Visualizing and quantifying 33P uptake and translocation by maize plants grown in soil.

Phosphorus (P) availability severely limits plant growth due to its immobility and inaccessibility in soils. Yet, visualization and measurements of P uptake from different root types or regions in soil are methodologically challenging. Here, we explored the potential of phosphor imaging combined with local injection of radioactive 33P to quantitatively visualize P uptake and translocation along roots of maize grown in soils. Rhizoboxes (20 × 40 × 1 cm) were filled with sandy field soil or quartz sand, with one maize plant per box. Soil compartments were created using a gravel layer to restrict P transfer. After 2 weeks, a compartment with the tip region of a seminal root was labeled with a NaH2 33PO4 solution containing 12 MBq of 33P. Phosphor imaging captured root P distribution at 45 min, 90 min, 135 min, 180 min, and 24 h post-labeling. After harvest, 33P levels in roots and shoots were quantified. 33P uptake exhibited a 50% increase in quartz sand compared to sandy soil, likely attributed to higher P adsorption to the sandy soil matrix than to quartz sand. Notably, only 60% of the absorbed 33P was translocated to the shoot, with the remaining 40% directed to growing root tips of lateral or seminal roots. Phosphor imaging unveiled a continuous rise in 33P signal in the labeled seminal root from immediate post-labeling until 24 h after labeling. The highest 33P activities were concentrated just above the labeled compartment, diminishing in locations farther away. Emerging laterals from the labeled root served as strong sinks for 33P, while a portion was also transported to other seminal roots. Our study quantitatively visualized 33P uptake and translocation dynamics, facilitating future investigations into diverse root regions/types and varying plant growth conditions. This improves our understanding of the significance of different P sources for plant nutrition and potentially enhances models of plant P uptake.

Topsoil dilution by subsoil admixture had less impact on soil organic carbon stock development than fertilizer form and erosion state.

Enhancing the agroecosystems carbon (C) sink function for climate mitigation faced challenges, particularly with traditional measures with limited suitability for increasing soil organic carbon (SOC) stocks. Inducing a SOC undersaturation in the topsoil by abrupt subsoil admixture is a way to create an additional C sink. However, the deep tillage traditionally used for this topsoil dilution was not always successful. It was due to a lack of knowledge and suitable approaches to record the effect of all relevant factors in SOC recovery, including soil conditions and fertilizer forms. We addressed these problems by establishing a three-factorial experiment: I) "moderate topsoil dilution," II) "N fertilization form," and III) "soil erosion state," representing three soil types in the hummocky ground moraine landscape of NE Germany. SOC dynamics were determined over a year of winter rye cropping using a novel robotic chamber system capable of measuring CO2 exchange on 36 experimental plots with a reduced methodological bias than previous measuring systems. The averaged net ecosystem carbon balance, a proxy for SOC stock change, indicated that topsoil dilution only reduced further SOC losses. The N fertilizer form had a significantly stronger and more differentiated effect. While the mineral N fertilization consistently produced only C sources, the organic fertilization, in combination with the diluted topsoil, led to a C sink. This C-sink function was, however, more pronounced in the eroded soil than in the non-eroded soil. Overall, the results have made clear that the impact of topsoil dilution on the further development of the SOC stock is only possible if the effect of other relevant factors, such as N fertilizer form and erosion state, are taken into account.

Impact of vegetation composition and seasonality on sensitivity of modelled CO2 exchange in temperate raised bogs.

Encroachment of vascular plants (VP) in temperate raised bogs, as a consequence of altered hydrological conditions and nutrient input, is widely observed. Effects of such vegetation shift on water and carbon cycles are, however, largely unknown and identification of responsible plant physiological traits is challenging. Process-based modelling offers the opportunity of gaining insights into ecosystem functioning beyond observations, and to infer decisive trait shifts of plant functional groups. We adapted the Soil-Vegetation-Atmosphere Transfer model pyAPES to a temperate raised bog site by calibration against measured peat temperature, water table and surface CO2 fluxes. We identified the most important traits determining CO2 fluxes by conducting Morris sensitivity analysis (MSA) under changing conditions throughout the year and simulated VP encroachment. We further investigated transferability of results to other sites by extending MSA to parameter ranges derived from literature review. We found highly variable intra-annual plant traits importance determining ecosystem CO2 fluxes, but only a partial shift of importance of photosynthetic processes from moss to VP during encroachment. Ecosystem respiration was dominated by peat respiration. Overall, carboxylation rate, base respiration rate and temperature sensitivity (Q10) were most important for determining bog CO2 balance and parameter ranking was robust even under the extended MSA.

Effects of birch encroachment, water table and vegetation on methane emissions from peatland microforms in a rewetted bog.

This study investigated the influence of vegetation and microforms on methane (CH4) balances of a rewetted bog in north-west Germany. The two study sites are in close proximity on the same former peat extraction area, one dominated by Sphagnum-mosses and the other one by a dense Betula pubescens stand with a high Eriophorum vaginatum cover. The contribution of microforms (hummocks/hollows) to CH4 emissions and the effect of Betula encroachment has been studied. Transparent and opaque chambers were used to measure CH4 fluxes every 3-4 weeks during daytime for one year. For the estimation of annual balances, three methods were compared and the method using water level and soil temperature as explanatory variables was selected. Fluxes were scaled to the site level. The annual emissions per site are and 7.1 ± 1.5 g CH4-C m-2 year-1 at the treed site and 36.1 ± 3.5 g CH4-C m-2 year-1 at the open site, mainly controlled by higher water levels. Highest annual emissions originated from hollows at the open site, but in the vegetation period, hummock emissions tend to be higher. At the tree site, emission differences between the microforms were less pronounced. There were no differences between fluxes from transparent and opaque chambers.

Interaction between beech and spruce trees in temperate forests affects water use, root water uptake pattern and canopy structure.

Beneficial and negative effects of species interactions can strongly influence water fluxes in forest ecosystems. However, little is known about how trees dynamically adjust their water use when growing with interspecific neighbours. Therefore, we investigated the interaction effects between Fagus sylvatica (European beech) and Picea abies (Norway spruce) on water-use strategies and aboveground structural characteristics. We used continuous in situ isotope spectroscopy of xylem and soil water to investigate source water dynamics and root water uptake depths. Picea abies exhibited a reduced sun-exposed crown area in equally mixed compared with spruce-dominated sites, which was further correlated to a reduction in sap flow of -14.5 ± 8.2%. Contrarily, F. sylvatica trees showed +13.3 ± 33.3% higher water fluxes in equally mixed compared with beech-dominated forest sites. Although a significantly higher crown interference by neighbouring trees was observed, no correlation of water fluxes and crown structure was found. High time-resolved xylem δ2H values showed a large plasticity of tree water use (-74.1 to -28.5‰), reflecting the δ2H dynamics of soil and especially precipitation water sources. Fagus sylvatica in equally mixed sites shifted water uptake to deeper soil layers, while uptake of fresh precipitation was faster in beech-dominated sites. Our continuous in situ water stable isotope measurements traced root water uptake dynamics at unprecedented temporal resolution, indicating highly dynamic use of water sources in response to precipitation and to neighbouring species competition. Understanding this plasticity may be highly relevant in the context of increasing water scarcity and precipitation variability under climate change.

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.

Challenges in studying water fluxes within the soil-plant-atmosphere continuum: A tracer-based perspective on pathways to progress.

Tracing and quantifying water fluxes in the hydrological cycle is crucial for understanding the current state of ecohydrological systems and their vulnerability to environmental change. Especially the interface between ecosystems and the atmosphere that is strongly mediated by plants is important to meaningfully describe ecohydrological system functioning. Many of the dynamic interactions generated by water fluxes between soil, plant and the atmosphere are not well understood, which is partly due to a lack of interdisciplinary research. This opinion paper reflects the outcome of a discussion among hydrologists, plant ecophysiologists and soil scientists on open questions and new opportunities for collaborative research on the topic "water fluxes in the soil-plant-atmosphere continuum" especially focusing on environmental and artificial tracers. We emphasize the need for a multi-scale experimental approach, where a hypothesis is tested at multiple spatial scales and under diverse environmental conditions to better describe the small-scale processes (i.e., causes) that lead to large-scale patterns of ecosystem functioning (i.e., consequences). Novel in-situ, high-frequency measurement techniques offer the opportunity to sample data at a high spatial and temporal resolution needed to understand the underlying processes. We advocate for a combination of long-term natural abundance measurements and event-based approaches. Multiple environmental and artificial tracers, such as stable isotopes, and a suite of experimental and analytical approaches should be combined to complement information gained by different methods. Virtual experiments using process-based models should be used to inform sampling campaigns and field experiments, e.g., to improve experimental designs and to simulate experimental outcomes. On the other hand, experimental data are a pre-requisite to improve our currently incomplete models. Interdisciplinary collaboration will help to overcome research gaps that overlap across different earth system science fields and help to generate a more holistic view of water fluxes between soil, plant and atmosphere in diverse ecosystems.

Tracing plant source water dynamics during drought by continuous transpiration measurements: An in-situ stable isotope approach.

The isotopic composition of xylem water (δX ) is of considerable interest for plant source water studies. In-situ monitored isotopic composition of transpired water (δT ) could provide a nondestructive proxy for δX -values. Using flow-through leaf chambers, we monitored 2-hourly δT -dynamics in two tropical plant species, one canopy-forming tree and one understory herbaceous species. In an enclosed rainforest (Biosphere 2), we observed δT -dynamics in response to an experimental severe drought, followed by a 2 H deep-water pulse applied belowground before starting regular rain. We also sampled branches to obtain δX -values from cryogenic vacuum extraction (CVE). Daily flux-weighted δ18 OT -values were a good proxy for δ18 OX -values under well-watered and drought conditions that matched the rainforest's water source. Transpiration-derived δ18 OX -values were mostly lower than CVE-derived values. Transpiration-derived δ2 HX -values were relatively high compared to source water and consistently higher than CVE-derived values during drought. Tracing the 2 H deep-water pulse in real-time showed distinct water uptake and transport responses: a fast and strong contribution of deep water to canopy tree transpiration contrasting with a slow and limited contribution to understory species transpiration. Thus, the in-situ transpiration method is a promising tool to capture rapid dynamics in plant water uptake and use by both woody and nonwoody species.

Fertilized graminoids intensify negative drought effects on grassland productivity.

Droughts can strongly affect grassland productivity and biodiversity, but responses differ widely. Nutrient availability may be a critical factor explaining this variation, but is often ignored in analyses of drought responses. Here, we used a standardized nutrient addition experiment covering 10 European grasslands to test if full-factorial nitrogen, phosphorus, and potassium addition affected plant community responses to inter-annual variation in drought stress and to the extreme summer drought of 2018 in Europe. We found that nutrient addition amplified detrimental drought effects on community aboveground biomass production. Drought effects also differed between functional groups, with a negative effect on graminoid but not forb biomass production. Our results imply that eutrophication in grasslands, which promotes dominance of drought-sensitive graminoids over forbs, amplifies detrimental drought effects. In terms of climate change adaptation, agricultural management would benefit from taking into account differential drought impacts on fertilized versus unfertilized grasslands, which differ in ecosystem services they provide to society.

Borehole Equilibration: Testing a New Method to Monitor the Isotopic Composition of Tree Xylem Water in situ.

Forest water use has been difficult to quantify. One promising approach is to measure the isotopic composition of plant water, e.g., the transpired water vapor or xylem water. Because different water sources, e.g., groundwater versus shallow soil water, often show different isotopic signatures, isotopes can be used to investigate the depths from which plants take up their water and how this changes over time. Traditionally such measurements have relied on the extraction of wood samples, which provide limited time resolution at great expense, and risk possible artifacts. Utilizing a borehole drilled through a tree's stem, we propose a new method based on the notion that water vapor in a slow-moving airstream approaches isotopic equilibration with the much greater mass of liquid water in the xylem. We present two empirical data sets showing that the method can work in practice. We then present a theoretical model estimating equilibration times and exploring the limits at which the approach will fail. The method provides a simple, cheap, and accurate means of continuously estimating the isotopic composition of the source water for transpiration.

Water Stable Isotopes in Ecohydrological Field Research: Comparison Between In Situ and Destructive Monitoring Methods to Determine Soil Water Isotopic Signatures.

Ecohydrological isotope based field research is often constrained by a lack of temporally explicit soil water data, usually related to the choice of destructive sampling in the field and subsequent analysis in the laboratory. New techniques based on gas permeable membranes allow to sample soil water vapor in situ and infer soil liquid water isotopic signatures. Here, a membrane-based in situ soil water vapor sampling method was tested at a grassland site in Freiburg, Germany. It was further compared with two commonly used destructive sampling approaches for determination of soil liquid water isotopic signatures: cryogenic vacuum extraction and centrifugation. All methods were tested under semi-controlled field conditions, conducting an experiment with dry-wet cycling and two isotopically different labeling irrigation waters. We found mean absolute differences between cryogenic vacuum extraction and in situ vapor measurements of 0.3-14.2‰ (δ18O) and 0.4-152.2‰ (δ2H) for soil liquid water. The smallest differences were found under natural abundance conditions of 2H and 18O, the strongest differences were observed after irrigation with labeled waters. Labeling strongly increased the isotopic variation in soil water: Mean soil water isotopic signatures derived by cryogenic vacuum extraction were -11.6 ± 10.9‰ (δ18O) and +61.9 ± 266.3‰ (δ2H). The in situ soil water vapor method showed isotopic signatures of -12.5 ± 9.4‰ (δ18O) and +169.3 ± 261.5‰ (δ2H). Centrifugation was unsuccessful for soil samples due to low water recovery rates. It is therefore not recommended. Our study highlights that the in situ soil water vapor method captures the temporal dynamics in the isotopic signature of soil water well while the destructive approach also includes the natural lateral isotopic heterogeneity. The different advantages and limitations of the three methods regarding setup, handling and costs are discussed. The choice of method should not only consider prevailing environmental conditions but the experimental design and goal. We see a very promising tool in the in situ soil water vapor method, capturing both temporal developments and spatial variability of soil water processes.

Investigating the root plasticity response of Centaurea jacea to soil water availability changes from isotopic analysis.

Root water uptake is a key ecohydrological process for which a physically based understanding has been developed in the past decades. However, due to methodological constraints, knowledge gaps remain about the plastic response of whole plant root systems to a rapidly changing environment. We designed a laboratory system for nondestructive monitoring of stable isotopic composition in plant transpiration of a herbaceous species (Centaurea jacea) and of soil water across depths, taking advantage of newly developed in situ methods. Daily root water uptake profiles were obtained using a statistical Bayesian multisource mixing model. Fast shifts in the isotopic composition of both soil and transpiration water could be observed with the setup and translated into dynamic and pronounced shifts of the root water uptake profile, even in well watered conditions. The incorporation of plant physiological and soil physical information into statistical modelling improved the model output. A simple exercise of water balance closure underlined the nonunique relationship between root water uptake profile on the one hand, and water content and root distribution profiles on the other, illustrating the continuous adaption of the plant water uptake as a function of its root hydraulic architecture and soil water availability during the experiment.

Nitrogen Loading Enhances Stress Impact of Drought on a Semi-natural Temperate Grassland.

Two important threats to the sustainable functioning of seminatural grasslands in temperate zones are (1) nutrient loading due to agricultural fertilization and pollution, and (2) the increase of extreme drought events due to climate change. These threats may cause substantial shifts in species diversity and abundance and considerably affect the carbon and water balance of ecosystems. The synergistic effects between those two threats, however, can be complex and are poorly understood. Here, we experimentally investigated the effects of nitrogen addition and extreme drought (separately and in combination) on a seminatural temperate grassland, located in Freiburg (South Germany). To study the grassland response, we combined eddy-covariance techniques with open gas exchange systems. Open gas exchange chambers were connected to an infrared gas analyzer and water isotope spectrometer, which allowed the partitioning of net ecosystem exchange and evapotranspiration. Vegetation parameters were described by species richness, species abundance, and leaf area index. Our results suggest that grassland communities, strongly weakened in their stress response by nitrogen loading, can substantially lose their carbon sink function during drought. While nitrogen addition caused a significant loss in forb species (-25%), precipitation reduction promoted a strong dominance of grass species at season start. Consequently, the grass-dominated and species-poor community suffered from a strong above-ground dieback during the dry summer months, likely caused by lower water use efficiency and weaker drought adaptations of the species community. Over the growing season (April-September), the carbon sequestration of the studied grassland was reduced by more than 60% as a consequence of nitrogen addition. Nitrogen addition in combination with precipitation reduction decreased carbon sequestration by 73%. Eutrophication can severely threaten the resilient functioning of grasslands, in particular when drought periods will increase as predicted by future climate scenarios. Our findings emphasize the importance of preserving high diversity of grasslands to strengthen their resistance against extreme events such as droughts.

A pool-weighted perspective on the two-water-worlds hypothesis.

The 'two-water-worlds' hypothesis is based on stable isotope differences in stream, soil and xylem waters in dual isotope space. It postulates no connectivity between bound and mobile soil waters, and preferential plant water uptake of bound soil water sources. We tested the pool-weighted impact of isotopically distinct water pools for hydrological cycling, the influence of species-specific water use and the degree of ecohydrological separation. We combined stable isotope analysis (δ18 O and δ2 H) of ecosystem water pools of precipitation, groundwater, soil and xylem water of two distinct species (Quercus suber, Cistus ladanifer) with observations of soil water contents and sap flow. Shallow soil water was evaporatively enriched during dry-down periods, but enrichment faded strongly with depth and upon precipitation events. Despite clearly distinct water sources and water-use strategies, both species displayed a highly opportunistic pattern of root water uptake. Here we offer an alternative explanation, showing that the isotopic differences between soil and plant water vs groundwater can be fully explained by spatio-temporal dynamics. Pool weighting the contribution of evaporatively enriched soil water reveals only minor annual impacts of these sources to ecosystem water cycling (c. 11% of bulk soil water and c. 14% of transpired water).

Water fluxes mediated by vegetation: emerging isotopic insights at the soil and atmosphere interfaces.

Plants mediate water fluxes within the soil-vegetation-atmosphere continuum. This water transfer in soils, through plants, into the atmosphere can be effectively traced by stable isotopologues of water. However, rapid dynamic processes have only recently gained attention, such as adaptations in root water uptake depths (within hours to days) or the imprint of transpirational fluxes on atmospheric moisture, particularly promoted by the development of real-time in-situ water vapour stable isotope observation techniques. We focus on open questions and emerging insights at the soil-plant and plant-atmosphere interfaces, as we believe that these are the controlling factors for ecosystem water cycling. At both interfaces, complex pictures of interacting ecophysiological and hydrological processes emerge: root water uptake dynamics depend on both spatiotemporal variations in water availability and species-specific regulation of adaptive root conductivity within the rooting system by, for example, modulating soil-root conductivity in response to water and nutrient demands. Similarly, plant water transport and losses are a fine-tuned interplay between species-specific structural and functional strategies of water use and atmospheric processes. We propose that only by explicitly merging insights from distinct disciplines - for example, hydrology, plant physiology and atmospheric sciences - will we gain a holistic picture of the impact of vegetation on processes governing the soil-plant-atmosphere continuum.

Terpenoid Emissions of Two Mediterranean Woody Species in Response to Drought Stress.

Drought is a major environmental constrain affecting plant performance and survival, particularly in Mediterranean ecosystems. Terpenoids may play a protective role under these conditions, however, observations of drought effects on plant terpenoid emissions are controversial ranging from decreased emissions to unaffected or increased release of terpenoids. In the present study we investigated terpenoid emissions of cork oak (Quercus suber) and gum rockrose (Cistus ladanifer) in response to summer drought stress in 2017. Pre-dawn leaf water potential (ΨPD) decreased from -0.64 to -1.72 MPa in Q. suber and from -1.69 to -4.05 MPa in C. ladanifer, indicating a transition from mild to severe drought along summer. Total terpenoid emissions decreased with drought, but differed significantly between species (p < 0.001) and in response to ΨPD, air temperature and assimilation rates. C. ladanifer emitted a large variety of >75 compounds comprising monoterpenes, sesquiterpenes and even diterpenes, which strongly decreased from 1.37 ± 0.23 µg g-1h-1 to 0.40 ± 0.08 µg g-1h-1 (p < 0.001) in response to drought. Total emission rates were positively correlated to air temperature (p < 0.001). C. ladanifer behavior points toward terpenoid leaf storage depletion and reduced substrate availability for terpenoid synthesis with increasing drought, most likely accelerated by high air temperatures. Q. suber emitted mainly monoterpenes and emissions declined significantly from June (0.50 ± 0.08 µg g-1h-1) to August (0.29 ± 0.02 µg g-1h-1) (p < 0.01). Emission rates were weakly correlated with net assimilation rates (R2 = 0.19, p < 0.001), but did not respond strongly to ΨPD and air temperature. Early onset of drought in 2017 most likely reduced plant metabolism in Q. suber, resulting in diminished, but stable terpenoid fluxes. Calculation of standard emission factors (at 30°C) revealed contrasting emission patterns of decreasing, unaffected, or increasing fluxes of single terpenoid compounds. Unaffected or drought-enhanced emissions of compounds such as α-pinene, camphene or manoyl oxide may point toward a specific role of these terpenoids in abiotic stress adaptation. In conclusion, these results suggest a strong negative, but species- and compound-specific effect of severe drought on terpenoid fluxes in Mediterranean ecosystems.

Quantifying differences in water and carbon cycling between paddy and rainfed rice (Oryza sativa L.) by flux partitioning.

Agricultural crops play an important role in the global carbon and water cycle. Global climate change scenarios predict enhanced water scarcity and altered precipitation pattern in many parts of the world. Hence, a mechanistic understanding of water fluxes, productivity and water use efficiency of cultivated crops is of major importance, i.e. to adapt management practices. We compared water and carbon fluxes of paddy and rainfed rice by canopy scale gas exchange measurements, crop growth, daily evapotranspiration, transpiration and carbon flux modeling. Throughout a monsoon rice growing season, soil evaporation in paddy rice contributed strongly to evapotranspiration (96.6% to 43.3% from initial growth to fully developed canopy and amounted to 57.9% of total water losses over the growing seasons. Evaporation of rainfed rice was significantly lower (by 65% on average) particularly before canopy closure. Water use efficiency (WUE) was significantly higher in rainfed rice both from an agronomic (WUEagro, i.e. grain yield per evapotranspiration) and ecosystem (WUEeco, i.e. gross primary production per evapotranspiration) perspective. However, our results also show that higher WUE in rainfed rice comes at the expense of higher respiration losses compared to paddy rice (26% higher on average). Hence, suggestions on water management depend on the regional water availability (i.e. Mediterranean vs. Monsoon climate) and the balance between higher respiratory losses versus a potential reduction in CH4 and other greenhouse gas emissions. Our results suggest that a shift from rainfed/unsaturated soil to waterlogged paddy conditions after closure of the rice canopy might be a good compromise towards a sustainable use of water while preserving grain yield, particularly for water-limited production areas.

Impact of Leaf Traits on Temporal Dynamics of Transpired Oxygen Isotope Signatures and Its Impact on Atmospheric Vapor.

Oxygen isotope signatures of transpiration (δ E ) are powerful tracers of water movement from plant to global scale. However, a mechanistic understanding of how leaf morphological/physiological traits effect δ E is missing. A laser spectrometer was coupled to a leaf-level gas-exchange system to measure fluxes and isotopic signatures of plant transpiration under controlled conditions in seven distinct species (Fagus sylvatica, Pinus sylvestris, Acacia longifolia, Quercus suber, Coffea arabica, Plantago lanceolata, Oxalis triangularis). We analyzed the role of stomatal conductance (gs ) and leaf water content (W) on the temporal dynamics of δ E following changes in relative humidity (rH). Changes in rH were applied from 60 to 30% and from 30 to 60%, which is probably more than covering the maximum step changes occurring under natural conditions. Further, the impact of gs and W on isotopic non-steady state isofluxes was analyzed. Following changes in rH, temporal development of δ E was well described by a one-pool modeling approach for most species. Isofluxes of δ E were dominantly driven by stomatal control on E, particularly for the initial period of 30 min following a step change. Hence, the deviation of isofluxes from isotopic steady state can be large, even though plants transpire near to isotopic steady state. Notably, not only transpiration rate and stomatal conductance, but also the leaf traits stomatal density (as a measure of gmax) and leaf water content are significantly related to the time constant (τ) and non-steady-state isofluxes. This might provide an easy-to-access means of a priori assumptions for the impact of isotopic non-steady-state transpiration in various ecosystems. We discuss the implications of our results from leaf to ecosystem scale.