Different model assumptions about plant hydraulics and photosynthetic temperature acclimation yield diverging implications for tropical forest gross primary production under warming.

Tropical forest photosynthesis can decline at high temperatures due to (1) biochemical responses to increasing temperature and (2) stomatal responses to increasing vapor pressure deficit (VPD), which is associated with increasing temperature. It is challenging to disentangle the influence of these two mechanisms on photosynthesis in observations, because temperature and VPD are tightly correlated in tropical forests. Nonetheless, quantifying the relative strength of these two mechanisms is essential for understanding how tropical gross primary production (GPP) will respond to climate change, because increasing atmospheric CO2 concentration may partially offset VPD-driven stomatal responses, but is not expected to mitigate the effects of temperature-driven biochemical responses. We used two terrestrial biosphere models to quantify how physiological process assumptions (photosynthetic temperature acclimation and plant hydraulic stress) and functional traits (e.g., maximum xylem conductivity) influence the relative strength of modeled temperature versus VPD effects on light-saturated GPP at an Amazonian forest site, a seasonally dry tropical forest site, and an experimental tropical forest mesocosm. By simulating idealized climate change scenarios, we quantified the divergence in GPP predictions under model configurations with stronger VPD effects compared with stronger direct temperature effects. Assumptions consistent with stronger direct temperature effects resulted in larger GPP declines under warming, while assumptions consistent with stronger VPD effects resulted in more resilient GPP under warming. Our findings underscore the importance of quantifying the role of direct temperature and indirect VPD effects for projecting the resilience of tropical forests in the future, and demonstrate that the relative strength of temperature versus VPD effects in models is highly sensitive to plant functional parameters and structural assumptions about photosynthetic temperature acclimation and plant hydraulics.

Mesophyll airspace unsaturation drives C4 plant success under vapor pressure deficit stress.

A fundamental assumption in plant science posits that leaf air spaces remain vapor saturated, leading to the predominant view that stomata alone control leaf water loss. This concept has been pivotal in photosynthesis and water-use efficiency research. However, recent evidence has refuted this longstanding assumption by providing evidence of unsaturation in the leaf air space of C3 plants under relatively mild vapor pressure deficit (VPD) stress. This phenomenon represents a nonstomatal mechanism restricting water loss from the mesophyll. The potential ubiquity and physiological implications of this phenomenon, its driving mechanisms in different plant species and habitats, and its interaction with other ecological adaptations have. In this context, C4 plants spark particular interest for their importance as crops, bundle sheath cells' unique anatomical characteristics and specialized functions, and notably higher water-use efficiency relative to C3 plants. Here, we confirm reduced relative humidities in the substomatal cavity of the C4 plants maize, sorghum, and proso millet down to 80% under mild VPD stress. We demonstrate the critical role of nonstomatal control in these plants, indicating that the role of the CO2 concentration mechanism in CO2 management at a high VPD may have been overestimated. Our findings offer a mechanistic reconciliation between discrepancies in CO2 and VPD responses reported in C4 species. They also reveal that nonstomatal control is integral to maintaining an advantageous microclimate of relatively higher CO2 concentrations in the mesophyll air space of C4 plants for carbon fixation, proving vital when these plants face VPD stress.

Nearly instantaneous stem diameter response to fluctuations in the atmospheric water demand.

Changes in vapour pressure deficit can lead to the depletion and replenishment of stem water pools to buffer water potential variations in the xylem. Yet, the precise velocity at which stem water pools track environmental cues remains poorly explored. Nine eucalyptus seedlings grown in a glasshouse experienced high-frequency environmental oscillations and their stem radial variations (ΔR) were monitored at a 30-s temporal resolution in upper and lower stem locations and on the bark and xylem. The stem ΔR response to vapour pressure deficit changes was nearly instantaneous (<1 min), while temperature lagged behind stem ΔR. No temporal differences in the stem ΔR response were observed between locations. Punctual gravimetric measurements confirmed the synchrony between transpiration and stem ΔR dynamics. These results indicate (i) that stem-stored water can respond to the atmospheric evaporative demand much faster than commonly assumed and (ii) that the origin of the water released to the transpiration stream seems critical in determining time lags in stem water pool dynamics. Near-zero time lags may be explained by the high elasticity of eucalyptus woody tissues and the predominant water use from the xylem, circumventing the hydraulic radial barriers to water flow from/to the outer tissues.

Tropospheric ozone pollution increases the sensitivity of plant production to vapor pressure deficit across diverse ecosystems in the Northern Hemisphere.

Tropospheric ozone (O3) pollution often accompanies droughts and heatwaves, which could collectively reduce plant productivity. Previous research suggested that O3 pollution can alter plant responses to drought by interfering with stomatal closure while drought can reduce stomatal conductance and provide protection against O3 stress. However, the interactions between O3 pollution and drought stress remain poorly understood at ecosystem scales with diverse plant functional types. To address this research gap, we used 10-year (2012-2021) satellite near-infrared reflectance of vegetation (NIRv) observations, reanalysis data of vapor pressure deficit (VPD), soil moisture (SM), and air temperature (Ta), along with O3 measurements and reanalysis data across the Northern Hemisphere to statistically disentangle the interconnections between NIRv, VPD, SM, and Ta under varying O3 levels. We found that high O3 concentrations significantly exacerbate the sensitivity of NIRv to VPD while have no notable impacts on the sensitivity of NIRv to Ta or SM for all plant functional types, indicating an enhanced combined impact of VPD and O3 on plants. Specifically, the sensitivity of NIRv to VPD increased by >75 % when O3 anomalies increased from the lowest 10 to the highest 10 percentiles across diverse plant functional types. This is likely because long-term exposure to high O3 concentrations can inhibit stomatal closure and photosynthetic enzyme activities, resulting in reduced water use efficiency and photosynthetic efficiency. This study highlights the need to consider O3 in understanding plant responses to climate factors and that O3 can alter plant responses to VPD independently of Ta and SM.

Hydrogen isotope fractionation in plants with C3, C4, and CAM CO2 fixation.

Measurements of stable isotope ratios in organic compounds are widely used tools for plant ecophysiological studies. However, the complexity of the processes involved in shaping hydrogen isotope values (δ2H) in plant carbohydrates has limited its broader application. To investigate the underlying biochemical processes responsible for 2H fractionation among water, sugars, and cellulose in leaves, we studied the three main CO2 fixation pathways (C3, C4, and CAM) and their response to changes in temperature and vapor pressure deficit (VPD). We show significant differences in autotrophic 2H fractionation (εA) from water to sugar among the pathways and their response to changes in air temperature and VPD. The strong 2H depleting εA in C3 plants is likely driven by the photosynthetic H+ production within the thylakoids, a reaction that is spatially separated in C4 and strongly reduced in CAM plants, leading to the absence of 2H depletion in the latter two types. By contrast, we found that the heterotrophic 2H-fractionation (εH) from sugar to cellulose was very similar among the three pathways and is likely driven by the plant's metabolism, rather than by isotopic exchange with leaf water. Our study offers new insights into the biochemical drivers of 2H fractionation in plant carbohydrates.

Dry inside: progressive unsaturation within leaves with increasing vapour pressure deficit affects estimation of key leaf gas exchange parameters.

Climate change not only leads to higher air temperatures but also increases the vapour pressure deficit (VPD) of the air. Understanding the direct effect of VPD on leaf gas exchange is crucial for precise modelling of stomatal functioning. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a VPD range of 0.8-3.6 kPa, while maintaining constant temperatures without soil water limitation. In addition to applying the standard assumption of saturated vapour pressure inside leaves (ei), we inferred ei from oxygen isotope discrimination of CO2 and water vapour. ei desaturated progressively with increasing VPD, consistently across species, resulting in an intercellular relative humidity as low as 0.73 ± 0.11 at the highest tested VPD. Assuming saturation of ei overestimated the extent of reductions in stomatal conductance and CO2 mole fraction inside leaves in response to increasing VPD compared with calculations that accounted for unsaturation. In addition, a significant decrease in mesophyll conductance with increasing VPD only occurred when the unsaturation of ei was considered. We suggest that the possibility of unsaturated ei should not be overlooked in measurements related to leaf gas exchange and in stomatal models, especially at high VPD.

Coping with extremes: Responses of Quercus robur L. and Fagus sylvatica L. to soil drought and elevated vapour pressure deficit.

Climate change, particularly droughts and heat waves, significantly impacts global photosynthesis and forest ecosystem sustainability. To understand how trees respond to and recover from hydrological stress, we investigated the combined effects of soil moisture and atmospheric vapour pressure deficit (VPD) on seedlings of the two major European broadleaved tree species Fagus sylvatica (FS) and Quercus robur (QR). The experiment was conducted under natural forest gap conditions, while soil water availability was strictly manipulated. We monitored gas exchange (net photosynthesis, stomatal conductance and transpiration rates), nonstructural carbohydrates (NSC) concentration in roots and stomatal morphometry (size and density) during a drought period and recovery. Our comparative empirical study allowed us to distinguish and quantify the effects of soil drought and VPD on stomatal behavior, going beyond theoretical models. We found that QR conserved water more conservatively than FS by reducing transpiration and regulating stomatal conductance under drought. FS maintained higher stomatal conductance and transpiration at elevated VPD until soil moisture became critically low. QR showed higher intrinsic water use efficiency than FS. Stomata density and size also likely played a role in photosynthetic rate and speed of recovery, especially since QR with its seasonal adjustments in stomatal traits (smaller, more numerous stomata in summer leaves) responded and recovered faster compared to FS. Our focal species showed different responses in NSC content under drought stress and recovery, suggesting possible different evolutionary pathways in coping with stress. QR mobilized soluble sugars, while FS relied on starch mobilization to resist drought. Although our focal species often co-occur in mixed forests, our study showed that they have evolved different physiological, morphological and biochemical strategies to cope with drought stress. This suggests that ongoing climate change may alter their competitive ability and adaptive potential in favor of one of the species studied.

Decoding stomatal characteristics regulating water use efficiency at leaf and plant scales in rice genotypes.

MAIN CONCLUSION: Stomatal traits in rice genotypes affect water use efficiency. Low-frequency small-size stomata correlate with whole plant efficiency, while low-frequency large-size stomata show intrinsic efficiency and responsiveness to vapour pressure deficit. Leaf surface and the patterning of the epidermal layer play a vital role in determining plant growth. While the surface helps in determining radiation interception, epidermal pattern of stomatal factors strongly regulate gas exchange and water use efficiency (WUE). This study focuses on identifying distinct stomatal traits among rice genotypes to comprehend their influence on WUE. Stomatal frequency ranged from 353 to 687 per mm2 and the size varied between 128.31 and 339.01 µm2 among 150 rice germplasm with significant variability in abaxial and adaxial surfaces. The cumulative water transpired and WUE determined at the outdoor phenomics platform, over the entire crop growth period as well as during specific hours of a 24 h-day did not correlate with stomatal frequency nor size. However, genotypes with low-frequency and large-size stomata recorded higher intrinsic water use efficiency (67.04 µmol CO2 mol-1 H2O) and showed a quicker response to varying vapour pressure deficit that diurnally ranged between 0.03 and 2.17 kPa. The study demonstrated the role of stomatal factors in determining physiological subcomponents of WUE both at single leaf and whole plant levels. Differential expression patterns of stomatal regulatory genes among the contrasting groups explained variations in the epidermal patterning. Increased expression of ERECTA, TMM and YODA genes appear to contribute to decreased stomatal frequency in low stomatal frequency genotypes. These findings underscore the significance of stomatal traits in breeding programs and strongly support the importance of these genes that govern variability in stomatal architecture in future crop improvement programs.

Enhanced decreases in rice evapotranspiration in response to elevated atmospheric carbon dioxide under warmer environments.

A short period of exposure to elevated CO2 is known to decrease evapotranspiration via stomatal closure. Based on theoretical evaluation of a canopy transpiration model, we hypothesized that this decrease in the evapotranspiration of rice under elevated CO2 was greater under higher temperature conditions due to an increased sensitivity of transpiration to changes in CO2 induced by the greater vapour pressure deficit. In a temperature gradient chamber-based experiment, a 200 ppm increase in CO2 concentration led to 0.4 mm (-7%) and 1.5 mm (-15%) decreases in 12 h evapotranspiration under ambient temperature and high temperature (+3.7°C) conditions, respectively. Model simulations revealed that the greater vapour pressure deficit under higher temperature conditions explained the variations in the reduction of evapotranspiration observed under elevated CO2 levels between the temperature treatments. Our study suggests the utility of a simple modelling framework for mechanistic understanding of evapotranspiration and crop energy balance system under changing environmental conditions.

The multidimensionality of plant drought stress: The relative importance of edaphic and atmospheric drought.

Drought threatens plant growth and related ecosystem services. The emergence of plant drought stress under edaphic drought is well studied, whilst the importance of atmospheric drought only recently gained momentum. Yet, little is known about the interaction and relative contribution of edaphic and atmospheric drought on the emergence of plant drought stress. We conducted a gradient experiment, fully crossing gravimetric water content (GWC: maximum water holding capacity-permanent wilting point) and vapour pressure deficit (VPD: 1-2.25 kPa) using five wheat varieties from three species (Triticum monococcum, T. durum & T. aestivum). We quantified the occurrence of plant drought stress on molecular (abscisic acid), cellular (stomatal conductance), organ (leaf water potential) and stand level (evapotranspiration). Plant drought stress increased with decreasing GWC across all organizational levels. This effect was magnified nonlinearly by VPD after passing a critical threshold of soil water availability. At around 20%GWC (soil matric potential 0.012 MPa), plants lost their ability to regulate leaf water potential via stomata regulation, followed by the emergence of hydraulic dysfunction. The emergence of plant drought stress is characterized by changing relative contributions of soil versus atmosphere and their non-linear interaction. This highly non-linear response is likely to abruptly alter plant-related ecosystem services in a drying world.

Stomatal CO2 responses at sub- and above-ambient CO2 levels employ different pathways in Arabidopsis.

Stomatal pores that control plant CO2 uptake and water loss affect global carbon and water cycles. In the era of increasing atmospheric CO2 levels and vapor pressure deficit (VPD), it is essential to understand how these stimuli affect stomatal behavior. Whether stomatal responses to sub-ambient and above-ambient CO2 levels are governed by the same regulators and depend on VPD remains unknown. We studied stomatal conductance responses in Arabidopsis (Arabidopsis thaliana) stomatal signaling mutants under conditions where CO2 levels were either increased from sub-ambient to ambient (400 ppm) or from ambient to above-ambient levels under normal or elevated VPD. We found that guard cell signaling components involved in CO2-induced stomatal closure have different roles in the sub-ambient and above-ambient CO2 levels. The CO2-specific regulators prominently affected sub-ambient CO2 responses, whereas the lack of guard cell slow-type anion channel SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) more strongly affected the speed of above-ambient CO2-induced stomatal closure. Elevated VPD caused lower stomatal conductance in all studied genotypes and CO2 transitions, as well as faster CO2-responsiveness in some studied genotypes and CO2 transitions. Our results highlight the importance of experimental setups in interpreting stomatal CO2-responsiveness, as stomatal movements under different CO2 concentration ranges are controlled by distinct mechanisms. Elevated CO2 and VPD responses may also interact. Hence, multi-factor treatments are needed to understand how plants integrate different environmental signals and translate them into stomatal responses.

The stomatal response to vapor pressure deficit drives the apparent temperature response of photosynthesis in tropical forests.

As temperature rises, net carbon uptake in tropical forests decreases, but the underlying mechanisms are not well understood. High temperatures can limit photosynthesis directly, for example by reducing biochemical capacity, or indirectly through rising vapor pressure deficit (VPD) causing stomatal closure. To explore the independent effects of temperature and VPD on photosynthesis we analyzed photosynthesis data from the upper canopies of two tropical forests in Panama with Generalized Additive Models. Stomatal conductance and photosynthesis consistently decreased with increasing VPD, and statistically accounting for VPD increased the optimum temperature of photosynthesis (Topt) of trees from a VPD-confounded apparent Topt of c. 30-31°C to a VPD-independent Topt of c. 33-36°C, while for lianas no VPD-independent Topt was reached within the measured temperature range. Trees and lianas exhibited similar temperature and VPD responses in both forests, despite 1500 mm difference in mean annual rainfall. Over ecologically relevant temperature ranges, photosynthesis in tropical forests is largely limited by indirect effects of warming, through changes in VPD, not by direct warming effects of photosynthetic biochemistry. Failing to account for VPD when determining Topt misattributes the underlying causal mechanism and thereby hinders the advancement of mechanistic understanding of global warming effects on tropical forest carbon dynamics.

A medida que aumenta la temperatura, disminuye la absorción neta de carbono en los bosques tropicales, sin embargo, aún no se conocen bien los mecanismos que la subyacen. Las altas temperaturas pueden limitar la fotosíntesis directamente, por ejemplo, reduciendo la eficiencia de los procesos bioquímicos, pero también de forma indirecta a través del aumento del déficit de presión de vapor (DPV) que resulta en el cierre estomático. Para explorar los efectos independientes de la temperatura y el DPV en la fotosíntesis, analizamos datos de la absorción neta de carbono del dosel de dos bosques tropicales en Panamá utilizando modelos aditivos generalizados. La conductancia estomática y la fotosíntesis disminuyó consistentemente con el aumento de DPV, y considerando el DPV en modelas estadísticas, la temperatura óptima de la fotosíntesis (Topt) aumentó, de un Topt aparente influida por la DVP de c. 30­31°C a un Topt independiente del DPV de c. 33­36°C. Los árboles y las lianas mostraron respuestas similares a la temperatura y a la DVP en ambos bosques, a pesar de la diferencia de 1500 mm en la precipitación media anual. La fotosíntesis en los bosques tropicales está limitada en gran medida por los efectos indirectos del aumento de la temperatura, a través de cambios en el DPV y no por los efectos directos en los procesos bioquímicos. Si no se tiene en cuenta el DPV al determinar el Topt, se atribuye erróneamente el mecanismo causal subyacente y, por lo tanto, se obstaculiza el avance en la comprensión de los efectos del calentamiento global en la dinámica del carbono.

Impacts of elevated temperature and vapour pressure deficit on leaf gas exchange and plant growth across six tropical rainforest tree species.

Elevated air temperature (Tair) and vapour pressure deficit (VPDair) significantly influence plant functioning, yet their relative impacts are difficult to disentangle. We examined the effects of elevated Tair (+6°C) and VPDair (+0.7 kPa) on the growth and physiology of six tropical tree species. Saplings were grown under well-watered conditions in climate-controlled glasshouses for 6 months under three treatments: (1) low Tair and low VPDair, (2) high Tair and low VPDair, and (3) high Tair and high VPDair. To assess acclimation, physiological parameters were measured at a set temperature. Warm-grown plants grown under elevated VPDair had significantly reduced stomatal conductance and increased instantaneous water use efficiency compared to plants grown under low VPDair. Photosynthetic biochemistry and thermal tolerance (Tcrit) were unaffected by VPDair, but elevated Tair caused Jmax25 to decrease and Tcrit to increase. Sapling biomass accumulation for all species responded positively to an increase in Tair, but elevated VPDair limited growth. This study shows that stomatal limitation caused by even moderate increases in VPDair can decrease productivity and growth rates in tropical species independently from Tair and has important implications for modelling the impacts of climate change on tropical forests.

Global convergence in terrestrial gross primary production response to atmospheric vapor pressure deficit.

Atmospheric vapor pressure deficit (VPD) increases with climate warming and may limit plant growth. However, gross primary production (GPP) responses to VPD remain a mystery, offering a significant source of uncertainty in the estimation of global terrestrial ecosystems carbon dynamics. In this study, in-situ measurements, satellite-derived data, and Earth System Models (ESMs) simulations were analysed to show that the GPP of most ecosystems has a similar threshold in response to VPD: first increasing and then declining. When VPD exceeds these thresholds, atmospheric drought stress reduces soil moisture and stomatal conductance, thereby decreasing the productivity of terrestrial ecosystems. Current ESMs underscore CO2 fertilization effects but predict significant GPP decline in low-latitude ecosystems when VPD exceeds the thresholds. These results emphasize the impacts of climate warming on VPD and propose limitations to future ecosystems productivity caused by increased atmospheric water demand. Incorporating VPD, soil moisture, and canopy conductance interactions into ESMs enhances the prediction of terrestrial ecosystem responses to climate change.

Stomatal response to VPD in C4 plants with different biochemical sub-pathways.

C4 NAD-malic enzyme (NAD-ME) species occurs in drier regions and exhibit different drought responses compared to C4 NADP-malic enzyme (NADP-ME) species. However, a physiological mechanism explaining the geographical discrepancies remains uncertain. This study examined gas exchange patterns that might explain different distributions observed between two subtypes of C4 photosynthesis. We measured the response of leaf gas exchange to vapour pressure deficit (VPD) and CO2 in plants from six distinct C4 clades having closely related NAD-ME and NADP-ME species using a Li-Cor 6400 gas exchange system. We found that NAD-ME species exhibited greater relative reductions in stomatal conductance with increases in VPD than NADP-ME species but observed no consistent subtype differences in C4 cycle activity as indicated by the initial slope of the A response to intercellular CO2 concentration. Based on these results, we hypothesise the greater response of gs to increasing VPD may enable NAD-ME plants to outperform NADP-ME plants in hot, dry environments where VPD is normally high.

The poorly-explored stomatal response to temperature at constant evaporative demand.

Changes in leaf temperature are known to drive stomatal responses, because the leaf-to-air water vapour gradient (Δw) increases with temperature if ambient vapour pressure is held constant, and stomata respond to changes in Δw. However, the direct response of stomata to temperature (DRST; the response when Δw is held constant by adjusting ambient humidity) has been examined far less extensively. Though the meagre available data suggest the response is usually positive, results differ widely and defy broad generalisation. As a result, little is known about the DRST. This review discusses the current state of knowledge about the DRST, including numerous hypothesised biophysical mechanisms, potential implications of the response for plant adaptation, and possible impacts of the DRST on plant-atmosphere carbon and water exchange in a changing climate.

Comparing Antoine parameter sources for accurate vapor pressure prediction across a range of temperatures.

Determining the vapor pressure of a substance at the relevant process temperature is a key component in conducting an exposure assessment to ascertain worker exposure. However, vapor pressure data at various temperatures relevant to the work environment is not readily available for many chemicals. The Antoine equation is a mathematical expression that relates temperature and vapor pressure. The objective of this analysis was to compare Antoine parameter data from 3 independent data sources; Hansen, Yaws, and Custom data and identify the source that generates the most accurate vapor pressure values with the least bias, relative to the referent data set from the CRC Handbook of Chemistry and Physics. Temperatures predicted from 3 different Antoine sources across a range of vapor pressures for 59 chemicals are compared to the reference source. The results show that temperatures predicted using Antoine parameters from the 3 sources are not statistically significantly different, indicating that all 3 sources could be useful. However, the Yaws dataset will be used in the SDM 2.0 because the data is readily available and robust.

The impacts of rising vapour pressure deficit in natural and managed ecosystems.

An exponential rise in the atmospheric vapour pressure deficit (VPD) is among the most consequential impacts of climate change in terrestrial ecosystems. Rising VPD has negative and cascading effects on nearly all aspects of plant function including photosynthesis, water status, growth and survival. These responses are exacerbated by land-atmosphere interactions that couple VPD to soil water and govern the evolution of drought, affecting a range of ecosystem services including carbon uptake, biodiversity, the provisioning of water resources and crop yields. However, despite the global nature of this phenomenon, research on how to incorporate these impacts into resilient management regimes is largely in its infancy, due in part to the entanglement of VPD trends with those of other co-evolving climate drivers. Here, we review the mechanistic bases of VPD impacts at a range of spatial scales, paying particular attention to the independent and interactive influence of VPD in the context of other environmental changes. We then evaluate the consequences of these impacts within key management contexts, including water resources, croplands, wildfire risk mitigation and management of natural grasslands and forests. We conclude with recommendations describing how management regimes could be altered to mitigate the otherwise highly deleterious consequences of rising VPD.

Impacts of elevated CO2 levels and temperature on photosynthesis and stomatal closure along an altitudinal gradient are counteracted by the rising atmospheric vapor pressure deficit.

The efficiency of water use in plants, a critical ecophysiological parameter closely related to water and carbon cycles, is essential for understanding the interactions between plants and their environment. This study investigates the effects of ongoing climate change and increasing atmospheric CO2 concentration on intrinsic (stomata-based; iWUE) and evaporative (transpiration-based; eWUE) water use efficiency in oak trees along a naturally small altitudinal gradient (130-630 m a.s.l.) of Vihorlat Mountains (eastern Slovakia, Central Europe). To assess changes in iWUE and eWUE values over the past 60 years (1961-2020), stable carbon isotope ratios in latewood cellulose (δ13Ccell) of annually resolved tree rings were analyzed. Such an approach was sensitive enough to distinguish tree responses to growth environments at different altitudes. Our findings revealed a rising trend in iWUE, particularly in oak trees at low and middle altitudes. However, this increase was negligible at high altitudes. Warmer and drier conditions at lower altitudes likely led to significant stomatal closure and enhanced efficiency in photosynthetic CO2 uptake due to rising CO2 concentration. Conversely, the increasing intracellular-to-ambient CO2 ratio (Ci/Ca) at higher altitudes indicated lower efficiency in photosynthetic CO2 uptake. In contrast to iWUE, eWUE showed no increasing trends over the last 60 years. This suggests that the positive impacts of elevated CO2 concentrations and temperature on photosynthesis and stomatal closure are counteracted by the rising atmospheric vapor pressure deficit (VPD). These differences underscore the importance of the correct interpretation of stomata-based and transpiration-based WUEs and highlight the necessity of atmospheric VPD correction when applying tree-ring δ13C-derived WUE at ecosystem and global levels.