In-situ measurements of dissolved gases in xylem sap as tracers in plant physiology.

Trees transport gases from below ground into the atmosphere through the process of transpiration. Tracing gases transported through this mechanism continuously and under field conditions remains an experimental challenge. Here we measured gases dissolved in tree sap in-situ and in real time, aiming to simultaneously analyse the transport of several gases (He, Ar, Kr, N2, O2, CO2) from the soil, through the trees, into the atmosphere. We constructed and inserted custom-made semi-permeable membrane probes in the xylem of a fir tree and measured gas abundances at different heights using a portable gas equilibrium membrane-inlet mass spectrometer ('miniRUEDI'). With this method we were able to continuously measure the abundances of He, Ar, Kr, N2, O2, CO2 in sap over several weeks. We observed diurnal variations of CO2 and O2 concentrations that reflected tree physiological activities. As a proof of concept that trees do uptake dissolved gases in soil water, we irrigated the tree with He-enriched water in a tracer experiment, and were able to determine upwards sap flow velocity. Measurements of inert gases together with reactive species as CO2 and O2 allows to separate physical transport and exchange of gases derived from the soil or the atmosphere from biological reactions. We discuss the opportunities that our technique provides for continuous in-situ measurements of gases in tree sap.

Numerical modeling of PFAS movement through the vadose zone: Influence of plant water uptake and soil organic carbon distribution.

In this study, we investigated the effects of soil organic carbon (SOC) distribution and water uptake by plant roots on PFAS movement in the vadose zone with a deep groundwater table under temperate, humid climate conditions. Two series of numerical simulations were performed with the HYDRUS computer code, representing the leaching of historical PFOS contamination and the infiltration of water contaminated with PFOA, respectively. We considered soil profiles with three distributions of SOC (no SOC, realistic SOC distribution decreasing with depth, and uniform SOC equal to the content measured in topsoil), three root distributions (bare soil, grassland, and forest), and three soil textures (sand, sandy loam, and loam). The SOC distribution had a profound impact on the velocity of PFOS movement. The apparent retardation factor for realistic SOC distribution was twice as large as for the scenario with no SOC and more than three times smaller than for the scenario with uniformly high SOC content. We also showed that the root distribution in soil profoundly impacts the simulations of PFAS migration through soil. Including the root zone significantly slows down the movement of PFAS, primarily due to increased evapotranspiration and reduced downward water flux. Another effect of water uptake by plant roots is an increase of PFAS concentrations in soil water (evapo-concentration). The evapo-concentration and the slowdown of PFAS movement due to root water uptake are more significant in fine-textured soils than in sand.

Drought effects on trait space of winter wheat are independent of land management.

Investigating plant responses to climate change is key to develop suitable adaptation strategies. However, whether changes in land management can alleviate increasing drought threats to crops in the future is still unclear. We conducted a management × drought experiment with winter wheat (Triticum aestivum L.) to study plant water and vegetative traits in response to drought and management (conventional vs organic farming, with intensive vs conservation tillage). Water traits (root water uptake pattern, stem metaxylem area, leaf water potential, stomatal conductance) and vegetative traits (plant height, leaf area, leaf Chl content) were considered simultaneously to characterise the variability of multiple traits in a trait space, using principal component analysis. Management could not alleviate the drought impacts on plant water traits as it mainly affected vegetative traits, with yields ultimately being affected by both management and drought. Trait spaces were clearly separated between organic and conventional management as well as between drought and control conditions. Moreover, changes in trait space triggered by management and drought were independent from each other. Neither organic management nor conservation tillage eased drought impacts on winter wheat. Thus, our study raised concerns about the effectiveness of these management options as adaptation strategies to climate change.

Dynamic modeling of stem water content during the dormant period in walnut trees.

Water content (WC) is a key variable in plant physiology even during the winter period. To simulate stem WC during the dormant season, a series of experiments were carried out on walnut trees under controlled conditions. In the field, WC was significantly correlated with soil temperature at 50 cm depth (R2 = 0.526). In the greenhouse, WC remained low as long as soil temperature was kept cold (<+5 °C) and increased after the soil temperature was warmed to +15 °C regardless of the date. Stem dehydration rate was significantly influenced by the WC and evaporative demand. A parsimonious model with functions describing the main experimental results was calibrated and validated with field data from 13 independent winter dynamics in Juglans regia L. orchards. Three functions of water uptake were tested, and these gave equivalent accuracies (root-mean-square error (RMSE) = 0.127-8; predictive root-mean-square error = 0.116). However, only a sigmoid function describing the relationship between the root water uptake and soil temperature gave values in agreement with the experimental results. Finally, the simulated WC provided a similar accuracy in predicting frost hardiness compared with the measured WC (RMSE ca 3 °C) and was excellent in spring (RMSE ca 2 °C). This model may be a relevant tool for predicting the risk of spring frost in walnut trees. Its genericity should be tested in other fruit and forest tree species.

The role of arbuscular mycorrhizal symbiosis in improving plant water status under drought.

Arbuscular mycorrhizal fungi (AMF) have been presumed to ameliorate crop tolerance to drought. Here, we review the role of AMF in maintaining water supply to plants from drying soils and the underlying biophysical mechanisms. We used a soil-plant hydraulic model to illustrate the impact of several AMF mechanisms on plant responses to edaphic drought. The AMF enhance the soil's capability to transport water and extend the effective root length, thereby attenuating the drop in matric potential at the root surface during soil drying. The synthesized evidence and the corresponding simulations demonstrate that symbiosis with AMF postpones the stress onset limit, which is defined as the disproportionality between transpiration rates and leaf water potentials, during soil drying. The symbiosis can thus help crops survive extended intervals of limited water availability. We also provide our perspective on future research needs and call for reconciling the dynamic changes in soil and root hydraulics in order to better understand the role of AMF in plant water relations in the face of climate changes.

Coordination of available soil water content and root distribution modifies water source apportionment of the shrub plant Caragana korshinskii.

Stable isotopes have been widely used to identify root water uptake (RWU) by classifying potential water sources as distinct endmembers and evaluating their contributions to xylem water. However, the estimated contributions of endmembers (mainly soil layers) are usually based on variations in soil water isotopes alone. Available soil water and root distributions are key limiting factors of RWU but are rarely considered in water source apportionment. Thus, we have compared the relative contributions of distinct soil layers based on mean soil water isotope values, and values weighted by both available soil water content (AWC) and root weight density (RWD), to RWU of Caragana korshinskii. We derived these values (hereafter mean and weighted contributions, respectively) using three Bayesian mixing models (SIAR, simmr and MixSIAR) at three sites with different water conditions. We calculated the differences between the mean and weighted contributions (DC) and the accumulation of the absolute value of DC (AADC) to analyse the differences between them and their relationships with AWC and RWD. Both the weighted and mean contributions varied with sites and models. We obtained the following AADC values: 27, 8 and 11 % for Sites 1-3, respectively, using SIAR; 39, 13 and 14 %, respectively, using simmr; 68, 40 and 25 %, respectively, using MixSIAR. We detected a significant correlation between DC and RWD when AWC ≤ 6 %, as well as a significant correlation between DC and AWC when AWC > 6 %, indicating that the influence of RWD on DC depended on soil water conditions. Based on our findings, endmembers weighted by AWC and RWD altered the proportion of water source allocation relative to non-weighted endmembers, while the magnitude of the effect was related to the model used. Thus, we suggest careful consideration of the characterisation of endmember isotopes and model selection when partitioning plant water sources using δ2H and δ18O.

The effect of root hairs on root water uptake is determined by root-soil contact and root hair shrinkage.

The effect of root hairs on water uptake remains controversial. In particular, the key root hair and soil parameters that determine their importance have been elusive. We grew maize plants (Zea mays) in microcosms and scanned them using synchrotron-based X-ray computed microtomography. By means of image-based modelling, we investigated the parameters determining the effectiveness of root hairs in root water uptake. We explicitly accounted for rhizosphere features (e.g. root-soil contact and pore structure) and took root hair shrinkage of dehydrated root hairs into consideration. Our model suggests that > 85% of the variance in root water uptake is explained by the hair-induced increase in root-soil contact. In dry soil conditions, root hair shrinkage reduces the impact of hairs substantially. We conclude that the effectiveness of root hairs on root water uptake is determined by the hair-induced increase in root-soil contact and root hair shrinkage. Although the latter clearly reduces the effect of hairs on water uptake, our model still indicated facilitation of water uptake by root hairs at soil matric potentials from -1 to -0.1 MPa. Our findings provide new avenues towards a mechanistic understanding of the role of root hairs on water uptake.

Transpiration response to soil drying versus increasing vapor pressure deficit in crops: physical and physiological mechanisms and key plant traits.

The water deficit experienced by crops is a function of atmospheric water demand (vapor pressure deficit) and soil water supply over the whole crop cycle. We summarize typical transpiration response patterns to soil and atmospheric drying and the sensitivity to plant hydraulic traits. We explain the transpiration response patterns using a soil-plant hydraulic framework. In both cases of drying, stomatal closure is triggered by limitations in soil-plant hydraulic conductance. However, traits impacting the transpiration response differ between the two drying processes and act at different time scales. A low plant hydraulic conductance triggers an earlier restriction in transpiration during increasing vapor pressure deficit. During soil drying, the impact of the plant hydraulic conductance is less obvious. It is rather a decrease in the belowground hydraulic conductance (related to soil hydraulic properties and root length density) that is involved in transpiration down-regulation. The transpiration response to increasing vapor pressure deficit has a daily time scale. In the case of soil drying, it acts on a seasonal scale. Varieties that are conservative in water use on a daily scale may not be conservative over longer time scales (e.g. during soil drying). This potential independence of strategies needs to be considered in environment-specific breeding for yield-based drought tolerance.

Deep roots mitigate drought impacts on tropical trees despite limited quantitative contribution to transpiration.

Deep rooting is considered a central drought-mitigation trait with vast impact on ecosystem water cycling. Despite its importance, little is known about the overall quantitative water use via deep roots and dynamic shifts of water uptake depths with changing ambient conditions. Knowledge is especially sparse for tropical trees. Therefore, we conducted a drought, deep soil water labeling and re-wetting experiment at Biosphere 2 Tropical Rainforest. We used in situ methods to determine water stable isotope values in soil and tree water in high temporal resolution. Complemented by soil and stem water content and sap flow measurements we determined percentages and quantities of deep-water in total root water uptake dynamics of different tree species. All canopy trees had access to deep-water (max. uptake depth 3.3 m), with contributions to transpiration ranging between 21 % and 90 % during drought, when surface soil water availability was limited. Our results suggest that deep soil is an essential water source for tropical trees that delays potentially detrimental drops in plant water potentials and stem water content when surface soil water is limited and could hence mitigate the impacts of increasing drought occurrence and intensity as a consequence of climate change. Quantitatively, however, the amount of deep-water uptake was low due to the trees' reduction of sap flow during drought. Total water uptake largely followed surface soil water availability and trees switched back their uptake depth dynamically, from deep to shallow soils, following rainfall. Total transpiration fluxes were hence largely driven by precipitation input.

Deep-water uptake under drought improved due to locally increased root conductivity in maize, but not in faba bean.

Moderate soil drying can cause a strong decrease in the soil-root system conductance. The resulting impact on root water uptake depends on the spatial distribution of the altered conductance relatively to remaining soil water resources, which is largely unknown. Here, we analyzed the vertical distribution of conductance across root systems using a novel, noninvasive sensor technology on pot-grown faba bean and maize plants. Withholding water for 4 days strongly enhanced the vertical gradient in soil water potential. Therefore, roots in upper and deeper soil layers were affected differently: In drier, upper layers, root conductance decreased by 66%-72%, causing an amplification of the drop in leaf water potential. In wetter, deeper layers, root conductance increased in maize but not in faba bean. The consequently facilitated deep-water uptake in maize contributed up to 21% of total water uptake at the end of the measurement. Analysis of root length distributions with MRI indicated that the locally increased conductance was mainly caused by an increased intrinsic conductivity and not by additional root growth. Our findings show that plants can partly compensate for a reduced root conductance in upper, drier soil layers by locally increasing root conductivity in wetter layers, thereby improving deep-water uptake.

Stress-induced deeper rooting introgression enhances wheat yield under terminal drought.

Water scarcity is the primary environmental constraint affecting wheat growth and production and is increasingly exacerbated due to climatic fluctuation, which jeopardizes future food security. Most breeding efforts to improve wheat yields under drought have focused on above-ground traits. Root traits are closely associated with various drought adaptability mechanisms, but the genetic variation underlying these traits remains untapped, even though it holds tremendous potential for improving crop resilience. Here, we examined this potential by re-introducing ancestral alleles from wild emmer wheat (Triticum turgidum ssp. dicoccoides) and studied their impact on root architecture diversity under terminal drought stress. We applied an active sensing electrical resistivity tomography approach to compare a wild emmer introgression line (IL20) and its drought-sensitive recurrent parent (Svevo) under field conditions. IL20 exhibited greater root elongation under drought, which resulted in higher root water uptake from deeper soil layers. This advantage initiated at the pseudo-stem stage and increased during the transition to the reproductive stage. The increased water uptake promoted higher gas exchange rates and enhanced grain yield under drought. Overall, we show that this presumably 'lost' drought-induced mechanism of deeper rooting profile can serve as a breeding target to improve wheat productiveness under changing climate.

Plant water uptake modelling: added value of cross-disciplinary approaches.

In recent years, research interest in plant water uptake strategies has rapidly increased in many disciplines, such as hydrology, plant ecology and ecophysiology. Quantitative modelling approaches to estimate plant water uptake and spatiotemporal dynamics have significantly advanced through different disciplines across scales. Despite this progress, major limitations, for example, predicting plant water uptake under drought or drought impact at large scales, remain. These are less attributed to limitations in process understanding, but rather to a lack of implementation of cross-disciplinary insights into plant water uptake model structure. The main goal of this review is to highlight how the four dominant model approaches, that is, Feddes approach, hydrodynamic approach, optimality and statistical approaches, can be and have been used to create interdisciplinary hybrid models enabling a holistic system understanding that, among other things, embeds plant water uptake plasticity into a broader conceptual view of soil-plant feedbacks of water, nutrient and carbon cycling, or reflects observed drought responses of plant-soil feedbacks and their dynamics under, that is, drought. Specifically, we provide examples of how integration of Bayesian and hydrodynamic approaches might overcome challenges in interpreting plant water uptake related to different travel and residence times of different plant water sources or trade-offs between root system optimization to forage for water and nutrients during different seasons and phenological stages.

Construction of root tip density function and root water uptake characteristics in alpine meadows.

Accurate calculation of root water uptake (RWU) is the key to improving vegetation water use efficiency and identifying water cycle evolution patterns, and root tips play an important role in RWU. However, most of the current RWU models in the alpine meadow are calculated based on the root length density (RLD) function. In this study, a large number of roots, soil hydraulic conductivity, and physicochemical property indices were obtained by continuous field prototype observation experiments for up to 2 years. It was found that the RLD and root tip density (RTD) in alpine meadows decrease by 16.2% and 14.6%, respectively, in the wilting stage compared to the regreening stage. The RTD distribution function of the alpine meadow was constructed, and the RWU model was established accordingly. The results show that the RTD function is more accurate than the RLD function to reflect the RWU pattern. Compared with RLD, the simulated RWU model constructed by using RTD as the root index that can effectively absorb water increased by 24.64% on average, and the simulated values were more consistent with the actual situation. It can be seen that there is an underestimation of RWU calculated based on the RLD function, which leads to an underestimation of the effect of climate warming on evapotranspiration. The simulation results of the RWU model based on RTD showed that the RWU rate in the regreening stage increased by 30.24% on average compared with that in the wilting stage. Meanwhile, the top 67% of the rhizosphere was responsible for 86.76% of the total RWU on average. This study contributes to the understanding of the alpine meadow water cycle system and provides theoretical support for the implementation of alpine meadow vegetation protection and restoration projects.

Comparative cryogenic extraction rehydration experiments reveal isotope fractionation during root water uptake in Gramineae.

Determining whether isotope fractionation occurs during root water uptake is a prerequisite for using stem or xylem water isotopes to trace water sources. However, it is unclear whether isotope fractionation occurs during root water uptake in gramineous crops. We conducted prevalidation experiments to estimate the isotope measurement bias associated with cryogenic vacuum distillation (CVD). Next, we assessed isotope fractionation during root water uptake in two common agronomic crops, wheat (Triticum aestivum L.) and maize (Zea mays L.), under flooding after postdrought stress conditions. Cryogenic vacuum distillation caused significant depletion of 2 H but negligible effects on 18 O for both soil and stem water. Surprisingly CVD caused depletion of 2 H and enrichment of 18 O in root water. Stem and root water δ18 O were more than soil water δ18 O, even considering the uncertainty of CVD. Soil water 18 O was depleted compared with irrigation water 18 O in the pots with plants but enriched relative to irrigation water 18 O in the pots without plants. These results indicate that isotope fractionation occurred during wheat and maize root water uptake after full irrigation and led to a heavy isotope enrichment in stem water. Therefore, the xylem/stem water isotope approach widely used to trace water sources should be carefully evaluated.

Soil-plant interactions modulated water availability of Swiss forests during the 2015 and 2018 droughts.

Central Europe has been experiencing unprecedented droughts during the last decades, stressing the decrease in tree water availability. However, the assessment of physiological drought stress is challenging, and feedback between soil and vegetation is often omitted because of scarce belowground data. Here we aimed to model Swiss forests' water availability during the 2015 and 2018 droughts by implementing the mechanistic soil-vegetation-atmosphere-transport (SVAT) model LWF-Brook90 taking advantage of regionalized depth-resolved soil information. We calibrated the model against soil matric potential data measured from 2014 to 2018 at 44 sites along a Swiss climatic and edaphic drought gradient. Swiss forest soils' storage capacity of plant-available water ranged from 53 mm to 341 mm, with a median of 137 ± 42 mm down to the mean potential rooting depth of 1.2 m. Topsoil was the primary water source. However, trees switched to deeper soil water sources during drought. This effect was less pronounced for coniferous trees with a shallower rooting system than for deciduous trees, which resulted in a higher reduction of actual transpiration (transpiration deficit) in coniferous trees. Across Switzerland, forest trees reduced the transpiration by 23% (compared to potential transpiration) in 2015 and 2018, maintaining annual actual transpiration comparable to other years. Together with lower evaporative fluxes, the Swiss forests did not amplify the blue water deficit. The 2018 drought, characterized by a higher and more persistent transpiration deficit than in 2015, triggered widespread early wilting across Swiss forests that was better predicted by the SVAT-derived mean soil matric potential in the rooting zone than by climatic predictors. Such feedback-driven quantification of ecosystem water fluxes in the soil-plant-atmosphere continuum will be crucial to predicting physiological drought stress under future climate extremes.

Quantification of root water uptake and redistribution using neutron imaging: a review and future directions.

Quantifying root water uptake is essential to understanding plant water use and responses to different environmental conditions. However, non-destructive measurement of water transport and related hydraulics in the soil-root system remains a challenge. Neutron imaging, with its high sensitivity to hydrogen, has become an unparalleled tool to visualize and quantify root water uptake in vivo. In combination with isotopes (e.g., deuterated water) and a diffusion-convection model, root water uptake and hydraulic redistribution in root and soil can be quantified. Here, we review recent advances in utilizing neutron imaging to visualize and quantify root water uptake, hydraulic redistribution in roots and soil, and root hydraulic properties of different plant species. Under uniform soil moisture distributions, neutron radiographic studies have shown that water uptake was not uniform along the root and depended on both root type and age. For both tap (e.g., lupine [Lupinus albus L.]) and fibrous (e.g., maize [Zea mays L.]) root systems, water was mainly taken up through lateral roots. In mature maize, the location of water uptake shifted from seminal roots and their laterals to crown/nodal roots and their laterals. Under non-uniform soil moisture distributions, part of the water taken up during the daytime maintained the growth of crown/nodal roots in the upper, drier soil layers. Ultra-fast neutron tomography provides new insights into 3D water movement in soil and roots. We discuss the limitations of using neutron imaging and propose future directions to utilize neutron imaging to advance our understanding of root water uptake and soil-root interactions.

Dry Season Transpiration and Soil Water Dynamics in the Central Amazon.

With current observations and future projections of more intense and frequent droughts in the tropics, understanding the impact that extensive dry periods may have on tree and ecosystem-level transpiration and concurrent carbon uptake has become increasingly important. Here, we investigate paired soil and tree water extraction dynamics in an old-growth upland forest in central Amazonia during the 2018 dry season. Tree water use was assessed via radial patterns of sap flow in eight dominant canopy trees, each a different species with a range in diameter, height, and wood density. Paired multi-sensor soil moisture probes used to quantify volumetric water content dynamics and soil water extraction within the upper 100 cm were installed adjacent to six of those trees. To link depth-specific water extraction patterns to root distribution, fine root biomass was assessed through the soil profile to 235 cm. To scale tree water use to the plot level (stand transpiration), basal area was measured for all trees within a 5 m radius around each soil moisture probe. The sensitivity of tree transpiration to reduced precipitation varied by tree, with some increasing and some decreasing in water use during the dry period. Tree-level water use scaled with sapwood area, from 11 to 190 L per day. Stand level water use, based on multiple plots encompassing sap flow and adjacent trees, varied from ∼1.7 to 3.3 mm per day, increasing linearly with plot basal area. Soil water extraction was dependent on root biomass, which was dense at the surface (i.e., 45% in the upper 5 cm) and declined dramatically with depth. As the dry season progressed and the upper soil dried, soil water extraction shifted to deeper levels and model projections suggest that much of the water used during the month-long dry-down could be extracted from the upper 2-3 m. Results indicate variation in rates of soil water extraction across the research area and, temporally, through the soil profile. These results provide key information on whole-tree contributions to transpiration by canopy trees as water availability changes. In addition, information on simultaneous stand level dynamics of soil water extraction that can inform mechanistic models that project tropical forest response to drought.

Root System Scale Models Significantly Overestimate Root Water Uptake at Drying Soil Conditions.

Soil hydraulic conductivity (k soil ) drops significantly in dry soils, resulting in steep soil water potential gradients (ψ s ) near plant roots during water uptake. Coarse soil grid resolutions in root system scale (RSS) models of root water uptake (RWU) generally do not spatially resolve this gradient in drying soils which can lead to a large overestimation of RWU. To quantify this, we consider a benchmark scenario of RWU from drying soil for which a numerical reference solution is available. We analyze this problem using a finite volume scheme and investigate the impact of grid size on the RSS model results. At dry conditions, the cumulative RWU was overestimated by up to 300% for the coarsest soil grid of 4.0 cm and by 30% for the finest soil grid of 0.2 cm, while the computational demand increased from 19 s to 21 h. As an accurate and computationally efficient alternative to the RSS model, we implemented a continuum multi-scale model where we keep a coarse grid resolution for the bulk soil, but in addition, we solve a 1-dimensional radially symmetric soil model at rhizosphere scale around individual root segments. The models at the two scales are coupled in a mass-conservative way. The multi-scale model compares best to the reference solution (-20%) at much lower computational costs of 4 min. Our results demonstrate the need to shift to improved RWU models when simulating dry soil conditions and highlight that results for dry conditions obtained with RSS models of RWU should be interpreted with caution.

Root hydraulic phenotypes impacting water uptake in drying soils.

Soil drying is a limiting factor for crop production worldwide. Yet, it is not clear how soil drying impacts water uptake across different soils, species, and root phenotypes. Here we ask (1) what root phenotypes improve the water use from drying soils? and (2) what root hydraulic properties impact water flow across the soil-plant continuum? The main objective is to propose a hydraulic framework to investigate the interplay between soil and root hydraulic properties on water uptake. We collected highly resolved data on transpiration, leaf and soil water potential across 11 crops and 10 contrasting soil textures. In drying soils, the drop in water potential at the soil-root interface resulted in a rapid decrease in soil hydraulic conductance, especially at higher transpiration rates. The analysis reveals that water uptake was limited by soil within a wide range of soil water potential (-6 to -1000 kPa), depending on both soil textures and root hydraulic phenotypes. We propose that a root phenotype with low root hydraulic conductance, long roots and/or long and dense root hairs postpones soil limitation in drying soils. The consequence of these root phenotypes on crop water use is discussed.

Negative effects of low root temperatures on water and carbon relations in temperate tree seedlings assessed by dual isotopic labelling.

Low root zone temperatures restrict water and carbon (C) uptake and transport in plants and may contribute to the low temperature limits of tree growth. Here, we quantified the effects of low root temperatures on xylem conductance, photosynthetic C assimilation and phloem C transport in seedlings of four temperate tree species (two broad-leaved and two conifer species) by applying a simultaneous stable isotope labelling of 2H-enriched source water and 13C-enriched atmospheric CO2. Six days before the pulse labelling, the seedlings were transferred to hydroponic tubes and exposed to three different root temperatures (2, 7 and 15 °C), while all seedlings received the same, warm air temperatures (between 18 and 24 °C). Root cooling led to drought-like symptoms with reduced growth, leaf water potentials and stomatal conductance, indicating increasingly adverse conditions for water uptake and transport with decreasing root temperatures. Averaged across all four species, water transport to leaves was reduced by 40% at 7 °C and by 70% at 2 °C root temperature relative to the 15 °C treatment, while photosynthesis was reduced by 20 and 40% at 7 and 2 °C, respectively. The most severe effects were found on the phloem C transport to roots, which was reduced by 60% at 7 °C and almost ceased at 2 °C in comparison with the 15 °C root temperature treatment. This extreme effect on C transport was likely due to a combination of simultaneous reductions of phloem loading, phloem mass flow and root growth. Overall, the dual stable isotope labelling proved to be a useful method to quantify water and C relations in cold-stressed trees and highlighted the potentially important role of hydraulic constraints induced by low soil temperatures as a contributing factor for the climatic distribution limits of temperate tree species.