A new empirical framework to quantify the hydraulic effects of soil and atmospheric drivers on plant water status.

Metrics to quantify regulation of plant water status at the daily as opposed to the seasonal scale do not presently exist. This gap is significant since plants are hypothesised to regulate their water potential not only with respect to slowly changing soil drought but also with respect to faster changes in air vapour pressure deficit (VPD), a variable whose importance for plant physiology is expected to grow because of higher temperatures in the coming decades. We present a metric, the stringency of water potential regulation, that can be employed at the daily scale and quantifies the effects exerted on plants by the separate and combined effect of soil and atmospheric drought. We test our theory using datasets from two experiments where air temperature and VPD were experimentally manipulated. In contrast to existing metrics based on soil drought that can only be applied at the seasonal scale, our metric successfully detects the impact of atmospheric warming on the regulation of plant water status. We show that the thermodynamic effect of VPD on plant water status can be isolated and compared against that exerted by soil drought and the covariation between VPD and soil drought. Furthermore, in three of three cases, VPD accounted for more than 5 MPa of potential effect on leaf water potential. We explore the significance of our findings in the context of potential future applications of this metric from plant to ecosystem scale.

Drought-induced fiber water release and xylem embolism susceptibility of intact balsam poplar saplings.

Balsam poplar (Populus balsamifera L.) is a widespread tree species in North America with significant ecological and economic value. However, little is known about the susceptibility of saplings to drought-induced embolism and its link to water release from surrounding xylem fibers. Questions remain regarding localized mechanisms that contribute to the survival of saplings in vivo of this species under drought. Using X-ray micro-computed tomography on intact saplings of genotypes Gillam-5 and Carnduff-9, we found that functional vessels are embedded in a matrix of water-filled fibers under well-watered conditions in both genotypes. However, water-depleted fibers started to appear under moderate drought stress while vessels remained water-filled in both genotypes. Drought-induced xylem embolism susceptibility was comparable between genotypes, and a greater frequency of smaller diameter vessels in GIL-5 did not increase embolism resistance in this genotype. Despite having smaller vessels and a total vessel number that was comparable to CAR-9, stomatal conductance was generally higher in GIL-5 compared to CAR-9. In conclusion, our in vivo data on intact saplings indicate that differences in embolism susceptibility are negligible between GIL-5 and CAR-9, and that fiber water release should be considered as a mechanism that contributes to the maintenance of vessel functional status in saplings of balsam poplar experiencing their first drought event.

Ecologically driven selection of nonstructural carbohydrate storage in oak trees.

Leaf habit is a major axis of plant diversity that has consequences for carbon balance since the leaf is the primary site of photosynthesis. Nonstructural carbohydrates (NSCs) produced by photosynthesis can be allocated to storage and serve as a resiliency mechanism to future abiotic and biotic stress. However, how leaf habit affects NSC storage in an evolutionary context has not been shown. Using a comparative physiological framework and an analysis of evolutionary model fitting, we examined if variation in NSC storage is explained by leaf habit. We measured sugar and starch concentrations in 51 oak species (Quercus spp.) growing in a common garden and representing multiple evolutions of three different leaf habits (deciduous, brevideciduous and evergreen). The best fitting evolutionary models indicated that deciduous oak species are evolving towards higher NSC concentrations than their brevideciduous and evergreen relatives. Notably, this was observed for starch (the primary storage molecule) in the stem (a long-term C storage organ). Overall, our work provides insight into the evolutionary drivers of NSC storage and suggests that a deciduous strategy may confer an advantage against stress associated with a changing world. Future work should examine additional clades to further corroborate this idea.

Differences in grapevine rootstock sensitivity and recovery from drought are linked to fine root cortical lacunae and root tip function.

Structural changes during severe drought stress greatly modify the hydraulic properties of fine roots. Yet, the physiological basis behind the restoration of fine root water uptake capacity during water recovery remains unknown. Using neutron radiography (NR), X-ray micro-computed tomography (micro-CT), fluorescence microscopy, and fine root hydraulic conductivity measurements (Lpr ), we examined how drought-induced changes in anatomy and hydraulic properties of contrasting grapevine rootstocks are coupled with fine root growth dynamics during drought and return of soil moisture. Lacunae formation in drought-stressed fine roots was associated with a significant decrease in fine root Lpr for both rootstocks. However, lacunae formation occurred under milder stress in the drought-resistant rootstock, 110R. Suberin was deposited at an earlier developmental stage in fine roots of 101-14Mgt (i.e. drought susceptible), probably limiting cortical lacunae formation during mild stress. During recovery, we found that only 110R fine roots showed rapid re-establishment of elongation and water uptake capacity and we found that soil water status surrounding root tips differed between rootstocks as imaged with NR. These data suggest that drought resistance in grapevine rootstocks is associated with rapid re-establishment of growth and Lpr near the root tip upon re-watering by limiting competing sites along the root cylinder.

Predicting Stomatal Closure and Turgor Loss in Woody Plants Using Predawn and Midday Water Potential.

Knowledge about physiological stress thresholds provides crucial information about plant performance and survival under drought. In this study, we report on the triphasic nature of the relationship between plant water potential (Ψ) at predawn and midday and describe a method that predicts Ψ at stomatal closure and turgor loss exclusively from this water potential curve (WP curve). The method is based on a piecewise linear regression model that was developed to predict the boundaries (termed Θ1 and Θ2) separating the three phases of the curve and corresponding slope values. The method was tested for three economically important woody species. For all species, midday Ψ was much more negative than predawn Ψ during phase I (mild drought), reductions in midday Ψ were minor while predawn Ψ continued to decline during phase II (moderate drought), and midday and predawn Ψ reached similar values during phase III (severe drought). Corresponding measurement of leaf gas exchange indicated that boundary Θ1 between phases I and II coincided with Ψ at stomatal closure. Data from pressure-volume curves demonstrated that boundary Θ2 between phases II and III predicted Ψ at leaf turgor loss. The WP curve method described here is an advanced application of the Scholander-type pressure chamber to categorize plant dehydration under drought into three distinct phases and to predict Ψ thresholds of stomatal closure and turgor loss.

Salt stress reduces root water uptake in barley (Hordeum vulgare L.) through modification of the transcellular transport path.

The aim of the study was to understand the hydraulic response to salt stress of the root system of the comparatively salt-tolerant crop barley (Hordeum vulgare L.). We focused on the transcellular path of water movement across the root cylinder that involves the crossing of membranes. This path allows for selective water uptake, while excluding salt ions. Hydroponically grown plants were exposed to 100 mM NaCl for 5-7 days and analysed when 15-17 days old. A range of complementary and novel approaches was used to determine hydraulic conductivity (Lp). This included analyses at cell, root and plant level and modelling of water flow. Apoplastic barrier formation and gene expression level of aquaporins (AQPs) was analysed. Salt stress reduced the Lp of root system through reducing water flow along the transcellular path. This involved changes in the activity and gene expression level of AQPs. Modelling of root-Lp showed that the reduction in root-Lp did not require added hydraulic resistances through apoplastic barriers at the endodermis. The bulk of data points to a near-perfect semi-permeability of roots of control plants (solute reflection coefficient σ ~1.0). Roots of salt-stressed plants are almost as semi-permeable (σ > 0.8).

Spatiotemporal Coupling of Vessel Cavitation and Discharge of Stored Xylem Water in a Tree Sapling.

Water discharge from stem internal storage compartments is thought to minimize the risk of vessel cavitation. Based on this concept, one would expect that water storage compartments involved in the buffering of xylem tensions empty before the onset of vessel cavitation under drought stress, and potentially refill after soil saturation. However, scant in vivo data exist that elucidate this localized spatiotemporal coupling. In this study on intact saplings of American chestnut (Castanea dentata), x-ray computed microtomography (microCT) showed that the xylem matrix surrounding vessels releases stored water and becomes air-filled either concurrent to or after vessel cavitation under progressive drought stress. Among annual growth rings, the xylem matrix of the current year remained largely water-filled even under severe drought stress. In comparison, microtomography images collected on excised stems showed that applied pressures of much greater than 0 MPa were required to induce water release from the xylem matrix. Viability staining highlighted that water release from the xylem matrix was associated primarily with emptying of dead fibers. Refilling of the xylem matrix and vessels was detected in intact saplings when the canopy was bagged and stem water potential was close to 0 MPa, and in leafless saplings over the winter period. In conclusion, this study indicates that the bulk of water stored in the xylem matrix is released after the onset of vessel cavitation, and suggests that capillary water contributes to overall stem water storage under drought but is not used primarily for the prevention of drought-induced vessel cavitation in this species.

In vivo visualization of the final stages of xylem vessel refilling in grapevine (Vitis vinifera) stems.

Embolism removal is critical for restoring hydraulic pathways in some plants, as residual gas bubbles should expand when vessels are reconnected to the transpiration stream. Much of our understanding of embolism removal remains theoretical as a consequence of the lack of in vivo images of the process at high magnification. Here, we used in vivo X-ray micro-computed tomography (microCT) to visualize the final stages of xylem refilling in grapevine (Vitis vinifera) paired with scanning electron microscopy. Before refilling, vessel walls were covered with a surface film, but vessel perforation plate openings and intervessel pits were filled with air. Bubbles were removed from intervessel pits first, followed by bubbles within perforation plates, which hold the last volumes of air which eventually dissolve. Perforation plates were dimorphic, with more steeply angled scalariform plates in narrow diameter vessels, compared with the simple perforation plates in older secondary xylem, which may favor rapid refilling and compartmentalization of embolisms that occur in small vessels, while promoting high hydraulic conductivity in large vessels. Our study provides direct visual evidence of the spatial and temporal dynamics of the final stages of embolism removal.

Water uptake can occur through woody portions of roots and facilitates localized embolism repair in grapevine.

Water acquisition is thought to be limited to the unsuberized surface located close to root tips. However, there are recurring periods when the unsuberized surfaces are limited in woody root systems, and radial water uptake across the bark of woody roots might play an important physiological role in hydraulic functioning. Using X-ray microcomputed tomography (microCT) and hydraulic conductivity measurements (Lpr ), we examined water uptake capacity of suberized woody roots in vivo and in excised samples. Bark hydration in grapevine woody roots occurred quickly upon exposure to water (c. 4 h). Lpr measurements through the bark of woody roots showed that it is permeable to water and becomes more so upon wetting. After bark hydration, microCT analysis showed that absorbed water was utilized to remove embolism locally, where c. 20% of root xylem vessels refilled completely within 15 h. Embolism removal did not occur in control roots without water. Water uptake through the bark of woody roots probably plays an important role when unsuberized tissue is scarce/absent, and would be particularly relevant following large irrigation events or in late winter when soils are saturated, re-establishing hydraulic functionality before bud break.

In vivo quantification of plant starch reserves at micrometer resolution using X-ray microCT imaging and machine learning.

Starch is the primary energy storage molecule used by most terrestrial plants to fuel respiration and growth during periods of limited to no photosynthesis, and its depletion can drive plant mortality. Destructive techniques at coarse spatial scales exist to quantify starch, but these techniques face methodological challenges that can lead to uncertainty about the lability of tissue-specific starch pools and their role in plant survival. Here, we demonstrate how X-ray microcomputed tomography (microCT) and a machine learning algorithm can be coupled to quantify plant starch content in vivo, repeatedly and nondestructively over time in grapevine stems (Vitis spp.). Starch content estimated for xylem axial and ray parenchyma cells from microCT images was correlated strongly with enzymatically measured bulk-tissue starch concentration on the same stems. After validating our machine learning algorithm, we then characterized the spatial distribution of starch concentration in living stems at micrometer resolution, and identified starch depletion in live plants under experimental conditions designed to halt photosynthesis and starch production, initiating the drawdown of stored starch pools. Using X-ray microCT technology for in vivo starch monitoring should enable novel research directed at resolving the spatial and temporal patterns of starch accumulation and depletion in woody plant species.

Storage Compartments for Capillary Water Rarely Refill in an Intact Woody Plant.

Water storage is thought to play an integral role in the maintenance of whole-plant water balance. The contribution of both living and dead cells to water storage can be derived from rehydration and water-release curves on excised plant material, but the underlying tissue-specific emptying/refilling dynamics remain unclear. Here, we used x-ray computed microtomography to characterize the refilling of xylem fibers, pith cells, and vessels under both excised and in vivo conditions in Laurus nobilis In excised stems supplied with water, water uptake exhibited a biphasic response curve, and x-ray computed microtomography images showed that high water storage capacitance was associated with fiber and pith refilling as driven by capillary forces: fibers refilled more rapidly than pith cells, while vessel refilling was minimal. In excised stems that were sealed, fiber and pith refilling was associated with vessel emptying, indicating a link between tissue connectivity and water storage. In contrast, refilling of fibers, pith cells, and vessels was negligible in intact saplings over two time scales, 24 h and 3 weeks. However, those compartments did refill slowly when the shoot was covered to prevent transpiration. Collectively, our data (1) provide direct evidence that storage compartments for capillary water refill in excised stems but rarely under in vivo conditions, (2) highlight that estimates of capacitance from excised samples should be interpreted with caution, as certain storage compartments may not be utilized in the intact plant, and (3) question the paradigm that fibers play a substantial role in daily discharge/recharge of stem capacitance in an intact tree.

In Situ Visualization of the Dynamics in Xylem Embolism Formation and Removal in the Absence of Root Pressure: A Study on Excised Grapevine Stems.

Gas embolisms formed during drought can disrupt long-distance water transport through plant xylem vessels, but some species have the ability to remove these blockages. Despite evidence suggesting that embolism removal is linked to the presence of vessel-associated parenchyma, the underlying mechanism remains controversial and is thought to involve positive pressure generated by roots. Here, we used in situ x-ray microtomography on excised grapevine stems to determine if embolism removal is possible without root pressure, and if the embolism formation/removal affects vessel functional status after sample excision. Our data show that embolism removal in excised stems was driven by water droplet growth and was qualitatively identical to refilling in intact plants. When stem segments were rehydrated with H2O after excision, vessel refilling occurred rapidly (<1 h). The refilling process was substantially slower when polyethylene glycol was added to the H2O source, thereby providing new support for an osmotically driven refilling mechanism. In contrast, segments not supplied with H2O showed no refilling and increased embolism formation. Dynamic changes in liquid/wall contact angles indicated that the processes of embolism removal (i.e. vessel refilling) by water influx and embolism formation by water efflux were directly linked to the activity of vessel-associated living tissue. Overall, our results emphasize that root pressure is not required as a driving force for vessel refilling, and care should be taken when performing hydraulics measurements on excised plant organs containing living vessel-associated tissue, because the vessel behavior may not be static.

Mechanical Failure of Fine Root Cortical Cells Initiates Plant Hydraulic Decline during Drought.

Root systems perform the crucial task of absorbing water from the soil to meet the demands of a transpiring canopy. Roots are thought to operate like electrical fuses, which break when carrying an excessive load under conditions of drought stress. Yet the exact site and sequence of this dysfunction in roots remain elusive. Using in vivo x-ray computed microtomography, we found that drought-induced mechanical failure (i.e. lacunae formation) in fine root cortical cells is the initial and primary driver of reduced fine root hydraulic conductivity (Lpr) under mild to moderate drought stress. Cortical lacunae started forming under mild drought stress (-0.6 MPa Ψstem), coincided with a dramatic reduction in Lpr, and preceded root shrinkage or significant xylem embolism. Only under increased drought stress was embolism formation observed in the root xylem, and it appeared first in the fine roots (50% loss of hydraulic conductivity [P50] reached at -1.8 MPa) and then in older, coarse roots (P50 = -3.5 MPa). These results suggest that cortical cells in fine roots function like hydraulic fuses that decouple plants from drying soil, thus preserving the hydraulic integrity of the plant's vascular system under early stages of drought stress. Cortical lacunae formation led to permanent structural damage of the root cortex and nonrecoverable Lpr, pointing to a role in fine root mortality and turnover under drought stress.

Water Transport Properties of the Grape Pedicel during Fruit Development: Insights into Xylem Anatomy and Function Using Microtomography.

Xylem flow of water into fruits declines during fruit development, and the literature indicates a corresponding increase in hydraulic resistance in the pedicel. However, it is unknown how pedicel hydraulics change developmentally in relation to xylem anatomy and function. In this study on grape (Vitis vinifera), we determined pedicel hydraulic conductivity (kh) from pressure-flow relationships using hydrostatic and osmotic forces and investigated xylem anatomy and function using fluorescent light microscopy and x-ray computed microtomography. Hydrostatic kh (xylem pathway) was consistently 4 orders of magnitude greater than osmotic kh (intracellular pathway), but both declined before veraison by approximately 40% and substantially over fruit development. Hydrostatic kh declined most gradually for low (less than 0.08 MPa) pressures and for water inflow and outflow conditions. Specific kh (per xylem area) decreased in a similar fashion to kh despite substantial increases in xylem area. X-ray computed microtomography images provided direct evidence that losses in pedicel kh were associated with blockages in vessel elements, whereas air embolisms were negligible. However, vessel elements were interconnected and some remained continuous postveraison, suggesting that across the grape pedicel, a xylem pathway of reduced kh remains functional late into berry ripening.

Limitation of Cell Elongation in Barley (Hordeum vulgare L.) Leaves Through Mechanical and Tissue-Hydraulic Properties.

The aim of the present study was to assess the mechanical and hydraulic limitation of growth in leaf epidermal cells of barley (Hordeum vulgare L.) in response to agents which affect cellular water (mercuric chloride, HgCl(2)) and potassium (cesium chloride, CsCl; tetraethylammonium, TEA) transport, pump activity of plasma membrane H(+)-ATPase and wall acidification (fusicoccin, FC). Cell turgor (P) was measured with the cell pressure probe, and cell osmotic pressure (π) was analyzed through picoliter osmometry of single-cell extracts. A wall extensibility coefficient (M) and tissue hydraulic conductance coefficient (L) were derived using the Lockhart equation. There was a significant positive linear relationship between relative elemental growth rate and P, which fit all treatments, with an overall apparent yield threshold of 0.368 MPa. Differences in growth between treatments could be explained through differences in P. A comparison of L and M showed that growth in all except the FC treatment was co-limited through hydraulic and mechanical properties, though to various extents. This was accompanied by significant (0.17-0.24 MPa) differences in water potential (ΔΨ) between xylem and epidermal cells in the leaf elongation zone. In contrast, FC-treated leaves showed ΔΨ close to zero and a 10-fold increase in L.

Polarity of water transport across epidermal cell membranes in Tradescantia virginiana.

Using the automated cell pressure probe, small and highly reproducible hydrostatic pressure clamp (PC) and pressure relaxation (PR) tests (typically, applied step change in pressure = 0.02 MPa and overall change in volume = 30 pL, respectively) were applied to individual Tradescantia virginiana epidermal cells to determine both exosmotic and endosmotic hydraulic conductivity (L(p)(OUT) and L(p)(IN), respectively). Within-cell reproducibility of measured hydraulic parameters depended on the method used, with the PR method giving a lower average coefficient of variation (15.2%, 5.8%, and 19.0% for half-time, cell volume [V(o)], and hydraulic conductivity [L(p)], respectively) than the PC method (25.4%, 22.0%, and 24.2%, respectively). V(o) as determined from PC and PR tests was 1.1 to 2.7 nL and in the range of optically estimated V(o) values of 1.5 to 4.9 nL. For the same cell, V(o) and L(p) estimates were significantly lower (about 15% and 30%, respectively) when determined by PC compared with PR. Both methods, however, showed significantly higher L(p)(OUT) than L(p)(IN) (L(p)(OUT)/L(p)(IN) ≅ 1.20). Because these results were obtained using small and reversible hydrostatically driven flows in the same cell, the 20% outward biased polarity of water transport is most likely not due to artifacts associated with unstirred layers or to direct effects of externally applied osmotica on the membrane, as has been suggested in previous studies. The rapid reversibility of applied flow direction, particularly for the PR method, and the lack of a clear increase in L(p)(OUT)/L(p)(IN) over a wide range of L(p) values suggest that the observed polarity is an intrinsic biophysical property of the intact membrane/protein complex.

Grapevine species from varied native habitats exhibit differences in embolism formation/repair associated with leaf gas exchange and root pressure.

Drought induces xylem embolism formation, but grapevines can refill non-functional vessels to restore transport capacity. It is unknown whether vulnerability to embolism formation and ability to repair differ among grapevine species. We analysed in vivo embolism formation and repair using x-ray computed microtomography in three wild grapevine species from varied native habitats (Vitis riparia, V. arizonica, V. champinii) and related responses to measurements of leaf gas exchange and root pressure. Vulnerability to embolism formation was greatest in V. riparia, intermediate in V. arizonica and lowest in V. champinii. After re-watering, embolism repair was rapid and pronounced in V. riparia and V. arizonica, but limited or negligible in V. champinii even after numerous days. Similarly, root pressure measured after re-watering was positively correlated with drought stress severity for V. riparia and V. arizonica (species exhibiting embolism repair) but not for V. champinii. Drought-induced reductions in transpiration were greatest for V. riparia and least in V. champinii. Recovery of transpiration after re-watering was delayed for all species, but was greatest for V. champinii and most rapid in V. arizonica. These species exhibit varied responses to drought stress that involve maintenance/recovery of xylem transport capacity coordinated with root pressure and gas exchange responses.

Water uptake along the length of grapevine fine roots: developmental anatomy, tissue-specific aquaporin expression, and pathways of water transport.

To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lpr) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lpr and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaporins play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lpr, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential.

Do root hydraulic properties change during the early vegetative stage of plant development in barley (Hordeum vulgare)?

BACKGROUND AND AIMS: As annual crops develop, transpirational water loss increases substantially. This increase has to be matched by an increase in water uptake through the root system. The aim of this study was to assess the contributions of changes in intrinsic root hydraulic conductivity (Lp, water uptake per unit root surface area, driving force and time), driving force and root surface area to developmental increases in root water uptake. METHODS: Hydroponically grown barley plants were analysed during four windows of their vegetative stage of development, when they were 9-13, 14-18, 19-23 and 24-28 d old. Hydraulic conductivity was determined for individual roots (Lp) and for entire root systems (Lp(r)). Osmotic Lp of individual seminal and adventitious roots and osmotic Lp(r) of the root system were determined in exudation experiments. Hydrostatic Lp of individual roots was determined by root pressure probe analyses, and hydrostatic Lp(r) of the root system was derived from analyses of transpiring plants. KEY RESULTS: Although osmotic and hydrostatic Lp and Lp(r) values increased initially during development and were correlated positively with plant transpiration rate, their overall developmental increases (about 2-fold) were small compared with increases in transpirational water loss and root surface area (about 10- to 40-fold). The water potential gradient driving water uptake in transpiring plants more than doubled during development, and potentially contributed to the increases in plant water flow. Osmotic Lp(r) of entire root systems and hydrostatic Lp(r) of transpiring plants were similar, suggesting that the main radial transport path in roots was the cell-to-cell path at all developmental stages. CONCLUSIONS: Increase in the surface area of root system, and not changes in intrinsic root hydraulic properties, is the main means through which barley plants grown hydroponically sustain an increase in transpirational water loss during their vegetative development.

Leaf water relations and osmotic adjustment of Canada Western Red Spring wheat cultivars subjected to drought.

For wheat (Triticum aestivum ), sustained crop yield at limited soil water availability has been linked to osmotic adjustment (OA) - a physiological mechanism that aids maintenance of leaf hydration status, turgor (P ) and growth. 'Canada Western Red Spring' (CWRS) wheat cultivars are typically grown in rainfed areas with milder climates, but ongoing climate change is increasesing the frequency and intensity of drought events. The overarching goal of this study was to elucidate if commercially used CWRS cultivars ('Superb', 'Stettler', 'AAC Viewfield') have the ability for leaf OA. Measurements of leaf water relation parameters (water potential, Ψ ; solute potential, Ψ s ; stomatal conductance, g s ; relative water content, RWC) showed that all three cultivars reached zero P (= Ψ - Ψ s ) at a leaf Ψ of -1.1MPa. Prior to that, P maintenance in 'Superb' and 'AAC Viewfield' was associated with a significant reduction in leaf Ψ s and OA contributed 0.53MPa ('Superb') and 0.73MPa ('AAC Viewfield'). Our data analyses provided no support for the existence of OA in 'Stettler'. Under water deficit, leaf g s was significantly higher in 'AAC Viewfield' compared to 'Stettler'; it was intermediate in 'Superb'. Together, drought tolerance in CWRS wheat cultivars is most likely linked to the degree of OA.