the two vein orders (Figs 1, 4). In 5/23 samples, only one secondary vein was embolized and at different water potentials (including one control), while the other three samples were severely dehydrated (Fig. 4) when embolisms appeared in this vein order. No clear pattern of which vascular bundles in the midrib would embolize first was observed; however, all embolisms found in secondary veins originated from the lateral vascular bundles of the midrib. Embolisms appeared in tertiary veins for only 4/23 samples analyzed, with only one that was not water stressed. In one sample of Cabernet Sauvignon at Ψ w= –1.8 MPa, clear connections of embolisms were found in the three major veins (Fig. 4). Embolisms in minor veins (i.e. from the fourth to the sixth, including free-ending veins) only occurred at Ψ w< –1.25 MPa and were rare, despite the very negative Ψ w that some samples exhibited, ranging from –1.8 MPa to –2.8 MPa. Among these, the only sample that showed embolized conduits was at Ψ w= –2.8 MPa, exhibiting a total of nine embolized conduits in the fourth-order vein and one isolated embolism in a sixth-order vein (Fig. 4). No embolism was observed in the fifth-order vein of any sample. Two samples exhibited one isolated embolized conduit each in the fourth-order vein, and both had Ψ w> –1.5 MPa. Fig. 4. Open in new tabDownload slide Embolism during severe dehydration in leaves of Vitis vinifera cv. Cabernet Sauvignon. Paradermal view of 3D reconstructed grapevine leaves showing embolized xylem conduits and leaf venation (A–D). First embolisms begin to occur from around –1.3 MPa and become more common as water potential becomes more negative. Embolism occurs first in the midrib and then spreads to higher order veins through xylem branching, showing a high degree of connectivity between vessels. Water-filled xylem conduits are not shown. Panels (E–G) are from the same sample shown in (A). (E) Embolized conduit in the midrib with intervessel pit in detail as shown in (A) (arrow). (F) Isolated embolized conduit in a minor vein (fourth order) shown by the arrow, same as seen in (A). Scale bar=50 µm. Embolized conduits are dark, while water-filled are lighter and not always visible. (G) Longitudinal view of the section represented by the line in (A) showing lack of embolisms in different vein orders with a cross-section of a tertiary vein seen in detail (bright arrow). Panels (H–J) are from the same sample shown in (B). (H) 3D cross-section showing embolism spreading from the midrib to a secondary (darker arrow) and tertiary (brighter arrow) vein via xylem connectivity. (I) Free-ending veins (darker arrow) embedded in the mesophyll. (J) Longitudinal view of the section represented by the transverse line in (B), showing lack of embolisms in different vein orders. Panels (K–L) are from the same sample shown in (C). (K) Detail of a vessel element indicated by the darker arrow in (C). Scale bar=1 µm. (L) Longitudinal view of the section represented by the line in (C), showing lack of embolisms in different vein orders. Panels (M–O) are from the same sample shown in (D). (M) Free-ending veins inside the same areole of (D), indicated by darker arrows. (N) Detail of isolated embolism in tertiary and fourth-order veins indicated by the bright arrow in (D). The full embolism extent was comprised within the area shown. (O) Longitudinal view of the section represented by the line in (D), showing lack of embolisms in different vein orders. Scale bars=100 µm, except for (F) and (K). (This figure is available in color at JXB online.) Fig. 4. Open in new tabDownload slide Embolism during severe dehydration in leaves of Vitis vinifera cv. Cabernet Sauvignon. Paradermal view of 3D reconstructed grapevine leaves showing embolized xylem conduits and leaf venation (A–D). First embolisms begin to occur from around –1.3 MPa and become more common as water potential becomes more negative. Embolism occurs first in the midrib and then spreads to higher order veins through xylem branching, showing a high degree of connectivity between vessels. Water-filled xylem conduits are not shown. Panels (E–G) are from the same sample shown in (A). (E) Embolized conduit in the midrib with intervessel pit in detail as shown in (A) (arrow). (F) Isolated embolized conduit in a minor vein (fourth order) shown by the arrow, same as seen in (A). Scale bar=50 µm. Embolized conduits are dark, while water-filled are lighter and not always visible. (G) Longitudinal view of the section represented by the line in (A) showing lack of embolisms in different vein orders with a cross-section of a tertiary vein seen in detail (bright arrow). Panels (H–J) are from the same sample shown in (B). (H) 3D cross-section showing embolism spreading from the midrib to a secondary (darker arrow) and tertiary (brighter arrow) vein via xylem connectivity. (I) Free-ending veins (darker arrow) embedded in the mesophyll. (J) Longitudinal view of the section represented by the transverse line in (B), showing lack of embolisms in different vein orders. Panels (K–L) are from the same sample shown in (C). (K) Detail of a vessel element indicated by the darker arrow in (C). Scale bar=1 µm. (L) Longitudinal view of the section represented by the line in (C), showing lack of embolisms in different vein orders. Panels (M–O) are from the same sample shown in (D). (M) Free-ending veins inside the same areole of (D), indicated by darker arrows. (N) Detail of isolated embolism in tertiary and fourth-order veins indicated by the bright arrow in (D). The full embolism extent was comprised within the area shown. (O) Longitudinal view of the section represented by the line in (D), showing lack of embolisms in different vein orders. Scale bars=100 µm, except for (F) and (K). (This figure is available in color at JXB online.) Fig. 5. Open in new tabDownload slide Number of embolized xylem conduits per vein order in intact leaves of grapevine during drought, showing that embolisms initiated in the midrib and spread to higher order veins with increasing tensions in the xylem. Analyses were performed in full 3D reconstructions, so all veins could be individually analyzed. (A) Cabernet Sauvignon showing initiation of xylem embolism around Ψ leaf= –1.3 MPa in the midrib with embolisms spreading to the secondary vein at higher tensions. Few embolisms were observed in higher order veins and only at very negative water potential values. Only one embolized conduit was found in a sixth-order vein (i.e. free-ending vein) and in the most water-stressed sample (Ψ leaf= –2.8 MPa). (B) Chardonnay leaves showed slightly less initial vulnerability to drought-induced xylem embolism formation than Cabernet Sauvignon, with midrib embolism inception at more negative water potentials. Midrib embolism greatly increased for both cultivars when Ψ leaf was ≤ –1.5 MPa with a steeper slope for Chardonnay. The most stressed Chardonnay sample with resolution to resolve for minor vein embolisms was at Ψ leaf= –1.6 MPa. Best fitted curves are only for midrib embolisms and were three-parameter sigmoidal. For Cabernet Sauvignon y=59.2179/<1+e{–[Ψw–(2.2492)]/(–0.3760)}> and for Chardonnay y=59.3279/<1+e{–[Ψw–(2.0151)]/(–0.1496)}>. Statistical data are shown in each panel. For embolism analysis in midribs, n=41. A subset of n=23 was analyzed for embolisms in all vein orders within each scan (see the Materials and Methods for further details). The dashed vertical line represents Ψ TLP and the gray shadowed area the SE. First embolisms occurred only at around or past Ψ TLP. Fig. 5. Open in new tabDownload slide Number of embolized xylem conduits per vein order in intact leaves of grapevine during drought, showing that embolisms initiated in the midrib and spread to higher order veins with increasing tensions in the xylem. Analyses were performed in full 3D reconstructions, so all veins could be individually analyzed. (A) Cabernet Sauvignon showing initiation of xylem embolism around Ψ leaf= –1.3 MPa in the midrib with embolisms spreading to the secondary vein at higher tensions. Few embolisms were observed in higher order veins and only at very negative water potential values. Only one embolized conduit was found in a sixth-order vein (i.e. free-ending vein) and in the most water-stressed sample (Ψ leaf= –2.8 MPa). (B) Chardonnay leaves showed slightly less initial vulnerability to drought-induced xylem embolism formation than Cabernet Sauvignon, with midrib embolism inception at more negative water potentials. Midrib embolism greatly increased for both cultivars when Ψ leaf was ≤ –1.5 MPa with a steeper slope for Chardonnay. The most stressed Chardonnay sample with resolution to resolve for minor vein embolisms was at Ψ leaf= –1.6 MPa. Best fitted curves are only for midrib embolisms and were three-parameter sigmoidal. For Cabernet Sauvignon y=59.2179/<1+e{–[Ψw–(2.2492)]/(–0.3760)}> and for Chardonnay y=59.3279/<1+e{–[Ψw–(2.0151)]/(–0.1496)}>. Statistical data are shown in each panel. For embolism analysis in midribs, n=41. A subset of n=23 was analyzed for embolisms in all vein orders within each scan (see the Materials and Methods for further details). The dashed vertical line represents Ψ TLP and the gray shadowed area the SE. First embolisms occurred only at around or past Ψ TLP. K leaf appeared as very sensitive to dehydration for both cultivars, as the Ψ w at which Kleaf was lost by 50% (P50Kleaf) was –0.30 MPa for Cabernet Sauvignon and –0.29 MPa for Chardonnay, and 80% loss of Kleaf (P80Kleaf) occurred at Ψ w= –0.71 MPa and Ψ w= –0.67 MPa, respectively (Table 2; Fig. 6). These vulnerabilities of Kleaf to dehydration were mainly driven by the high maximum values of Kleaf at 0 MPa, which rarely occur in grapevines under natural conditions (Williams and Araujo, 2002; Williams and Baeza, 2007; Bota et al., 2016; Charrier et al., 2018). Considering Ψ w= –0.3 MPa as the highest value, the maximum Kleaf is 7.32 mmol H2O m–2 s–1 Pa–1 and 6.83 mmol m–2 s–1 MPa–1 for Cabernet Sauvignon and Chardonnay, respectively, and their vulnerabilities values change to P50Kleaf of Ψ w= –0.61 MPa and Ψ w= –0.56 MPa and P80Kleaf of Ψ w= –1.02 MPa and Ψ w= –0.95 MPa, respectively (Table 2). Table 2. Values of maximum Kleaf and gs for the less negative water potential considered in the vulnerabilities curves (Ψ highest), at either 0 MPa or –0.3 MPa Vitis vinifera cvs . Ψhighest (MPa) . K leaf . . . g s . . . ΨTLP . . . K leaf maximum . P50Kleaf . P80Kleaf . g s maximum . P50gs . P80gs . . . . (mmol H2O m–2 s–1 MPa–1) . (MPa) . (MPa) . (mol H2O m–2 s–1) . (MPa) . (MPa) . (MPa) . Cabernet Sauvignon 0 14.52 –0.30 –0.71 0.279 –0.60 –1.21 –1.37±0.05 –0.3 7.32 –0.61 –1.02 0.199 –0.85 –1.37 Chardonnay 0 13.90 –0.29 –0.67 0.153 –0.53 –1.17 -1.49±0.06 –0.3 6.83 –0.56 –0.95 0.104 –0.81 –1.40 Vitis vinifera cvs . Ψhighest (MPa) . K leaf . . . g s . . . ΨTLP . . . K leaf maximum . P50Kleaf . P80Kleaf . g s maximum . P50gs . P80gs . . . . (mmol H2O m–2 s–1 MPa–1) . (MPa) . (MPa) . (mol H2O m–2 s–1) . (MPa) . (MPa) . (MPa) . Cabernet Sauvignon 0 14.52 –0.30 –0.71 0.279 –0.60 –1.21 –1.37±0.05 –0.3 7.32 –0.61 –1.02 0.199 –0.85 –1.37 Chardonnay 0 13.90 –0.29 –0.67 0.153 –0.53 –1.17 -1.49±0.06 –0.3 6.83 –0.56 –0.95 0.104 –0.81 –1.40 Water potentials at which Kleaf and gs declined by 50% (P50) and 80% (P80) varied depending on the Ψ highest. Turgor loss points (Ψ TLP) with SE (n=5) are shown in the last column Open in new tab Table 2. Values of maximum Kleaf and gs for the less negative water potential considered in the vulnerabilities curves (Ψ highest), at either 0 MPa or –0.3 MPa Vitis vinifera cvs . Ψhighest (MPa) . K leaf . . . g s . . . ΨTLP . . . K leaf maximum . P50Kleaf . P80Kleaf . g s maximum . P50gs . P80gs . . . . (mmol H2O m–2 s–1 MPa–1) . (MPa) . (MPa) . (mol H2O m–2 s–1) . (MPa) . (MPa) . (MPa) . Cabernet Sauvignon 0 14.52 –0.30 –0.71 0.279 –0.60 –1.21 –1.37±0.05 –0.3 7.32 –0.61 –1.02 0.199 –0.85 –1.37 Chardonnay 0 13.90 –0.29 –0.67 0.153 –0.53 –1.17 -1.49±0.06 –0.3 6.83 –0.56 –0.95 0.104 –0.81 –1.40 Vitis vinifera cvs . Ψhighest (MPa) . K leaf . . . g s . . . ΨTLP . . . K leaf maximum . P50Kleaf . P80Kleaf . g s maximum . P50gs . P80gs . . . . (mmol H2O m–2 s–1 MPa–1) . (MPa) . (MPa) . (mol H2O m–2 s–1) . (MPa) . (MPa) . (MPa) . Cabernet Sauvignon 0 14.52 –0.30 –0.71 0.279 –0.60 –1.21 –1.37±0.05 –0.3 7.32 –0.61 –1.02 0.199 –0.85 –1.37 Chardonnay 0 13.90 –0.29 –0.67 0.153 –0.53 –1.17 -1.49±0.06 –0.3 6.83 –0.56 –0.95 0.104 –0.81 –1.40 Water potentials at which Kleaf and gs declined by 50% (P50) and 80% (P80) varied depending on the Ψ highest. Turgor loss points (Ψ TLP) with SE (n=5) are shown in the last column Open in new tab Fig. 6. Open in new tabDownload slide Leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) of Cabernet Sauvignon (A–C) and Chardonnay (B–D) during dehydration. Cabernet Sauvignon exhibited greater vulnerability to dehydration; however, both cultivars reached P80 before the initiation of embolism events. Both cultivars showed similar Kleaf values along the dehydration range (A, B). Cabernet Sauvignon showed absolute gs values around twice as high as Chardonnay along the curve, indicating that even at the same water stress level it has twice the amount of gas exchange as Chardonnay. Best fitted curves were three-parameter exponential: y=0.071 + 14.453e–(–2.302)Ψw, n=24 (A); y= –0.155 + 14.060e–(–2.331)Ψw, n=23 (B); y= –0.061 + 0.334e–(–0.884)Ψw, n=102 (C), and y= –0.011 + 0.1634e–(–1.180)Ψw, n=94 (D). Statistical data are shown in each panel. The dashed vertical line represents Ψ TLP and the gray shadowed area the SE. Fig. 6. Open in new tabDownload slide Leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) of Cabernet Sauvignon (A–C) and Chardonnay (B–D) during dehydration. Cabernet Sauvignon exhibited greater vulnerability to dehydration; however, both cultivars reached P80 before the initiation of embolism events. Both cultivars showed similar Kleaf values along the dehydration range (A, B). Cabernet Sauvignon showed absolute gs values around twice as high as Chardonnay along the curve, indicating that even at the same water stress level it has twice the amount of gas exchange as Chardonnay. Best fitted curves were three-parameter exponential: y=0.071 + 14.453e–(–2.302)Ψw, n=24 (A); y= –0.155 + 14.060e–(–2.331)Ψw, n=23 (B); y= –0.061 + 0.334e–(–0.884)Ψw, n=102 (C), and y= –0.011 + 0.1634e–(–1.180)Ψw, n=94 (D). Statistical data are shown in each panel. The dashed vertical line represents Ψ TLP and the gray shadowed area the SE. Similar to Kleaf, gs declined exponentially during dehydration in both cultivars (Fig. 6), with Cabernet Sauvignon exhibiting almost double the maximum gs of Chardonnay at Ψ w=0 MPa (0.28 mol H2O m–2 s–1 and 0.15 mol H2O m–2 s–1). The cultivars showed similar decreases of gs during dehydration, with P50gs of –0.60 MPa and –0.53 MPa and P80gs of –1.21 MPa and –1.17 MPa for Cabernet Sauvignon and Chardonnay, respectively. Starting the curve at Ψ w= –0.3 MPa, the maximum values of gs were 0.20 mol H2O m–2 s–1 and 0.10 mol H2O m–2 s–1 for Cabernet Sauvignon and Chardonnay, respectively, which also changed their vulnerability values. Cabernet Sauvignon thus exhibited P50gs= –0.85 MPa and P80gs= –1.37 MPa, while Chardonnay showed P50gs= –0.81 MPa and P80gs= –1.40 MPa (Table 2). Kleaf was more vulnerable to dehydration than gs for both cultivars and scenarios, with the offset between P50Kleaf and P50gs being ~0.25 MPa in all situations (Table 2). This difference slightly increased as Ψ w became more negative, being ~0.4 MPa between P80Kleaf and P80gs. The turgor loss point (Ψ TLP) was slightly less negative for Cabernet Sauvignon than for Chardonnay (Ψ TLP= –1.37 MPa ±SE 0.05 and Ψ TLP= –1.49 MPa ±SE 0.06, respectively). Our model simulations showed that measured changes in leaf thickness, VLA, and porosity, the anatomical parameters that can influence MOFLO 2.0 during dehydration (Supplementary Table S2), did not explain the observed hydraulic declines in either cultivar. In fact, Cabernet Sauvignon and Chardonnay exhibited contrasting responses to our model simulations; however, the response to these anatomical changes in both cultivars was only slight. Kox would increase in Cabernet Sauvignon and slightly decrease in Chardonnay during dehydration, mainly driven by changes in VLA, which increased in the former and decreased in the latter. Even with the simulated reduction in Kox for Chardonnay, anatomical changes were not enough to account for the observed hydraulic decline. We found that, in order to explain the observed declines in Kleaf of both cultivars, it was necessary to assume an 80% reduction in membrane permeability combined with a Casparian-like band in the BS cell walls (Fig. 7, bottom panel). Fig. 7. Open in new tabDownload slide The model MOFLO 2.0 was used to simulate the anatomical effects of dehydration on the outside-xylem hydraulic conductance (Kox). Cabernet Sauvignon (A) showed a slight increase in Kox during dehydration while Chardonnay (B) exhibited a slight reduction in Kox with dehydration. The anatomical changes were mainly driven by leaf shrinkage and did not explain the observed leaf hydraulic declines for either cultivar shown previously. However, if membrane permeability, Pm, is assumed to decline by 80% (‘–Pm’) at low water potential, and if all water transport across the bundle sheath is assumed to be forced across membranes by a hydrophobic cell wall barrier (Casparian strip; ‘+Casp’), then Kox is predicted to decline as observed experimentally (bottom plot). As xylem embolism only occurred after Kleaf was lost and Kleaf is composed of both Kx and Kox, and the model is for Kox only, we assume that Kleaf is the same as Kox. Fig. 7. Open in new tabDownload slide The model MOFLO 2.0 was used to simulate the anatomical effects of dehydration on the outside-xylem hydraulic conductance (Kox). Cabernet Sauvignon (A) showed a slight increase in Kox during dehydration while Chardonnay (B) exhibited a slight reduction in Kox with dehydration. The anatomical changes were mainly driven by leaf shrinkage and did not explain the observed leaf hydraulic declines for either cultivar shown previously. However, if membrane permeability, Pm, is assumed to decline by 80% (‘–Pm’) at low water potential, and if all water transport across the bundle sheath is assumed to be forced across membranes by a hydrophobic cell wall barrier (Casparian strip; ‘+Casp’), then Kox is predicted to decline as observed experimentally (bottom plot). As xylem embolism only occurred after Kleaf was lost and Kleaf is composed of both Kx and Kox, and the model is for Kox only, we assume that Kleaf is the same as Kox. Discussion We present direct evidence that xylem embolism formation is not responsible for the coordinated decline in Kleaf and gs in grapevines during drought. Neither Cabernet Sauvignon nor Chardonnay exhibited drought-induced embolism before substantial loss of Kleaf and gs occurred, pointing to the outside-xylem as being mainly responsible for increased hydraulic resistance and stomatal closure during leaf dehydration. Moreover, drought-induced leaf vein embolism initiated below Ψ TLP, indicating that the turgor loss was also not affected by embolism formation. We also identified patterns of embolism spread in the highly connected leaf xylem network of grapevines, while our model simulations of the outside-xylem pathways indicated that membrane permeability of the BS combined with the presence of an apoplastic barrier would account for the observed hydraulic decline during dehydration. This is the first study combining both direct embolism observation and leaf hydraulics during dehydration in grapevines, showing that their leaf xylem network is extremely resistant to drought-induced embolism in intact grapevine leaves of two different cultivars. Despite the importance of xylem integrity for hydraulic function and its impact on gas exchange and crop performance, so far the only study combining hydraulics and direct embolism observation was conducted on wheat by Corso et al. (2020) (Table 1). Xylem embolism has been commonly assigned as an important early response to dehydration in grapevines and is responsible for triggering stomatal closure (Zufferey et al., 2011; Jacobsen and Pratt, 2012). However, recent studies also noted the outside-xylem water pathways as the drivers of Kleaf decline during drought rather than leaf xylem embolism in several species (Bouche et al., 2016; Trifilò et al., 2016; Scoffoni et al., 2017b, 2018; Corso et al., 2020). The idea that V. vinifera stems and petioles are highly susceptible to embolism and the cause for stomatal closure in wine grape cultivars (Zufferey et al., 2011; Jacobsen and Pratt, 2012) has been challenged by other studies showing that they are not as vulnerable to dehydration-induced embolism in both organs (Choat et al., 2010; Brodersen et al., 2013; Hochberg et al., 2015b) as well as leaf laminas (Hochberg et al., 2017a). Petiole embolisms only occurred at tensions rarely experienced under field conditions and are more vulnerable to embolism events than stems, with embolism appearing in petioles at a Ψ w of approximately –1.1 MPa (Hochberg et al., 2015a). Recent findings with the same cultivars as we investigated herein showed that the inflection point for loss of hydraulic conductivity in petioles of Cabernet Sauvignon was around Ψ w= –1.0 MPa (Hochberg et al., 2015b) while embolism inception in Chardonnay leaves occurred at a Ψ w of approximately –1.4 MPa and after stomatal closure (Hochberg et al., 2017a). Our results are in good agreement with those studies in both embolism thresholds and shape of the curves; as xylem conduits of grapevine petioles extend to the midrib (Chatelet et al., 2006; Thorne et al., 2006), it is expected that petioles and midribs would exhibit similar vulnerabilities to embolism. The mild discrepancies in embolism thresholds between studies may be due to differences in growing conditons and/or plant size. Moreover, we found in both cultivars that embolism initiated after stomatal closure. Table 1. Meta-data of studies on crop species showing water potentials at which embolisms were initially observed during dehydration and Kleaf vulnerabilities to drought Authors . Species . Ψw of initial leaf xylem embolism observation (MPa) . K leaf . . . . . P50Kleaf . P80Kleaf . This study V. vinifera cv. Cabernet Sauvignon –1.3 –0.3 –0.71 V. vinifera cv. Chardonnay –1.2 –0.29 –0.67 Hochberg et al. (2019) V. vinifera cv. Chardonnay –0.45 >Ψ w> –1.4a – – Hochberg et al. (2017a) V. vinifera cv. Chardonnay < –1.4 – – Hochberg et al. (2017b) V. vinifera cv. Merlot –0.5>Ψ w> –0.7 – – Martorell et al. (2015) V. vinifera cv. Grenache – –1.51>Ψ w>–2.15 –1.81>Ψ w> –2.59 V. vinifera cv. Tempranillo – –1.58>Ψ w> –2.22 –1.87>Ψ w> –3.07 Trifilò et al. (2016) Vitis labrusca – –1.23* –2.07a Corso et al. (2020) Triticum aestivum –1.5>Ψ w> –1.8a –0.91 –1.7c Cardoso et al. (2020) Persea americana –0.9>Ψ w > –2.32 – – Wang et al. (2018) Oryza sativa – –0.78* –1.43a Skelton et al. (2017) Solanum lycopersicum –1.24 –1.54 –1.8b Hernandez-Santana et al. (2016) Olea europea cv. Arbequina – –1.52 –5.35 Prunus dulcis cv. Guara – –1.45 –4.21 Scoffoni et al. (2011) Helianthus annuus – –0.83 –1.16 Authors . Species . Ψw of initial leaf xylem embolism observation (MPa) . K leaf . . . . . P50Kleaf . P80Kleaf . This study V. vinifera cv. Cabernet Sauvignon –1.3 –0.3 –0.71 V. vinifera cv. Chardonnay –1.2 –0.29 –0.67 Hochberg et al. (2019) V. vinifera cv. Chardonnay –0.45 >Ψ w> –1.4a – – Hochberg et al. (2017a) V. vinifera cv. Chardonnay < –1.4 – – Hochberg et al. (2017b) V. vinifera cv. Merlot –0.5>Ψ w> –0.7 – – Martorell et al. (2015) V. vinifera cv. Grenache – –1.51>Ψ w>–2.15 –1.81>Ψ w> –2.59 V. vinifera cv. Tempranillo – –1.58>Ψ w> –2.22 –1.87>Ψ w> –3.07 Trifilò et al. (2016) Vitis labrusca – –1.23* –2.07a Corso et al. (2020) Triticum aestivum –1.5>Ψ w> –1.8a –0.91 –1.7c Cardoso et al. (2020) Persea americana –0.9>Ψ w > –2.32 – – Wang et al. (2018) Oryza sativa – –0.78* –1.43a Skelton et al. (2017) Solanum lycopersicum –1.24 –1.54 –1.8b Hernandez-Santana et al. (2016) Olea europea cv. Arbequina – –1.52 –5.35 Prunus dulcis cv. Guara – –1.45 –4.21 Scoffoni et al. (2011) Helianthus annuus – –0.83 –1.16 a Data obtained from different methods and averaged Kleaf vulnerabilities. b Indicates P88Kleaf. c Indicates P90Kleaf. Open in new tab Table 1. Meta-data of studies on crop species showing water potentials at which embolisms were initially observed during dehydration and Kleaf vulnerabilities to drought Authors . Species . Ψw of initial leaf xylem embolism observation (MPa) . K leaf . . . . . P50Kleaf . P80Kleaf . This study V. vinifera cv. Cabernet Sauvignon –1.3 –0.3 –0.71 V. vinifera cv. Chardonnay –1.2 –0.29 –0.67 Hochberg et al. (2019) V. vinifera cv. Chardonnay –0.45 >Ψ w> –1.4a – – Hochberg et al. (2017a) V. vinifera cv. Chardonnay < –1.4 – – Hochberg et al. (2017b) V. vinifera cv. Merlot –0.5>Ψ w> –0.7 – – Martorell et al. (2015) V. vinifera cv. Grenache – –1.51>Ψ w>–2.15 –1.81>Ψ w> –2.59 V. vinifera cv. Tempranillo – –1.58>Ψ w> –2.22 –1.87>Ψ w> –3.07 Trifilò et al. (2016) Vitis labrusca – –1.23* –2.07a Corso et al. (2020) Triticum aestivum –1.5>Ψ w> –1.8a –0.91 –1.7c Cardoso et al. (2020) Persea americana –0.9>Ψ w > –2.32 – – Wang et al. (2018) Oryza sativa – –0.78* –1.43a Skelton et al. (2017) Solanum lycopersicum –1.24 –1.54 –1.8b Hernandez-Santana et al. (2016) Olea europea cv. Arbequina – –1.52 –5.35 Prunus dulcis cv. Guara – –1.45 –4.21 Scoffoni et al. (2011) Helianthus annuus – –0.83 –1.16 Authors . Species . Ψw of initial leaf xylem embolism observation (MPa) . K leaf . . . . . P50Kleaf . P80Kleaf . This study V. vinifera cv. Cabernet Sauvignon –1.3 –0.3 –0.71 V. vinifera cv. Chardonnay –1.2 –0.29 –0.67 Hochberg et al. (2019) V. vinifera cv. Chardonnay –0.45 >Ψ w> –1.4a – – Hochberg et al. (2017a) V. vinifera cv. Chardonnay < –1.4 – – Hochberg et al. (2017b) V. vinifera cv. Merlot –0.5>Ψ w> –0.7 – – Martorell et al. (2015) V. vinifera cv. Grenache – –1.51>Ψ w>–2.15 –1.81>Ψ w> –2.59 V. vinifera cv. Tempranillo – –1.58>Ψ w> –2.22 –1.87>Ψ w> –3.07 Trifilò et al. (2016) Vitis labrusca – –1.23* –2.07a Corso et al. (2020) Triticum aestivum –1.5>Ψ w> –1.8a –0.91 –1.7c Cardoso et al. (2020) Persea americana –0.9>Ψ w > –2.32 – – Wang et al. (2018) Oryza sativa – –0.78* –1.43a Skelton et al. (2017) Solanum lycopersicum –1.24 –1.54 –1.8b Hernandez-Santana et al. (2016) Olea europea cv. Arbequina – –1.52 –5.35 Prunus dulcis cv. Guara – –1.45 –4.21 Scoffoni et al. (2011) Helianthus annuus – –0.83 –1.16 a Data obtained from different methods and averaged Kleaf vulnerabilities. b Indicates P88Kleaf. c Indicates P90Kleaf. Open in new tab Both Chardonnay and Cabernet Sauvignon showed similar vulnerability to embolism, where the larger xylem conduits of the leaf midrib were the first ones to embolize, most probably due to the fact that they have the larger diameters of the leaf xylem network (Scoffoni et al., 2017a). As the conduits in the midrib are the main suppliers of water coming from the petioles, embolized conduits in the midrib could have a strong impact on whole-leaf hydraulic supply. Despite the fact that midrib embolism inception in Cabernet Sauvignon occurred slightly earlier than in Chardonnay, the very few embolized conduits would not account for the observed decline in both Kleaf and gs due to the redundancy of midrib xylem conduits and the water potential values at which it happened. Midrib embolism initiated when Kleaf was reduced by 94% in Cabernet Sauvignon and 96% in Chardonnay, which is strong evidence that the outside-xylem pathways are responsible for the hydraulic impairment of grapevine leaves during drought, and not xylem embolism. As leaves experience the most negative tensions in the soil–plant–atmosphere continuum, in the light of the hydraulic segmentation hypothesis (Tyree and Ewers, 1991), the living cells of the outside-xylem would be the first and main circuit breaker of the plant water transport system, acting to prevent increased tensions that could induce embolism in the leaf xylem. This would be especially important in grapevines due to their wide and large xylem conduits spanning from leaves to stems (Choat et al., 2010). These findings indicate that the leaf xylem network of grapevine leaves is very resistant to drought-induced embolism formation despite its high degree of connectivity and the most negative tensions in the xylem being experienced by the minor veins. As water potentials became more negative after stomatal closure, loss of Kleaf, and inception of embolism in midribs of both cultivars, embolism spread mainly to secondary and a few tertiary veins. This clear pattern of embolism spread with further dehydration was observed in all samples that showed embolism in secondary veins, since all conduits observed in secondary veins were branching from the lateral vascular bundles of the midrib and in one sample an embolism from the midrib spread to a tertiary vein. Even though there was no clear pattern of which midrib vascular bundles are the first to embolize, embolism spread seems to occur downstream of the water potential gradient and towards leaf margins, as embolisms that did not branch to secondary veins continued in the midrib towards the leaf tip. These findings are in good agreement with those reported by Thorne et al. (2006) in two grapevine species, showing that air can move from the midrib up to tertiary veins and towards the leaf margins via open conduits. In a follow-up study, Chatelet et al. (2006) attributed the lack of air movement to minor veins to xylem anatomy, as tracheids would replace vessels below this vein order, thus explaining why air never reached leaf margins in both the aforementioned studies. We can thus infer that leaf margins are the most stressed part of the leaves and exposed to the greatest water potential gradients, therefore generating higher tensions downstream of the xylem network. Corroborating these ideas, the few isolated embolized conduits we observed in higher order veins (Figs 4, 5) were only found at very negative Ψ w values that are not experienced by plants in vineyards (Choat et al., 2010; Charrier et al., 2018). The lack of embolism in minor vein conduits can also be explained by their narrow diameters, which would require extremely high tensions to induce embolism, as predicted by the rare-pit hypothesis (Christman et al., 2009, 2012) and/or by having short conduits as well as more resistant pit membranes. Indeed, a recent study in dehydrating leaves of several species also showed few and isolated embolisms in minor veins (Scoffoni et al., 2017a). In Arabidopsis, simulations of hydraulic dysfunction of higher order veins by collapse would have a minor impact in Kleaf and even in Kx decline during dehydration (Scoffoni et al., 2018). Contrary to Zhang et al. (2016), we found no evidence of collapse of leaf minor vein conduits even at extreme dehydration. The similar vulnerabilities that both cultivars exhibited in Kleaf and gs aligned with the absence of xylem embolism indicate a tight regulation of Kleaf by the outside-xylem pathways and its close coordination with gs during dehydration. Both Chardonnay and Cabernet Sauvignon showed similar Kleaf vulnerabilities to drought as well as Kmax values, exhibiting a very sensitive response to early dehydration. We then tested how these vulnerabilities would change assuming Ψ highest= –0.3 MPa, since plants rarely experience smaller tensions in vineyards and those were the greatest water potentials in our gas exchange measurements. These simulations did not affect our findings, as xylem embolism occurred after Kleaf was almost zero and the turgor loss point has been reached in both cultivars, with P50Kleaf and P80Kleaf ~0.3 MPa more negative, reflecting changes in Kmax. These results highlight the importance of including very high water potentials near full hydration (i.e. close to Ψ w=0 MPa) in vulnerability curves for a more uniform and better understanding of how susceptible species are to dehydration, as proposed in the Ferrari argument by Scoffoni and Sack (2017). Recently, a modeling study proposed that the loss of soil hydraulic conductivity is the driver of stomatal closure in medium-wet to dry soils (Carminati and Javaux, 2020); however, Kleaf was not accounted for in their model and the outside-xylem pathways were not considered. Our findings in grapevines show that the complex water transport pathways in living cells of the outside-xylem are responsible for the coordinated Kleaf and gs decline. Moreover, stomata are in the leaves and exposed to atmospheric vapor pressure deficit (VPD), so they may close even if exposed to high VPD despite a wet soil. As gs decline was less sensitive than Kleaf during dehydration, other factors may also be playing a role in the slower gs decline such as water stored in the leaf as well as reduced soil and roots hydraulic conductivity. Indeed, a study in grapevine roots showed that cortical lacunae formation reduced root hydraulic conductivity before root shrinkage and xylem embolism during dehydration (Cuneo et al., 2016). Integrated studies that combine both leaf and root responses to dehydration taking into account their living tissues will be crucial for a more comprehensive understanding of mechanisms involved in whole-plant responses to drought that does not take the xylem as the sole plant water transport pathway. Stomatal conductance of both cultivars exhibited similar vulnerabilities to dehydration and little to no influence of xylem embolism; however, cultivars differed in their maximum and thus absolute values of gs along the curves. On average, gs of Cabernet Sauvignon was double that of Chardonnay, and this difference increased as Ψ w became more negative. Maximum values of gs were twice as large for Cabernet Sauvignon as for Chardonnay in either Ψ max scenario tested (Table 2) and, as curves for both cultivars behaved similarly, this trend continued along the dehydration range. The offsets of P50gs and P80gs between scenarios were ~0.2 MPa in both cultivars, similar to what was found for Kleaf. Even in the Ψ highest= –0.3 scenario when P80gs was at Ψ w= –1.4 MPa for both cultivars, still the few midrib embolisms found at this range would most probably not account for the observed declines in gs as leaf vein redundancy would buffer the few embolized conduits from affecting xylem hydraulic transport. Contrasting with our findings, Zufferey et al. (2011) reported that daily fluctuations in gs in a wine grape cultivar were due to petiole embolism formation and repair. Even though grapevines have the ability to repair embolisms to restore xylem hydraulic function (Holbrook et al., 2001; Knipfer et al., 2016), this requires that water potentials approach 0 MPa, which rarely occurs under field conditions especially for deficit-irrigated plants. Zufferey et al. (2011) also reported 90% loss of conductivity due to petiole embolisms by –1.3 MPa when using hydraulics on excised samples, while we found in intact plants that embolisms begin to appear in leaf midribs at a Ψ w of approximately –1.25 MPa. Another study in grapevine leaves showed that embolism only occurred after complete stomatal closure (Hochberg et al., 2017a), even though their results might have been affected by the low light intensity used in their experiments. The very close coordination that both cultivars showed between Kleaf and gs during dehydration indicates an intermediate behavior between anisohydric and isohydric that lead us to further reject those categories for the wine grape cultivars of this study. Much attention was given to classifying wine grape cultivars as one or other of these categories since Schultz (2003); however, a recent study with several field-grown wine grape cultivars showed that they exhibit a continuum of stomatal responses during drought and challenged the exclusive classification of wine grapes as either isohydric or anisohydric (Levin et al., 2019). Our findings that Kleaf strongly regulates gs agrees with our previous study in Arabidopsis (Scoffoni et al., 2018), while a more recent study concluded that Kleaf was not the direct driver of gs decline during dehydration in wheat (Corso et al., 2020). However, Schultz (2003) also observed a strong correlation between gs and estimated Kleaf in two field-grown wine grape cultivars despite their different hydraulic responses to drought. The close matching between Kleaf and gs decline during dehydration is of great advantage for crop species, especially grapevines that not only are commonly grown under hot and dry conditions but that also benefit from water deficits, differently from annual crop species. This intermediate behavior brings benefits from both isohydric behavior by maintaining leaf tissues hydration and anisohydric behavior by avoiding embolism formation and keeping gas exchange during dehydration (Scoffoni and Sack, 2017). While the former may contribute to a fast recovery of gas exchange upon rehydration (i.e. irrigation), the latter allows stomata to remain open, thus ensuring carbon capture during further dehydration while also protecting the hydraulic system from embolism events, especially if Kleaf increased resistance is driven by the outside-xylem (Scoffoni and Sack, 2017). Interestingly, we observed that despite the close matching between Kleaf and gs, with Kleaf being slightly more vulnerable to dehydration in all cases (Table 2), this small difference of ~0.26 MPa at P50 vulnerabilities changed to ~0.45 MPa when P80 values were compared. Even if small, this indicates that as dehydration increases wine grapes may exhibit a near-anisohydric behavior, which agrees with recent findings that wine grape cultivars may exhibit different behaviors depending on the degree of dehydration (Levin et al., 2019) and that stomata remain open until the brink of embolism events in the midrib. This larger difference in vulnerabilities between Kleaf and gs as dehydration becomes more severe would reduce leaf water supply more rapidly than it is lost, thus decreasing Ψ w and turgor, which would further contribute to amplify stomatal closure. Grapevines would then avoid embolism formation that may compromise long-term hydraulic function while also ensuring carbon capture at higher tensions. Even though cultivars had similar responses to dehydration, the slightly lower vulnerability that Cabernet Sauvignon exhibited in comparison with Chardonnay aligned with the higher gs values measured in the former, indicating that Cabernet Sauvignon may lose more water under well-watered conditions than Chardonnay. However, it may be an advantage in terms of energy balance in hotter conditions where Cabernet Sauvignon is preferentially grown, while Chardonnay is more commonly cultivated in cooler regions. Interestingly, both showed similar Ψ TLP, with Chardonnay being slightly more negative, and even this small difference is contrary to what would be expected. The absence of leaf xylem embolisms indicates that the living tissues of the outside-xylem are the down-regulators of Kleaf and thus of gs decline during mild dehydration in grapevine leaves. Our modeling results indicated that only an 80% reduction in membrane water permeability (perhaps through reduced aquaporin expression or activity) combined with the presence of a Casparian-like band in the BSs would account for the decline in Kleaf during drought for both Cabernet Sauvignon and Chardonnay. Indeed, deactivation of aquaporin expression has been attributed to reducing Kleaf during drought (Kim and Steudle, 2007; Shatil-Cohen et al., 2011; Pantin et al., 2013; Pou et al., 2013; Sade et al., 2014, 2015). In grapevines, a study with two wine grape cultivars including Chardonnay showed reduced aquaporin expression in leaves and petioles in response to dehydration (Shelden et al., 2017), and another showed that the expression of several aquaporin genes is down-regulated at moderate water stress and more strongly in leaves than in roots (Galmés et al., 2007). This tight control would not only avoid excessive water loss and prevent xylem embolism formation, but would also allow rapid recovery of gs, since it is more likely to occur if it depends on recovery of cell turgor and/or aquaporin activation (Martre et al., 2002; Cochard et al., 2007; Pou et al., 2013; Sade et al., 2015). As water leaves the xylem and crosses the parenchyma cells, it flows through the BS before entering the mesophyll. The presence of a suberized or lignified Casparian-like hydrophobic band in the BS would force the water to flow across the living cells (i.e. via the symplast or transmembrane) since the suberized cell walls would greatly reduce water flow through the apoplast (i.e. porous cell walls), similarly to roots. These apoplastic barriers have been shown in leaves of some species (Lersten, 1997; Mertz and Brutnell, 2014; Taneda et al., 2016). In grapevines, suberin accumulation in response to water stress was seen in roots (Vandeleur et al., 2009) and around vascular bundles in petioles (Hochberg et al., 2013). If existent, then water would preferentially flow across the BS cells and aquaporins would have a major role in leaf hydraulic conductance. As leaf dehydration progresses, aquaporin activity would be reduced and water flow greatly diminished. Water then would encounter great resistances to flow across cells without these water-specific membrane-bound channels and through the hydrophobic suberized Casparian-like band in the apoplast. Aquaporin deactivation was also attributed to stomatal closure in grapevines (Pou et al., 2013), and modeling results from recent studies indicate that the membrane permeability due to aquaporin activity would be the cause of the observed Kleaf decline during drought in five angiosperm species from different ecological habitats (Scoffoni et al., 2017b, 2018). In conclusion, our results reveal that the tensions required to induce embolism are rarely experienced in commercial vineyards. Additionally, we report that embolism formation in minor veins is negligible, only rarely occurring at Ψ w values that are extremely negative and the plant is most probably near death. The finding that grapevines are highly resistant to drought-induced embolism formation may help explain why grapevines thrive in hot and dry regions and water deficit is a widely utilized tool to grow grapes. Furthermore, upon rehydration, grapevines could rapidly recover hydraulic function, thus ensuring carbon gain. Finally, even though both cultivars studied showed similar vulnerabilities to drought-induced leaf embolism formation, leaf hydraulics, and Kleaf, they also exhibited very different values of gs. Therefore, their apparent different responses to drought could come from having different gas exchange values despite having very similar vulnerabilities. Future work should address the hydraulic role of living tissues of the outside-xylem in grapevine leaves as well as molecular components in them during drought. Supplementary data The following supplementary data are available at JXB online. Table S1. Leaf anatomical parameters measured from light microscopy of Cabernet Sauvignon and Chardonnay used in MOFLO 2.0. Table S2. Leaf anatomical parameters measured from microCT scans of intact leaves of Cabernet Sauvignon and Chardonnay across a dehydration range used in MOFLO 2.0. Fig. S1. Location on the leaf where scans were targeted and paradermal view of midrib and leaf venation embed in the mesophyll. Fig. S2. Raw flow data of leaf hydraulic conductance of Cabernet Sauvignon and Chardonnay using the Evaporative Flux Method (EFM). Acknowledgements We thank Dula Parkinson and Alastair McDowell for technical assistance, and the Advanced Light Source in Berkeley, California (Beamline 8.3.2) and the Foundation Plant Services (FPS) at Davis for donating plant material. This work was supported by the USDA-ARS Sustainable Vineyard Production Systems CRIS (2032-21220-006-00-D) and CAPES/Brazil. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract no. DE-AC02-05CH11231. We also thank Thorsten Knipfer, Italo Cuneo, Clarissa Reyes, and Daniel Ribeiro for technical assistance. CA was supported by a CAPES/Brazil PhD Scholarship. CS and LS were supported by the US National Science Foundation (award #1457279). TNB was supported by the National Science Foundation (award #1557906) the USDA National Institute of Food and Agriculture (Hatch project 1016439) and the Almond Board of California (18.HORT37). Author contributions CA and AJM conceived the study and designed the experiments. CA performed the experiments and analysed the data. TNB ran the modeling. CA and AJM wrote the manuscript with input from all authors. Data availability The data supporting the findings of this study are available from the corresponding author, Andrew McElrone, upon request. 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TI - Coordinated decline of leaf hydraulic and stomatal conductances under drought is not linked to leaf xylem embolism for different grapevine cultivars
JF - Journal of Experimental Botany
DO - 10.1093/jxb/eraa392
DA - 2020-12-31
UR - https://www.deepdyve.com/lp/oxford-university-press/coordinated-decline-of-leaf-hydraulic-and-stomatal-conductances-under-tKbgHBWPe0
SP - 7286
EP - 7300
VL - 71
IS - 22
DP - DeepDyve
ER -