Decoupling of stomatal conductance, transpiration and photosynthesis in terrestrial plants under elevated temperature: a meta-analysis

Domain: biology
Published: 2026-01-08
Editorial outcome: major_revision

Abstract
Elevated temperatures pose challenges to stomatal conductance, which regulates transpiration and photosynthesis. However, the coupling of stomatal conductance, transpiration and photosynthesis may shift with warming. Here, we synthesize evidence from a meta-analysis of 207 studies to assess leaf physiological responses to warming. On average, the responses of stomatal conductance are highly variable, exhibiting no consistent directional trend, whereas transpiration increases and photosynthesis decreases, reflecting a shift towards transpirational cooling. Stomatal conductance declines until temperatures exceed 5°C above ambient, where as transpiration remains stable under warming <3°C. Beyond these two thresholds, both stomatal conductance and transpiration increase with further warming. The sensitivity of stomatal conductance, photosynthesis, and water-use efficiency to warming varies substantially among plant functional types, with distinct responses across life forms, phylogenetic groups, and photosynthetic pathways. Higher mean annual temperature amplifies the positive responses of stomatal conductance and transpiration to warming, whereas greater mean annual precipitation mitigates the warming-induced declines in photosynthesis. Elevated CO2 exacerbates warming-induced declines in photosynthesis, while drought constrains transpirational cooling. Collectively, these findings highlight a progressive decoupling of stomatal conductance, transpiration and photosynthesis with warming, revealing complex trade-offs between plant water use, thermal regulation, and carbon assimilation.

Introduction
Stomata play a critical role in regulating transpiration (E) and photosynthesis (Anet)1. Plants are generally assumed to regulate stomatal behavior in order to maximize carbon gain per unit water lost, thereby maintaining a strong coupling between stomatal conductance (gs), E and A2,3. This coordination, however, tends to weaken under elevated temperatures, which may compromise the balance between water loss and carbon gain4,5, and violate predictions based on stomatal optimization assumptions. Global surface temperatures have risen rapidly in recent decades and are projected to increase by at least 1.5°C by the end of this century, accompanied by more frequent and intense heat waves6. These climatic changes challenge stomatal behavior, with profound implications for terrestrial carbon and water cycles7.

Elevated temperatures typically lead to an increase in vapor pressure deficit (VPD), which drives stomatal closure to minimize water loss, thereby decreasing Anet8,9. Importantly, this response is primarily triggered by VPD rather than air temperature alone10. As temperature rises, stomata may instead open, leading to a decoupling of gs and Anet, suggesting that leaf cooling via E becomes a more critical driver of stomatal regulation than the potential enhancement of Anet11–13. Although E is often assumed to rely primarily on gs, high temperatures can decouple E from gs as elevated VPD increases the driving force of leaf water loss through cuticular pathways14. Such increased water loss may reflect a physiological prioritization of thermal safety over strict water conservation15, or may result from mechanical failure of the cuticle or guard cells16,17. However, the thresholds underlying the decoupling of gs, E and Anet under rising temperatures remain poorly understood, hindering our ability to accurately predict plant responses to future climate scenarios.

The impacts of elevated temperatures on gs, E and Anet, as well as their coupling, may vary significantly across plant functional types. For instance, different growth forms, such as herbaceous and woody plants, exhibit distinct adaptive strategies shaped by key functional traits18, resulting in variation in thermoregulation patterns among plant growth forms19. Herbaceous species generally possess more acquisitive traits that enhance resource capture and promote rapid growth under favorable conditions, whereas woody plants, particularly trees, tend to adopt more conservative resource-use strategies20. As a result of these divergent strategies, woody and herbaceous plants may exhibit different responses to warming. However, a recent meta-analysis indicates that, compared to herbs, woody plants can experience greater biomass gains in response to climate warming21. Beyond growth form, life form, phylogeny, leaf habit, and photosynthetic pathway further influence functional traits and water-use strategies22–24. For instance, deep-rooted trees can sustain high water-use efficiency (WUE) under warming, while shallow-rooted shrubs are more vulnerable to water deficits caused by warming, owing to differences in water uptake depth and stomatal regulation25. These variations underscore the role of functional traits in shaping the coupling dynamics of leaf gas exchange and highlight the diversity in plant strategies for coping with warming across different functional groups. However, relatively few studies have directly compared functional types in this regard, leaving important gaps in understanding how plants with different strategies respond to rising temperatures at the leaf level.

The extent to which gs, Anet, and E remain coupled under elevated temperatures can be influenced by experimental protocols. The type of warming facility, such as open-top chambers (OTCs), infrared heaters, or growth chambers, can generate distinct microclimatic conditions that alter the balance between carbon gain and water loss. For instance, the lower wind speeds typically occurring within OTCs can increase boundary layer resistance, thereby affecting whole-plant transpiration26. In addition, warming exacerbates surface soil water stress, driving plants to rely more heavily on deeper water sources27. Warming-induced topsoil desiccation can lead to pronounced vertical decoupling of water and nutrient availability within the soil profile, which may reduce Anet and WUE because nutrients in the dried topsoil become inaccessible to plants28. Consequently, rooting conditions, including soil depth and rooting volume, further shape plant physiological responses to warming. As a result, observed responses of gs, Anet, and E to elevated temperatures may vary considerably across studies, reflecting methodological artifacts associated with experimental design. A clearer understanding of how these methodological factors interact is essential for accurately predicting plant functional responses to future warming scenarios.

In natural ecosystems, the coupling between gs, Anet, and E under elevated temperatures can be shaped by climatic contexts. Factors, such as background temperature regimes, humidity, and soil water availability, interactively determine how stomata respond to warming. For example, both the mean and variability of xylem-specific hydraulic conductivity peak in warm, humid tropical regions and decline in colder, drier climates29, potentially influencing plant water use. Photosynthesis in low-latitude ecosystems with high mean annual precipitation (MAP) shows greater sensitivity to moderate warming, due to narrower thermal tolerance ranges, compared with colder, high-latitude regions30. Together, these climate-driven physiological differences generate region-specific patterns in plant carbon-water relations, further influencing the trade-offs between water conservation, carbon gain, and leaf cooling.

Furthermore, the effect of warming on Anet and E will depend on concurrent changes in atmospheric CO2 and soil water availability, which regulate gs and ultimately affect Tleaf7,31. While warming increases evaporative demand because warmer air has a higher capacity to hold water vapor, elevated CO2 generally lowers E by decreasing gs7,32. It remains uncertain whether the water-saving effect of elevated CO2 can fully offset the increased evaporative demand driven by warming. Soil moisture can also modulate the impacts of warming. For instance, Reich et al.33 demonstrate that in boreal tree seedlings, warming increases Anet in moist soils but reduces it in drier soils, where stomatal limitations prevail. Additionally, trees with adequate water supply are generally better equipped to withstand transient extreme heat events34. Heat avoidance is predicted to be feasible only when soil water availability is sufficient to meet leaf water demand12. Drought conditions exacerbate thermal damage during heat waves, as drought-stressed plants exhibit reduced thermal safety margins and greater leaf damage compared to well-watered plants35. Understanding the interactions of warming with elevated CO2 and drought on plant water use and carbon uptake is essential for predicting vegetation responses to future climate scenarios and developing strategies to mitigate climate change impacts on ecosystems.

Previous meta-analyses have synthesized plant physiological responses to climate warming, including thermal acclimation of leaf respiration36, single and combined global-change drivers on gs37, Anet38 and leaf respiration30,39,40. To the best of our knowledge, no study has yet comprehensively investigated the complex mechanisms underlying stomatal regulation and its interrelations with Anet and E under warming. Changes in gs, Anet and E inevitably lead to variations in intrinsic WUE (WUEi = Anet/gs) and instantaneous WUE (WUEt = Anet/E). These gaps make it challenging to forecast ecosystem processes in the context of future global warming and heat waves. Here, we conducted a comprehensive meta-analysis to evaluate the effects of warming on stomatal behavior and its consequences for water loss, thermal regulation, carbon assimilation and WUE. The primary objectives of the present study were to: (1) quantify the overall effects of experimental warming on leaf physiological traits, and assess the degree of coupling or decoupling among gs, Anet, and E under elevated temperatures; (2) evaluate the role of warming amount and duration in shaping plant physiological responses, with particular attention to thresholds at which gs, Anet, and E decouple; (3) determine how plant functional types modulate leaf physiological responses to warming, thereby explaining cross-species variation in carbon-water trade-offs; (4) assess the influence of experimental protocols on leaf physiological responses, in order to identify potential methodological artifacts that may bias cross-study comparisons; (5) investigate how background climatic context mediates warming effects on leaf physiological responses; (6) explore the interactive effects of warming with elevated atmospheric CO2 or altered soil water availability, to better predict plant responses under multifactor global change scenarios. Such work would provide a mechanistic foundation for refining carbon assimilation and E processes in dynamic global vegetation models and Earth system models, improving predictions of terrestrial carbon and water fluxes under future climate scenarios17.

Results
Overall, warming effects on gs were highly variable, exhibiting no consistent directional trend across the full dataset (−1.4%; 95% CI: −7.1% to 4.7%; Fig. 1). Among all observations, 54.67% showed increases, 43.15% showed decreases, and 2.18% showed no significant change (Supplementary Fig. S1a), underscoring the context-dependent nature of gs responses. In contrast, warming generally enhanced E by an average of 26.3% (95% CI: 12.1%–42.2%; Fig. 1), although individual responses were variable (61.4% were positive, 1.75% were neutral, and 36.84% were negative; Supplementary Fig. S1b). On average, Anet declined (−9.6%; 95% CI: −13.5% to −5.6%; Fig. 1), with a majority of observations showing decreases (61.74%; Supplementary Fig. S1c). This decrease in Anet contributed to the reduction in WUEi (−11.0%; 95% CI: −16.4% to −5.2%; Fig. 1), which was observed in 57.49% of cases (Supplementary Fig. S1d). WUEt decreased even more markedly (−30.2%; 95% CI: −37.7% to −21.8%; Fig. 1), with reductions being observed in 75.56% of the data (Supplementary Fig. S1e). Tleaf increased consistently by 19.3% (95% CI: 13.6% to 25.3%; Fig. 1), with nearly all 642 observations (98.13%) reporting positive responses (Supplementary Fig. S1f). Meanwhile, Ψleaf declined (−16.7%; 95% CI: −22.0% to −11.1%; Fig. 1), with negative responses in 69.87% of observations (Supplementary Fig. S1g).

Substantial heterogeneity among studies was attributable to variation in experimental design. Warming treatments ranged from 0.5 to 20°C and employed diverse methodologies (e.g., open-top chambers, infrared heaters, greenhouses, growth chambers). These methodological differences significantly influenced observed responses and limited extrapolation to natural ecosystems. To address this, we conducted a refined analysis restricted to field-based experiments and moderate warming experiments (≤5°C). In field-based experiments, gs showed a significant mean decline (−7.0%; 95% CI: −12.8% to −0.8%; Supplementary Fig. S4a). In contrast, E showed no consistent directional response to warming (0.2%; 95% CI: −11.5% to 13.6%; Supplementary Fig. S4a). Anet declined (−7.4%; 95% CI: −12.7% to −1.8%; Supplementary Fig. S4a). The reductions in WUEi (−4.0%; 95% CI: −13.4% to 6.5%) and WUEt (−17.9%; 95% CI: −33.4% to 1.3%) were not statistically significant (Supplementary Fig. S4a). Tleaf exhibited a consistent positive response, increasing by 12.7% (95% CI: 6.6%–19.1%; Supplementary Fig. S4a), whereas Ψleaf decreased (−13.2%; 95% CI: −20.1% to −5.7%; Supplementary Fig. S4a). In moderate warming experiments, gs showed no significant mean change (−4.3%; 95% CI: −10.5% to 2.3%), while the increase in E was not significant (5.5%; 95% CI: −3.9% to 15.7%; Supplementary Fig. S6a). Anet declined significantly (−4.9%; 95% CI: −8.3% to −1.3%), leading to corresponding reductions in WUEi (−7.5%; 95% CI: −14.3% to −0.1%) and WUEt (−15.6%; 95% CI: −23.8% to −6.6%). Tleaf (11.3%; 95% CI: 6.2%–16.6%) and Ψleaf (−14.7%; 95% CI: −21.4% to −7.4%) changed in the same direction as in the full dataset but with reduced magnitudes (Supplementary Fig. S6a).

Random forest models applied to the full dataset explained a moderate-to-high proportion of the variance in effect sizes (R2=0.52–0.83; Supplementary Fig. S2a–g), with dominant predictors varying by specific trait. Plant functional type (PFT) emerged as the primary predictor for gs (Supplementary Fig. S2a), Anet (Supplementary Fig. S2c), WUEi (Supplementary Fig. S2d) and Ψleaf (Supplementary Fig. S2g), whereas warming facility/method most strongly influenced E (Supplementary Fig. S2b). Warming amount exerted the greatest control over WUEt (Supplementary Fig. S2e) and Tleaf (Supplementary Fig. S2f). Secondary predictors also varied among traits: warming duration for gs (Supplementary Fig. S2a) and Ψleaf (Supplementary Fig. S2g); PFT for E (Supplementary Fig. S2b), WUEt (Supplementary Fig. S2e) and Tleaf (Supplementary Fig. S2f); warming amount for Anet (Supplementary Fig. S2c) and WUEi (Supplementary Fig. S2d). Third-ranking predictors included warming amount for gs (Supplementary Fig. S2a) and E (Supplementary Fig. S2b), warming duration for Anet (Supplementary Fig. S2c), warming time for WUEi (Supplementary Fig. S2d) and Ψleaf (Supplementary Fig. S2g), and warming facility/method for WUEt (Supplementary Fig. S2e) and Tleaf (Supplementary Fig. S2f).

Warming amount and duration significantly influenced multiple leaf physiological traits (Fig. 2; Supplementary Tables S1 and S3). Threshold-dependent responses were evident for gs (Fig. 2a) and E (Fig. 2b). For gs, a negative relationship occurred when plants were warmed by less than 5°C (p=0.0067; Fig. 2a); beyond this threshold, a significant positive correlation emerged (p=0.0031; Fig. 2a). A similar threshold response was detected for E (Fig. 2b). Below 3°C, warming had no significant effect on E (p=0.3), while above it, the effect became strongly positive (p<0.001; Fig. 2b). In contrast, Anet responses showed a negative correlation with increasing temperatures (p<0.001; Fig. 2c). The responses of WUEi exhibited a linearly negative response to warming amount (p<0.001; Fig. 2d), while the responses of WUEt showed a nonlinear response, with a steep decline above 5°C of warming (p<0.001; Fig. 2e). The response of Tleaf to warming amount was also best described by a segmented regression model, with a breakpoint at approximately 16°C warming (Fig. 2f). Tleaf initially increased with warming (p<0.001) but declined beyond this point (p=0.0028; Fig. 2f), implying that plants might switch successfully to high transpirational cooling under extreme warming. However, data points above 16°C all originated from a single study, and more experiments with extreme warming would be needed to evaluate the consistency of these observations. The absolute increase in Tleaf also showed a significant positive relationship with warming amount (p<0.001; Supplementary Fig. S9). For Ψleaf, the response ratio remained relatively stable at low to moderate levels of warming, but declined sharply at higher warming levels (p=0.0051; Fig. 2g).

To further assess the temperature dependence of these responses, we classified warming levels (ΔT) into three categories: low (ΔT<3°C), moderate (3°C≤ΔT≤5°C) and high (ΔT>5°C; Supplementary Fig. S10 and Table S2). The impacts of the three temperature levels on E (test of moderators: QM=10.2, p=0.006) and Tleaf (QM=56.74, p<0.001) were significantly different (Supplementary Fig. S10). While low and moderate warming had little effect, high warming significantly enhanced both E and Tleaf (Supplementary Fig. S10). The effects of warming on Anet (QM=7.79, p=0.02) and WUEt (QM=62.06, p<0.001) were also temperature-dependent, with high warming causing stronger declines than low or moderate warming (Supplementary Fig. S10). The impacts of warming on gs (QM=3.05, p=0.22), WUEi (QM=2.55, p=0.28) and Ψleaf (QM=1.63, p=0.44) were not significantly different across the three categories (Supplementary Fig. S10).

Warming duration also influenced leaf physiological responses (Fig. 2h–n). For gs, a significant negative relationship with warming duration was detected (p=0.02; Fig. 2h), with effect size declining sharply at shorter durations and stabilizing thereafter. Similarly, the positive effect of warming on E was strongest in short-term experiments and diminished with increasing duration, stabilizing under long-term warming (p<0.001; Fig. 2i), whereas the responses of Anet (p=0.014; Fig. 2j), WUEi (p=0.012; Fig. 2k) and WUEt (p<0.001; Fig. 2l) showed positive correlations with warming duration.

Significant interactive effects between warming amount and duration were detected for gs (p=0.05; Fig. 3a), Anet (p=0.046; Fig. 3b), and Ψleaf (p=0.006; Fig. 3c). In all three traits, high warming levels were associated with steep declines as warming duration increased, whereas low warming treatments showed relatively stable or slightly positive responses over time (Fig. 3). Intermediate warming levels exhibited moderate declines, falling between the low and high treatments. These patterns indicate that the negative impacts of warming on leaf gas exchanges were most pronounced under prolonged, high-intensity warming, while mild warming exerts minimal long-term effects.

Across all plant functional groups, warming effects varied substantially in magnitude and direction among traits (Fig. 4 and Supplementary Table S10). For gs, the 95% CIs of both woody species and herbs overlapped with zero, indicating no consistent responses. The between-group difference was weak and only marginal (QM=2.76, p=0.097), with herbs tending to show a slight positive effect and woody species a negative one. Herbs and woody plants showed similar directional responses in E (QM=2.08, p=0.15), Anet (QM=1.03, p=0.31), WUEi (QM=1.32, p=0.25), WUEt (QM=0.06, p=0.81), Tleaf (QM=0.0016, p=0.97) and Ψleaf (QM=0.71, p=0.4; Fig. 4a).

A marginally significant difference in gs response to warming was observed between shrubs and trees (QM=3.65, p=0.056), with gs response in shrubs varying without a consistent trend, whereas trees showed a significant decreasing trend (Fig. 4b). Warming effects on WUEi differed significantly between the two groups (QM=11.31, p<0.001; Fig. 4b), with a significant reduction observed in shrubs but not in trees (Fig. 4b). By contrast, no significant differences were detected between shrubs and trees for E (QM=0.8, p=0.37), Anet (QM=0.75, p=0.39), WUEt (QM=2.51, p=0.11) and Ψleaf (QM=0.94, p=0.33; Fig. 4b).

Broadleaf and coniferous trees exhibited significantly different responses to warming across several leaf physiological traits. In coniferous trees, warming significantly reduced gs (QM=16.09, p<0.001), Anet (QM=58.99, p<0.001) and WUEt (QM=4.81, p=0.028), whereas no significant changes were observed in broadleaf trees (Fig. 4c). Furthermore, the warming-induced increase in Tleaf was more pronounced in coniferous trees than in broadleaf trees (QM=5.41, p=0.02; Fig. 4c). The warming-induced decrease in Ψleaf was stronger in broadleaf trees than in coniferous trees (QM=5.57, p=0.018; Fig. 4c). No significant differences were found between broadleaf and coniferous trees in their responses of E (QM=0.62, p=0.43) or WUEi (QM=0.14, p=0.71; Fig. 4c).

Within the broadleaf group, evergreen species exhibited a stronger reduction in Anet under warming compared to deciduous species, whereas deciduous species showed no significant change (QM=10.85, p<0.001; Fig. 4d). Although changes in WUEi were not statistically significant in either group, WUEi tended to increase in deciduous species but decrease in evergreen species, resulting in a significant difference between the two groups (QM=9.97, p=0.0016; Fig. 4d). Similarly, a warming-induced decrease in WUEt was observed in evergreen species but not in deciduous species, and this difference was marginally significant (QM=3.81, p=0.051; Fig. 4d). Ψleaf decreased significantly in deciduous but not in evergreen species (QM=5.79, p=0.016; Fig. 4d and Supplementary Table S10). No significant differences between deciduous and evergreen species were found in their responses of gs (QM=0.069, p=0.79) or E (QM=0.54, p=0.46; Fig. 4d).

Photosynthetic pathway also played a key role in shaping plant responses to warming (Fig. 4e and Table S10). In C4 herbs, gs increased significantly under warming, while no such effect was observed in C3 herbs (QM=27.27, p<0.001; Fig. 4e). In contrast, Anet (QM=118.36, p<0.001) and WUEi (QM=14.76, p<0.001) decreased significantly in C3 herbs but remained unaffected in C4 herbs (Fig. 4e). The photosynthetic pathway did not significantly alter the responses of E (QM=0.51, p=0.48), WUEt (QM=1.16, p=0.28), Tleaf (QM=0.42, p=0.52) and Ψleaf (QM=0.47, p=0.49) to warming (Fig. 4e). Additionally, no significant differences were observed between annual and perennial herbs in most leaf traits, except for Anet, which declined under warming in perennial herbs but remained unchanged in annuals (QM=17.58, p<0.001; Fig. 4f).

To further investigate potential sources of variation in leaf physiological responses to warming, we assessed the effects of rooting condition, warming timing, and warming facility/method on effect sizes (Fig. 5 and Supplementary Table S14). Rooting condition had a significant influence on the warming responses of several traits (Fig. 5a). Although the warming effect on gs was slightly negative in free-rooted systems, it was neutral to positive in potted systems, with marginally significant differences between them (QM=2.96, p=0.085; Fig. 5a). Compared with free-rooted plants, potted plants exhibited significantly higher warming-induced increases in E (QM=5.13, p=0.024; Fig. 5a). Unexpectedly, potted plants also had a greater increase in Tleaf compared with free-rooted plants, albeit only marginally significant (QM=3.77, p=0.052; Fig. 5a). Warming timing also influenced trait responses (Fig. 5b). While 24 h, nighttime, and daytime warming had no individually significant effects on gs, their responses differed significantly among categories (QM=8.83, p=0.012; Fig. 5b): daytime warming tended to suppress gs, whereas nighttime warming tended to enhance it. Daytime warming reduced Anet more strongly than either 24 h or nighttime warming (QM=29.4, p<0.001; Fig. 5b). For other traits, such as E (QM=0.95, p=0.62), WUEi (QM=0.53, p=0.77) and Tleaf (QM=2.34, p=0.31), warming responses were relatively consistent across timing categories, with no significant between-group differences (Fig. 5b). Warming facility/method introduced further variability (Fig. 5c). Growth chamber experiments produced stronger warming-induced increases in E compared with OTCs and IR heaters (QM=6.25, p=0.044; Fig. 5c). Declines in WUEi (QM=16.66, p<0.001; Fig. 5c) and WUEt (QM=11.54, p=0.003; Fig. 5c) were not significant in heater-based experiments but were significant in growth chamber and OTC studies.

To assess how background climate modulates leaf physiological responses to warming, we analyzed relationships between response ratios and MAT (Fig. 6a–g and Supplementary Table S18), MAP (Fig. 6h–n and Supplementary Table S19) and AI (Fig. 6o–u and Supplementary Table S20) using a subset of studies performed under field conditions. Along the MAT gradient, threshold-type responses were observed for several traits. For gs, the warming effect was independent of MAT below 10.9°C (p=0.34), but above this threshold the warming response of gs increased with MAT (p<0.001; Fig. 6a). E showed a clear V-shaped response: the effect of warming decreased with MAT up to 11.3°C (p=0.0011) and increased at higher MAT (p=0.0054; Fig. 6b). A similar pattern was observed for Anet; the slopes on both sides of the breakpoint (MAT=12.5°C) were relatively small but significant (p<0.001; Fig. 6c). The warming effects on WUEi declined with MAT below 18.6°C (p=0.0075) and showed no clear trend at higher MAT (p=0.55; Fig. 6d). The warming effects on WUEt also declined with MAT at cooler sites (p<0.001) but became non-significant above the breakpoint (18.6°C; p=0.84; Fig. 6e). The responses of Tleaf showed no detectable dependence on MAT (p=0.077; Fig. 6f). The responses of Ψleaf peaked at 15.4°C: the warming effect increased toward the breakpoint (p<0.001) and then declined slightly at higher MAT (p=0.027; Fig. 6g).

The warming effect on gs was not related to MAP (p=0.19; Fig. 6h). In contrast, the warming effect on E was negatively associated with MAP (p<0.001; Fig. 6i), whereas the warming effects on Anet (p<0.001) and WUEi (p=0.0016) were positively correlated with MAP. The warming effect on WUEt showed no significant relationship below 354.4 mm MAP (p=0.079) but increased significantly above this threshold (p<0.001; Fig. 6l). Tleaf also exhibited a threshold-type response: no relationship below a breakpoint at 1413 mm (p=0.71), but a decline in the warming effect at higher MAP (p<0.001; Fig. 6m). The warming effect on Ψleaf showed no clear dependence on MAP (p=0.14; Fig. 6n).

The warming effect on gs remained unrelated to AI (p=0.27; Fig. 6o). The warming effect on E showed a negative linear relationship (p<0.001; Fig. 6p). The warming effect on Anet increased with AI up to a breakpoint at AI=0.49 (p=0.003) after which no significant trend was detected (p=0.55; Fig. 6q). The warming effect on WUEi increased with AI at the dry end (p=0.0084) and then plateaued (p=0.9; Fig. 6r), and the warming effect on WUEt similarly rose at low AI (p=0.0014) and then leveled off beyond an AI of 1.2 (p=0.94; Fig. 6s). The warming effects on Tleaf (Fig. 6t) and Ψleaf (Fig. 6u) were stable at low AI (p=0.55 and p=0.71, respectively) but declined in wetter climates (p<0.001 and p=0.0025, respectively).

To explore how other climate factors modulate leaf physiological responses to warming, we assessed the effect sizes of multiple traits under different atmospheric CO2 concentrations and soil moisture regimes (Fig. 7 and Supplementary Table S21). gs showed no consistent responses to warming under either aCO2 or eCO2. However, the magnitude of the responses differed between the two CO2 treatments (QM=5.95, p=0.015; Fig. 7a). Although warming elevated E in all treatments, the difference between aCO2 and eCO2 was not significant (QM=0.47, p=0.49; Fig. 7a). Warming led to decreased Anet across all treatments, with Anet being decreased to a greater extent under eCO2 (QM=10.74, p=0.001; Fig. 7a). Warming tended to reduce WUEi under aCO2, whereas the responses of WUEi were minimal under eCO2 (QM=18.72, p<0.001; Fig. 7a). No significant differences were found between aCO2 and eCO2 for the responses of WUEt (QM=1.4, p=0.24; Fig. 7a). Warming increased Tleaf at both CO2 levels, with a more pronounced increase observed under aCO2 (QM=5.79, p=0.016; Fig. 7a). Warming reduced Ψleaf under both CO2 levels, with a significantly larger decline under eCO2 (QM=6.55, p=0.011; Fig. 7a).

gs showed no consistent responses to warming under either well-watered or drought treatments. Nevertheless, differences in the magnitude of the gs response to warming were evident, with a significant overall difference between the two watering treatments (QM=5.8, p=0.016; Fig. 7b). Warming enhanced E in both well-watered and drought-stressed plants, but the increase was significantly greater under well-watered conditions (QM=22.95, p<0.001; Fig. 7b). Warming reduced Anet in both water treatments, but no significant difference was detected between well-watered and drought groups (QM=0.24, p=0.62; Fig. 7b). Warming significantly reduced WUEi in both water treatments, with a larger decrease under drought conditions (QM=49.02, p<0.001; Fig. 7b). The negative impacts of warming on WUEt were comparable in both well-watered and drought-stressed plants (QM=0.009, p=0.98; Fig. 7b). Warming increased Tleaf in both water treatments, with a marginally significant group difference (QM=3.6, p=0.058; Fig. 7b), with a greater warming-induced increase in Tleaf under drought conditions. Ψleaf declined under both treatments, but the decrease was significantly more pronounced under well-watered conditions (QM=40.61, p<0.001; Fig. 7b).

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I really enjoyed reviewing this interesting and timely paper reporting the results of a meta-analysis of the published literature on the responses of leaf gas exchange, temperature and water potential to warming at global scale. I think the main findings of the study are compelling and well supported by both theory and experimental evidence, as there is increasing evidence and awareness about the substantial decoupling of leaf stomatal conductance, transpiration and photosynthesis in terrestrial vegetation in response to elevated temperatures. This is certainly an important research topic that merits careful meta-analysis of the published literature to search for emergent patterns and thresholds and to gain further insights into plant functioning in a warmer world. The meta-analysis of warming interactions with other key global change drivers (elevated CO2 and drought) is also very timely and relevant, so I believe the topic of this study will be of interest to a wide readership. However, I think there is still plenty of room for further improvement in the current version of the manuscripts, so I offer several suggestions and comments that I hope the authors will find relevant and useful to enhance the scope, strength and novelty of the paper.\nFirst of all, I think it would be highly relevant, interesting and useful to also assess the impacts of warming on water use efficiency, including both intrinsic and instantaneous WUE (WUEi=A/gs and WUEt=A/E, respectively), as this are key parameters that reflect the trade-off between carbon assimilation and water loss in leaves. There is substantial evidence in the literature indicating that even moderate experimental warming can drastically reduce both WUEi and WUEt at leaf level (e.g. see papers by León-Sánchez et al and Querejeta et al), and this is a critically important aspect of plant responses to warming that is overlooked in the present meta-analysis. So I would strongly recommend to explicitly include these two additional parameters in the meta-analysis to further enhance the novelty and relevance of the paper and to gain further insights into plant physiological responses to warming (and interactions with drought and eCO2). According to the current results of the meta-analysis, decreased Anet coupled with increased gs and E should translate into sizeable reductions in both WUEi and WUEt under warming at global scale, right?. Moreover, WUEi and WUEt could be more sensitive and responsive to warming than either Anet, gs or E and might thus respond significantly to warming at lower Temp increase thresholds than the former variables (i.e. lower than the 3-5º C thresholds reported for E and gs in the current version of the manuscript). WUEi and/or WUEt can be easily calculated for all studies reporting Anet and gs and/or E.\nAnother aspect that deserves further clarification and re-analysis of the data is that the 184 primary research articles considered in the meta-analysis appear to include both manipulative field studies and greenhouse/pot/growth chamber studies indistinctively, which I think may be inappropriate and might be significantly biasing the main results encountered. So I would strongly recommend to re-analyse the results of the subset of manipulative field studies separately from the subset of pot/growth chamber studies, in order to identify and avoid any biases driven by “pot effects” (e.g. limitations on gs and E induced by constrained soil and rooting volume in pots and growth chambers). For example, plants exposed to experimental warming and increasing evaporative demand in the field could grow deeper or more extensive root systems to meet their increased demand for water, but this adaptive response to warming would be significantly constrained in potted plants with access to limited soil and rooting volume. Other aspects requiring further clarification are if the meta-analysis includes both daytime and night-time warming experiments (and if they should be analysed separately given their potentially very different impacts on daytime leaf gas exchange), or if plant responses to warming are significantly affected by the experimental method used to achieve warming in the field (e.g. Open Top Chambers vs heating cables, infrared heating or other methods). These important aspects require further clarification in the M&M section and re-analysis of the dataset in the Results section.\nI also recommend to further explore with more detail the strikingly wide and diverse range of responses of both gs and E to warming below the 3-5ºC thresholds encountered in the meta-analysis, for several important reasons. First and foremost, this is precisely the range of temperature increase that is expected with ongoing climate warming across most of the planet, so it is critically important to gain further insights into plant responses to this range of warming (0.5-5 C) that is the most realistic and relevant for plant functioning under a climate change scenario. Given that the literature surveyed reports a remarkably diverse variety of gs and E responses to 0.5-5ºC warming, ranging from large decreases to no change to large increases (see Figs 1 and 2), I think it would be helpful to re-analyse the data in search of any discernible patterns that might help identify the potential key drivers of this extreme variability of plant responses (e.g. MAT, MAP, Aridity, EVTP, latitude?) within this range of relatively moderate warming (0.5 to 5ºC) that is most realistic and ecologically relevant in a climate change scenario. To me, it is very interesting and relevant (but relatively unsurprising) that gs and E increase and Anet decreases significantly above 3—5ºC warming (up to 17ºC warming in Fig2, which is rather extreme!). But I think it would be much more interesting and relevant to gain further insight into the factors governing the strikingly wide range of gs and E responses to more moderate and realistic warming below the above thresholds (which are remarkably wide and include anything from large increases to large decreases to no change in gs and E with warming in Figs 1 and 2).\nOther comments\nFigure 1 shows that the number of observations included in the meta-analysis differs widely among response variables, which might significantly affect the robustness and reliability of the conclusions drawn for each response variable (most robust for Anet, but less robust for other parameters). This aspect deserves a brief mention and discussion in the Discussion section.\nIn Figure 1 panel a), I think a negative sign is missing from the percent reduction in leaf water potential, right?\nL21: I think the primary research articles included in the meta-analysis should be properly acknowledged, credited and cited in the main text (or alternatively in a Data Sources appendix, as in the recent meta-analysis by He et al 2024 ; He, P., Sardans, J., Wang, X., Ma, C., Man, L., Peñuelas, J., ... & Li, M. H. (2024). Nutritional changes in trees during drought‐induced mortality: A comprehensive meta‐analysis and a field study.Global Change Biology, 30(1), e17133.).\nL56, L59 and elsewhere throughout the text: I think the strong assertions made here (“…gs remained unchanged… gs remained stable…under elevated temperatures” are rather inaccurate and misleading, as gs actually shows a remarkably wide range of responses to warming that includes highly significant increases and decreases across the different studies included in the meta-analysis, also below the 5ºC threshold identified in the analysis. I think this questionable wording should be totally rephrased to more accurately reflect and acknowledge the real picture, which is much more complex and nuanced than suggested by these and similar sentences scattered throughout the text.\nL79: this sentence implicitly acknowledges that the temperature elevation expected from ongoing anthropogenic climate warming for the next decades is more moderate than the 3-5 degrees Celsius thresholds identified in the meta-analysis for gs and E, so I think it is critically important to gain further insight into the key factors governing the large variability of gs and E responses observed below these thresholds (within the 0.5-5 degrees C temperature elevation range).\nL91-92 Actually E depends primarily on BOTH gs and VPD, right?\nL106, L115: A recent paper published in Nat Comm might be relevant here: Muñoz-Gálvez et al (2025). Trait coordination and trade-offs constrain the diversity of water use strategies in Mediterranean woody plants. Nature Communications, 16(1), 4103.\nL158-167: Once again, it appears that both field experiments and pot/growth chamber experiments were pooled together in the meta-analysis. I strongly recommend to re-analyse these two distinct subsets of studies separately also, to make sure that the overall patterns emerging from the meta-analysis are not biased by any potential “pot effects” (e.g. limited soil and rooting volumes in pots potentially constraining and biasing the responses of plant gs and E to warming).\nL168-169: Please include also WUEi and WUEt in the meta-analysis.\nL171: Many of these environmental factors (e.g. latitude, MAP) would only be relevant for/apply to field experiments (but not to greenhouse/growth chamber experiments), right?\nL182-184: It would also be interesting to distinguish between trees and shrubs, as they often show different water use strategies (see Muñoz-Gálvez et al 2025).\nL260-262: Please expand Fig S3 to highlight similar graphs of response of gs, Anet, WUEi, WUEt, leaf water potential and leaf temperature to MAT, MAP and AI (aridity index?).\nL265-273: Please rephrase the inaccurate and misleading statement that “Globally, elevated temperature had no significant effect on gs”, in order to more accurately portray the complex and diverse range of highly significant and non-significant gs responses to warming described in the literature. Please expand this section to better highlight the wide range of positive, negative and neutral responses of gs and E to warming, particularly in the low/moderate range of warming (0.5 to 5 degrees Celsius). Please re-analyse the data to try to elucidate the key drivers (MAT, MAP, AI?) of this strikingly large variation of gs and E responses to warming among studies.\nL285-286: This result is very counterintuitive, as one would expect stronger negative impacts on Anet with increasing temperature elevation. Do you have a plausible explanation for this rather counterintuitive finding of the meta-analysis? Same would apply to L300-302.\nL299-300: This finding makes no sense to me, as I don’t understand why low temperature elevation had a significant impact on Tleaf but moderate temperature elevation didn’t…\nL339-343: I don’t understand what you mean here, please clarify, elaborate and expand with a more straightforward and explicit interpretation of the observed differences. Same for L350-352.\nL362-364: This statement appears to be at odds with the general patterns described in L56-57 and FigS3. If E decreases with warming in temperate/boreal regions and remains unchanged in subtropical/tropical regions, where is it that E increases significantly under warming? Please clarify this apparent contradiction in the manuscript.\nL370: Another inaccurate and potentially misleading statement that should be rephrased and carefully modulated: “… elevated temperature did not impact gs significantly across all studies”. Figure 2a shows a much more complex picture than suggested by this overly simplistic statement, with both highly significant increases and decreases in gs reported often in the literature.\nL371-395: This section is very interesting, plausible and compelling, and well supported by the literature. Same applies to the findings and patterns reported in L333-334, L303-307, L431-438, L440-455, among several others. Figure 3 is also very nice and totally consistent with theory and experimental evidence.\nL398-405: Please elaborate on this important point and try to better highlight it by providing suitable examples from the literature (e.g examples/references of large increases and large decreases in gs and E under warming).\nL415-430: I think this section is also very nice and relevant, although I have some reservations regarding the excessively high thresholds encountered for significant gs and E increases under warming (5 and 3 degrees, respectively). Also please rephrase the inaccurate statement “…when temperature increases were below 5ºC, elevated temperature did not affect gs…”, as Figure 2 clearly shows that many field and pot studies have reported highly significant increases (e.g. Leon-Sanchez et al 2016, 2018) or decreases (Wertin et al 2017) in gs under low/moderate experimental warming (2-3 degrees), as becomes evident from Figure 2. Same applies to L428 (gs responses to low-moderate warming vary widely among studies and are certainly not “stable”!). Same for L529-530 of the Conclusions section!\nL430: Do you mean trade-offs between hydraulic safety and thermal regulation? Please rephrase.\nL449-455: I think enhanced photorespiration with warming also contributes significantly to the observed pattern described in this paragraph, in addition to enhanced dark respiration, right?\nL468-482: Nice and compelling results, see the extensive work by Brent Helliker and co-workers on this topic for further support to this interpretation.\nL506-506-525: Very nice and compelling section, see work by Leon-Sanchez et al 2018-2020 and Aparecido et al 2020 for further support for your interpretation of the interactions between warming and drought.\nL526-554: The Conclusions section is very nice, compelling and timely, and elegantly summarizes the main findings of the study. My only reservation is that I think the warming thresholds reported for significant enhancement of gs and E (5 and 3 degrees) appear to be unrealistically high in my own experience, as I have often encountered large increases in gs and E in the field with low/moderate experimental warming (2-3ºC). I think that including WUEi and WUEt in the meta-analysis, and reanalysing field studies separately from pot/growth chamber studies might help clarify this (in my opinion) controversial and inconclusive aspect of the study (i.e. the high warming thresholds reported).\nL166-167: I think there are some highly relevant studies with suitable and useful data that are currently missing from the 184 references list used for the meta-analysis (see Supp Material), and that could/should be also included to expand its scope and the strength of the conclusions. Some of them are even cited in the main text, but are missing from the 184 references list in the Supp Information section, such as:\nUrban (2017). Stomatal conductance increases with rising temperature. Plant signaling & behavior, 12(8), e1356534.\nUrban (2017). Increase in leaf temperature opens stomata and decouples net photosynthesis from stomatal conductance in Pinus taeda and Populus deltoides x nigra. Journal of experimental botany, 68(7), 1757-1767.\nLeón‐Sánchez et al (2020). Altered leaf elemental composition with climate change is linked to reductions in photosynthesis, growth and survival in a semi‐arid shrubland. Journal of Ecology, 108(1), 47-60.\nMarchin (2016). Stomatal acclimation to vapour pressure deficit doubles transpiration of small tree seedlings with warming. Plant, Cell & Environment, 39(10), 2221-2234.\nFinally, I would like to recommend the authors to have a look at a very recent paper that I believe is highly relevant to the topic of the present paper, and that should be useful for a more fine-tuned interpretation of the patterns and main findings encountered in the meta-analysis.\nEvans, M. E., Hu, J., & Michaletz, S. T. (2025). Scaling plant responses to heat: From molecules to the biosphere. Science, 388(6752), 1167-1173.", "reviewer_2": "This manuscript presents an exhaustive literature review of elevated temperature studies with terrestrial plants using meta-analysis. The analysis appears to be well done, and quantitatively summarizes the literature in a concise manner. A few major concerns are:\n1) The Introduction does not describe what other meta-analyses have been done concerning elevated temperature in terrestrial plants. Thus, it is not entirely clear what is novel about the current manuscript. Such information is critical. Some examples of previous meta analyses include\nZhang, J., Deng, L., Jiang, H. et al. The effects of elevated CO2, elevated O3, elevated temperature, and drought on plant leaf gas exchanges: a global meta-analysis of experimental studies. Environ Sci Pollut Res 28, 15274–15289 (2021).\nhttps://doi.org/10.1007/s11356-020-11728-6.\nLiang, J, Xia, J, Liu, L. Global patterns of the responses of leaf-level photosynthesis and respiration in terrestrial plants to experimental warming. Journal of Plant Ecology 6: 437 – 447 (2013). DOI 10.1093/jpe/rtt003\nWang, D., Heckathorn, S.A., Wang, X. et al. A meta-analysis of plant physiological and growth responses to temperature and elevated CO2 . Oecologia 169, 1–13 (2012). https://doi.org/10.1007/s00442-011-2172-0\n2) Broad statements are often made based on regressions that sometimes explain less than 10% of the variability between the values of a given gas exchange variable and the magnitude in temperature difference between treatment and control. Something similar occurs between gas exchange variables and the duration of the temperature treatment. It would be helpful for the reasons behind the very low r2 values and the implications of this to be mentioned in the Discussion section.\n3) The Conclusions section contains a lengthy summary of the Results. This could possibly be shortened and more emphasis on why the results are important could be mentioned. Currently, the text only indicates that the results would help with modeling.\nA list of minor comments is given below:\n1) The title appears to be missing a word? Change “associated” to “under” or “in response to”?\n2) Acknowledgements (lines 21) – improve grammar “…..contributed to this in the present meta-analysis.” Change to “contributed to the present meta-analysis”\n3) Abstract (lines 56-57) – is the increase in E under elevated temperature related to a concurrent increase in air VPD? If so, this should be clarified briefly in the text.\n4) Abstract (line 60) – again, is the E increase related to a concurrent increase in VPD?\n5) Abstract (lines 62-64) – Under elevated CO2, was the amplification of the Anet response to temperature due to decreasing gs under elevated CO2? If so, please rephrase the text.\n6) Intro – Not much information on what other meta-analyses of elevated termperature have been done are given. Please add relevant studies at some point in the Introduction. This would be important for understanding the novelty of the current manuscript.\n7) Intro (line 73) – “stomata” is plural. So, the phrase should be “stomata play” rather than “stomata plays”?\n8) Intro (line 77) – “this coupling can become variable…..” What does this mean? Please revise the text.\n9) Intro (lines 85-86) – the authors state, “Elevated temperature typically leads to an increase in vapor pressure deficit (VPD), which drives stomatal closure to minimize water loss”. How much of an increase in temperature is the text referring to? It is awkward that the Abstract states that no increase in gs occurs with moderate elevated temperature increase, but here “stomatal closure” is referred to.\n10) Intro (line 148) – Does the word “global” mean “overall”? Please change if this interpretation is correct.\n11) Intro (last paragraph) – Would one or more hypotheses be helpful? Or, maybe it is felt that it is unnecessary because the study is a quantitative review of the existing literature?\n12) Intro (line 155) – fix the formatting of “{Blonder, 2023 #37641}”\n13) M+M (lines 158-159) – Please give the web addresses of the sites that were used, and the dates that they were accessed.\n14) M+M (line 164) – Please re-phrase the following “maintenance of the same initial conditions.” It is not entirely clear what this means.\n15) Figure S1 – Is a “register” something different from the two databases used? If not, please delete “registers”.\n16) M+M (line 168) – change “parameters” to “variables”?\n17) M+M (lines 205-206) – it is common that the number of replications is not given in many studies, or the authors sometimes incorrectly state the number of replications due to pseudoreplication issues. How were such difficulties handled? Were only studies used where the number of replicates was given?\n18) M+M (lines 226-230) – were possibly different effects in growth chambers, greenhouses, and field studies contemplated? If not, could this lead to a bias in the results obtained?\n19) Results (line 267) – change “globally” to “overall”?\n20) Results (lines 286-286)- The authors state, “, the impacts on A (p < 0.001; Figure 2c; Table S1) decreased significantly with ΔT,”. Maybe the text is not clear? If the response is linear, why would the “impact” decrease as deltaT increases?\n21) Supplementary material – A title of “Stomatal regulation at elevated temperature: Trade-offs between water use, transpirational cooling and photosynthesis” is given at the top of the document. What does this refer to?\n22) Figure 4 (legend) – the text states, “The dashed horizontal line marks the threshold for significant temperature effects.” This is unclear. The dashed line is at zero. Please change if necessary.\n23) Figure 7c – move asterisk.\n24) Discussion (lines 389-390) – the text states, “reported that decreased with rising temperatures until 42 °C,”. Is something missing here? E decreased?\n25) Discussion (line 406) – change “minimize” to “minimizing”?\n26) Discussion (line 417) – change “conflict” to “conflictive”?\n27) Discussion (line 544) – spelling of “decupling”?\n28) Discussion (line 555) – uncategorized references?\n29) Conclusions – most of the text reads as a summary of the results. Please shorten this aspect of the Conclusions. More emphasis could be placed on the implications of the study. Just stating that the results are interesting for modeling purposes seems rather limited."}, "author_response": "Response: We sincerely thank you for the thoughtful and constructive comments. We truly appreciate your positive assessment of our work.\nFirst of all, I think it would be highly relevant, interesting and useful to also assess the impacts of warming on water use efficiency, including both intrinsic and instantaneous WUE (WUEi=A/gs and WUEt=A/E, respectively), as this are key parameters that reflect the trade-off between carbon assimilation and water loss in leaves. There is substantial evidence in the literature indicating that even moderate experimental warming can drastically reduce both WUEi and WUEt at leaf level (e.g. see papers by León-Sánchez et al and Querejeta et al), and this is a critically important aspect of plant responses to warming that is overlooked in the present meta-analysis. So I would strongly recommend to explicitly include these two additional parameters in the meta-analysis to further enhance the novelty and relevance of the paper and to gain further insights into plant physiological responses to warming (and interactions with drought and eCO2). According to the current results of the meta-analysis, decreased Anet coupled with increased gs and E should translate into sizeable reductions in both WUEi and WUEt under warming at global scale, right?. Moreover, WUEi and WUEt could be more sensitive and responsive to warming than either Anet, gs or E and might thus respond significantly to warming at lower Temp increase thresholds than the former variables (i.e. lower than the 3-5º C thresholds reported for E and gs in the current version of the manuscript). WUEi and/or WUEt can be easily calculated for all studies reporting Anet and gs and/or E.\nResponse: Thank you for this constructive suggestion. Following your recommendation, we now quantify both intrinsic (WUE) and instantaneous WUE (WUE) in the meta-i t analysis. When not reported explicitly, we derived WUE (A /g ) and WUE (A /E) i net s t net from studies that provided A together with either g or E. The overall results show net s significant warming-induced declines in both WUE and WUE, indicative of a partial i t decoupling between carbon assimilation and water fluxes under warming. We have revised the Methods to describe calculation procedures, and we have updated the Results and Discussion to highlight these responses and their implications.\nAnother aspect that deserves further clarification and re-analysis of the data is that the 184 primary research articles considered in the meta-analysis appear to include both manipulative field studies and greenhouse/pot/growth chamber studies indistinctively, which I think may be inappropriate and might be significantly biasing the main results encountered. So I would strongly recommend to re-analyse the results of the subset of manipulative field studies separately from the subset of pot/growth chamber studies, in order to identify and avoid any biases driven by “pot effects” (e.g. limitations on gs and E induced by constrained soil and rooting volume in pots and growth chambers). For example, plants exposed to experimental warming and increasing evaporative demand in the field could grow deeper or more extensive root systems to meet their increased demand for water, but this adaptive response to warming would be significantly constrained in potted plants with access to limited soil and rooting volume. Other aspects requiring further clarification are if the meta-analysis includes both daytime and night-time warming experiments (and if they should be analysed separately given their potentially very different impacts on daytime leaf gas exchange), or if plant responses to warming are significantly affected by the experimental method used to achieve warming in the field (e.g. Open Top Chambers vs heating cables, infrared heating or other methods). These important aspects require further clarification in the M&M section and re-analysis of the dataset in the Results section.\nResponse: Thank you for highlighting this important source of potential bias. We agree that combining field studies with greenhouse/pot/growth-chamber experiments can confound inference via pot effects. Accordingly, we reclassified all records by study setting, re-analysed the field-only subset, and added moderator analyses for rooting condition (field/in-ground vs potted), warming timing (daytime, night-time, 24-h), and warming facility (open-top chambers, infrared heaters, growth chamber/glasshouse/greenhouse). In the field-only subset (Fig. S4), g declined under s warming, whereas E showed no consistent directional change; nevertheless, the g -E s decoupling persisted. The apparent influences of warming magnitude and duration (Fig. S12), plant functional type (Fig. S15) and experimental protocols (Fig. S18) partly shifted in this subset, some patterns changed, whereas others remained consistent with the full dataset. We have added these results in the supporting information. We further used random-forest models to quantify the relative importance of drivers of leaf-level responses, including plant functional type, warming magnitude and duration, warming timing and facility, and site climate (MAT, MAP, AI). Moderator tests (Fig. 6) indicate that rooting condition, warming timing, and facility type each significantly modify leaf-level responses to warming. We have revised the Methods to detail study classification, and we have updated the Results and Discussion to present and interpret these contrasts.\nFig. S4 Effect sizes of (a) and the importance of predictors for leaf physiological trait responses to warming (b-f) in field-based studies.\nI also recommend to further explore with more detail the strikingly wide and diverse range of responses of both gs and E to warming below the 3-5ºC thresholds encountered in the meta-analysis, for several important reasons. First and foremost, this is precisely the range of temperature increase that is expected with ongoing climate warming across most of the planet, so it is critically important to gain further insights into plant responses to this range of warming (0.5-5 C) that is the most realistic and relevant for plant functioning under a climate change scenario. Given that the literature surveyed reports a remarkably diverse variety of gs and E responses to 0.5-5ºC warming, ranging from large decreases to no change to large increases (see Figs 1 and 2), I think it would be helpful to re-analyse the data in search of any discernible patterns that might help identify the potential key drivers of this extreme variability of plant responses (e.g. MAT, MAP, Aridity, EVTP, latitude?) within this range of relatively moderate warming (0.5 to 5ºC) that is most realistic and ecologically relevant in a climate change scenario. To me, it is very interesting and relevant (but relatively unsurprising) that gs and E increase and Anet decreases significantly above 3—5ºC warming (up to 17ºC warming in Fig2, which is rather extreme!). But I think it would be much more interesting and relevant to gain further insight into the factors governing the strikingly wide range of gs and E responses to more moderate and realistic warming below the above thresholds (which are remarkably wide and include anything from large increases to large decreases to no change in gs and E with warming in Figs 1 and 2).\nResponse: Thank you for suggesting a deeper examination of the 0.5-5 °C warming range. We re-analysed all records with warming amount ≤ 5 °C (Fig. S6, Fig. S11 and Fig. S15) and repeated the analysis for the field-only subset under moderate warming (warming amount ≤ 5 °C; Fig. S6 and Fig. S8). These re-analyses broadly corroborate the full dataset. Specifically, in the full dataset g declined with moderate warming, and s in the ΔT ≤ 5 °C subset a larger number of g effect sizes was negative. For E, the full s dataset showed no significant change under ΔT ≤ 5 °C, and the subset likewise exhibited no consistent directional response. To explain the heterogeneity in leaf level responses within this range, we coupled meta-regressions with random-forest models that considered plant functional type, warming amount and duration, warming timing and facility, and site climate (MAT, MAP, AI). We have added these results and methodological details to the Supporting Information, and in the Discussion (Evidence for decoupling between g and E was also apparent in the subset analyses of field s studies and of experiments with moderate warming (≤ 5°C). In the subset of field experiments, g decreased but E showed no consistent changes (Fig. S4a). The s divergence between the full dataset and the field subset likely reflects the smaller warming magnitude in the field subset: the full dataset had mean/median ΔT of 4.57/4.00 °C, compared with 2.8/2.5 °C in the field subset; notably, E increased only when warming exceeded a 3.0 °C threshold. In experiments with moderate warming (≤ 5 °C), neither g nor E exhibited consistent overall responses, but g tended to decline s s while E tended to rise, consistent with partial decoupling whereby increases in VPD under warming elevated E even as g weakly decreased. Lines409-419)\nFigure 1 shows that the number of observations included in the meta-analysis differs widely among response variables, which might significantly affect the robustness and reliability of the conclusions drawn for each response variable (most robust for Anet, but less robust for other parameters). This aspect deserves a brief mention and discussion in the Discussion section.\nResponse: Thank you for pointing this out. We agree that unequal sample sizes among the different response variables may limit the strength of our conclusions for those parameters. Especially the relatively low number of E observations, because in many gas‐exchange studies, E is measured alongside A and g , but relatively few of those net s studies report data for E, which explains its underrepresentation in our meta‐analysis. To address this, we have added the following statement to the Discussion: “Notably, the number of observations across traits in our dataset was uneven. While A was well net represented, E was reported in far fewer studies, despite being commonly measured alongside A and g . This imbalance limits the robustness of conclusions regarding E. net s We recommend that future studies consistently report E together with A and g , and net s also include measurements of T and Ψ to enable more comprehensive assessments leaf leaf of the trade-offs among carbon uptake, water use, and thermal regulation. (lines 435-440)”.\nIn Figure 1 panel a), I think a negative sign is missing from the percent reduction in leaf water potential, right?\nResponse: Sorry for the mistakes. We have corrected this in the revised figure.\nL21: I think the primary research articles included in the meta-analysis should be properly acknowledged, credited and cited in the main text (or alternatively in a Data Sources appendix, as in the recent meta-analysis by He et al 2024 ; He, P., Sardans, J., Wang, X., Ma, C., Man, L., Peñuelas, J., ... & Li, M. H. (2024). Nutritional changes in trees during drought‐induced mortality: A comprehensive meta‐analysis and a field study.Global Change Biology, 30(1), e17133.).\nResponse: Thank you for this suggestion. We are deeply grateful to the authors of the primary studies, without whom this meta-analysis would not have been possible, and we agree that they should receive appropriate credit. In the revised manuscript, we have clarified in the Methods section that “Our search yielded 207 peer-reviewed articles, published from 1993 to 2024, that met these criteria (Data S1; Reference S1)”. We have provided the full list of primary research articles included in the meta-analysis in the Supporting Information (Reference S1). We are unsure whether items listed in the Supporting Information are counted as formal citations; if needed, we will consult the journal’s editorial office upon acceptance to ensure proper attribution.\nL56, L59 and elsewhere throughout the text: I think the strong assertions made here (“…gs remained unchanged… gs remained stable…under elevated temperatures” are rather inaccurate and misleading, as gs actually shows a remarkably wide range of responses to warming that includes highly significant increases and decreases across the different studies included in the meta-analysis, also below the 5ºC threshold identified in the analysis. I think this questionable wording should be totally rephrased to more accurately reflect and acknowledge the real picture, which is much more complex and nuanced than suggested by these and similar sentences scattered throughout the text.\nResponse: Thanks for the insightful comment. We agree that our initial wording overstated the consistency of g responses to warming. To address this, we have revised s the text to more accurately reflect the diversity of responses reported in the literature. Specifically, we now state that “g responses were highly variable, exhibiting no s consistent directional trend”, which acknowledges the wide range of both increases and decreases observed across studies.\nL79: this sentence implicitly acknowledges that the temperature elevation expected from ongoing anthropogenic climate warming for the next decades is more moderate than the 3-5 degrees Celsius thresholds identified in the meta-analysis for gs and E, so I think it is critically important to gain further insight into the key factors governing the large variability of gs and E responses observed below these thresholds (within the 0.5-5 degrees C temperature elevation range).\nResponse: Thank you for the insightful comment. We agree that understanding the drivers of the high variability in g and E responses within the moderate warming range s (0.5-5 °C) is critical. Based on your previous suggestion, we have conducted analyses focusing on subsets of data from moderate warming experiments (≤ 5 °C), including both the full dataset and field-based studies. We also performed random forest analyses, which identified key predictors such as plant functional type, experimental protocols and climate variables, which help explain the observed variability in g and E under s moderate warming conditions.\nL91-92 Actually E depends primarily on BOTH gs and VPD, right?\nResponse: Yes. This sentence has been revised as “Although E is often assumed to rely primarily on g , high temperatures can decouple E from g as elevated VPD increases s s the driving force of leaf water loss through cuticular pathways (lines84-87)”.\nL106, L115: A recent paper published in Nat Comm might be relevant here: Muñoz-Gálvez et al (2025). Trait coordination and trade-offs constrain the diversity of water use strategies in Mediterranean woody plants. Nature Communications, 16(1), 4103.\nResponse: Thank you for pointing us to this recent Nature Communications paper by Muñoz-Gálvez et al. (2025). We agree this work is relevant to our topic. We have now cited it in the Introduction “For instance, deep-rooted trees can sustain high water-use efficiency (WUE) under warming, while shallow-rooted shrubs are more vulnerable to water deficits caused by warming, owing to differences in water uptake depth, stomatal regulation, and WUE 25 (lines104-107)” and Discussion “These results are consistent with recent findings that deep-rooted trees, owing to their greater access to soil water and tighter stomatal regulation, are less vulnerable to warming-induced water deficits and can maintain higher WUE. In contrast, shallow-rooted shrubs are more susceptible (lines 489-492)”.\nL158-167: Once again, it appears that both field experiments and pot/growth chamber experiments were pooled together in the meta-analysis. I strongly recommend to re-analyse these two distinct subsets of studies separately also, to make sure that the overall patterns emerging from the meta-analysis are not biased by any potential “pot effects” (e.g. limited soil and rooting volumes in pots potentially constraining and biasing the responses of plant gs and E to warming).\nResponse: We thank the reviewer for this valuable suggestion. Following the recommendation, we have conducted separate analyses for field experiments, and also compared the results between free rooting experiments and pot chamber experiments (Fig. 5a). These contents were included in Results (lines 313-321) and discussion (lines 525-543).\nL168-169: Please include also WUEi and WUEt in the meta-analysis.\nResponse: Thanks for your suggestion. WUE and WUE were included.\ni t\nL171: Many of these environmental factors (e.g. latitude, MAP) would only be relevant for/apply to field experiments (but not to greenhouse/growth chamber experiments), right?\nResponse: Yes. To ensure environmental variables such as MAP, MAT, and AI were appropriately interpreted, we restricted our analyses of these factors to a subset of studies conducted under field conditions only.\nL182-184: It would also be interesting to distinguish between trees and shrubs, as they often show different water use strategies (see Muñoz-Gálvez et al 2025).\nResponse: Thank you for your valuable suggestion. We have now distinguished between trees and shrubs in our analyses. The results revealed clear differences in their water use strategies (Fig. 4b), which have been incorporated into the revised Results (lines 268-276) and Discussion sections (lines 488-495).\nL260-262: Please expand Fig S3 to highlight similar graphs of response of gs, Anet, WUEi, WUEt, leaf water potential and leaf temperature to MAT, MAP and AI (aridity index?).\nResponse: Thank you for your constructive comment. We have expanded Fig. S3 as suggested to include similar graphs showing the responses of g , E A , T , T , WUE s net leaf leaf i and WUE to MAT, MAP, and AI (Fig. 6 in the revised manuscript). The updated figure t is provided, and corresponding descriptions have been added to the revised Results (Lines 336-369) and Discussion (Lines 557-576) section.\nL265-273: Please rephrase the inaccurate and misleading statement that “Globally, elevated temperature had no significant effect on gs”, in order to more accurately portray the complex and diverse range of highly significant and non-significant gs responses to warming described in the literature. Please expand this section to better highlight the wide range of positive, negative and neutral responses of gs and E to warming, particularly in the low/moderate range of warming (0.5 to 5 degrees Celsius). Please re-analyse the data to try to elucidate the key drivers (MAT, MAP, AI?) of this strikingly large variation of gs and E responses to warming among studies.\nResponse: Thanks for your advice. This part has been revised as “Overall, warming effects on g were highly variable, exhibiting no consistent directional trend across the s full dataset (-1.4%; 95% CI: -7.1% to 4.7%; Fig. 1). Among all observations, 54.67% showed increases, 43.15% showed decreases, and 2.18% showed no significant change (Fig. S3a), underscoring the context-dependent nature of g responses. In contrast, s warming generally enhanced E by an average of 26.3% (95% CI: 12.1% to 42.2%; Fig. 1), although individual responses were variable (61.4% were positive, 1.75% were neutral, and 36.84% were negative; Fig. S3b). On average, A declined (-9.6%; 95% net CI: -13.5% to -5.6%; Fig. 1), with a majority of observations showing decreases (61.74%; Fig. S3c). This decrease in A contributed to the observed reduction in WUE net i (-11.0%; 95% CI: -16.4% to -5.2%; Fig. 1), which was observed in 57.49% of cases (Fig. S3d). WUE decreased even more markedly (-30.2%; 95% CI: -37.7% to -21.8%; t Fig. 1), with declines being observed in 75.56% of the data (Fig. S3e). T increased leaf consistently by 19.3% (95% CI: 13.6% to 25.3%; Fig. 1), with nearly all observations (98.13%) reporting positive responses (Fig. S3f). Meanwhile, Ψ declined (-16.7%; leaf 95% CI: -22.0% to -11.1%; Fig. 1), with negative responses in 69.87% of observations (Fig. S3g). lines 181-196”\nTo further elucidate the drivers of this large variability, we implemented a random forest model including plant functional type (PFT), warming duration, warming facility, rooting condition, warming intensity, mean annual temperature (MAT), mean annual precipitation (MAP), and aridity index (AI).\nL285-286: This result is very counterintuitive, as one would expect stronger negative impacts on Anet with increasing temperature elevation. Do you have a plausible explanation for this rather counterintuitive finding of the meta-analysis? Same would apply to L300-302.\nResponse: Thank you for pointing this out. We apologize for the confusion caused by our wording. You are correct. The effects of elevated temperature on A became net increasingly negative with greater temperature elevation. This was our intended meaning but was not clearly conveyed in the original text. In the revised manuscript, we have clarified this point and provided additional analyses. Specifically, using five different models, we analyzed the relationships between leaf physiological traits and warming amount, and found that A showed a significant negative correlation with net increasing warming amount, indicating that the negative impact of warming on A net became stronger with increasing warming magnitude.\nRegarding the original L300-302, the results changed slightly after we incorporated additional studies in the revised dataset. The updated analysis shows that “The effects of warming on A (Q = 7.79, p = 0.02) and WUE (Q = 62.06, p < net M t M 0.001) were also temperature-dependent, with high warming causing stronger declines than low or moderate warming (Fig. S11; Table S2) lines235-238”. However, the impact of temperature elevationon Ψ (QM = 1.63, p = 0.44) were not significantly leaf different across the three categories (Fig. S11; Table S2), despite >5℃ warming decreased Ψ the most. We suspect that these non-linear patterns may be partly driven leaf by the non-linear responses of g and E.\ns\nL299-300: This finding makes no sense to me, as I don’t understand why low temperature elevation had a significant impact on Tleaf but moderate temperature elevation didn’t…\nResponse: We appreciate your careful observation. We agree that this result appears counterintuitive. This is likely because the threshold-response of E. When warming amount < 3 °C, E showed no up-regulation, so transpirational cooling did not offset the added heat load and T increased significantly. By contrast, at 3-5 °C, E started to rise, leaf providing partial evaporative cooling that dampened the T response; the mean effect leaf was therefore smaller and not statistically significant. One additional factor likely contributed: smaller sample sizes for T , especially in the low- and moderate-warming leaf groups.\nL339-343: I don’t understand what you mean here, please clarify, elaborate and expand with a more straightforward and explicit interpretation of the observed differences. Same for L350-352.\nResponse: Thank you for your comment. Although warming did not significantly change g within either treatment group (ambient or elevated CO conditions; well- s 2 watered or drought), the responses in the two groups differed significantly from each other. This is a common outcome in meta-analysis: the between-group comparison (QM test) evaluates whether the mean effect sizes differ between groups, regardless of whether each group’s mean effect size is individually significant. In our case, warming have no significant effect on g regardless of CO conditions or water conditions, s 2 because of the variability of g responses. However, the magnitude of the response s varied, leading to a statistically significant difference between the two treatment categories. We have revised the results (lines 373-376 and lines 386-389).\nL362-364: This statement appears to be at odds with the general patterns described in L56-57 and FigS3. If E decreases with warming in temperate/boreal regions and remains unchanged in subtropical/tropical regions, where is it that E increases significantly under warming? Please clarify this apparent contradiction in the manuscript.\nResponse: We thank the reviewer for pointing out this important clarification. The apparent discrepancy arises because our main analysis was based on field experiments, which may not fully capture the continuous influence of climate along broader gradients. In addition, by classifying sites only into two broad categories (temperate/boreal vs. subtropical/tropical), some regional variability may have been obscured. In the revised manuscript, regression analysis of E against mean annual temperature (MAT) indicates that in regions with relatively high MAT, E tends to increase with warming (Fig. 6).\nL370: Another inaccurate and potentially misleading statement that should be rephrased and carefully modulated: “…elevated temperature did not impact gs significantly across all studies”. Figure 2a shows a much more complex picture than suggested by this overly simplistic statement, with both highly significant increases and decreases in gs reported often in the literature.\nResponse: Thank you for pointing out this inaccuracy. We agree that the original statement was overly simplistic and did not capture the variability in g responses across s studies. We have rephrased this sentence to better reflect the observed complexity, acknowledging that warming causes both significant increases and decreases in g in s different studies. The revised text now reads: “Based on a comprehensive dataset compiled from global warming experiments, we found that warming effects on g varied s substantially among studies, with both significant increases and decreases reported, resulting in no consistent overall effect when averaged across all observations (Fig. 1a)”.\nL371-395: This section is very interesting, plausible and compelling, and well supported by the literature. Same applies to the findings and patterns reported in L333-334, L303-307, L431-438, L440-455, among several others. Figure 3 is also very nice and totally consistent with theory and experimental evidence.\nResponse: We greatly appreciate your positive feedback on these sections and figures. We are pleased that the interpretation and literature support were clear and consistent with theoretical and experimental evidence.\nL398-405: Please elaborate on this important point and try to better highlight it by providing suitable examples from the literature (e.g examples/references of large increases and large decreases in gs and E under warming).\nResponse: Thanks for your suggestion. In the revised manuscript, we have elaborated on this point and added examples from the literature that illustrate the contrasting stomatal responses to warming. For instance, large decreases in g under warming have s been reported (Wujeska-Klause, Bossinger & Tausz, 2015; Lamba et al., 2018). In contrast, substantial increases in g have been documented under warming to stimulate s transpirational cooling (Urban et al., 2017; Marchin et al., 2022).\nL415-430: I think this section is also very nice and relevant, although I have some reservations regarding the excessively high thresholds encountered for significant gs and E increases under warming (5 and 3 degrees, respectively). Also please rephrase the inaccurate statement “…when temperature increases were below 5ºC, elevated temperature did not affect gs…”, as Figure 2 clearly shows that many field and pot studies have reported highly significant increases (e.g. Leon-Sanchez et al 2016, 2018) or decreases (Wertin et al 2017) in gs under low/moderate experimental warming (2-3 degrees), as becomes evident from Figure 2. Same applies to L428 (gs responses to low-moderate warming vary widely among studies and are certainly not “stable”!). Same for L529-530 of the Conclusions section!\nResponse: Thank you very much for this helpful clarification. We agree that our original wording understated the large variability in g responses under low-moderate warming. s In the revised manuscript, we have removed the inaccurate statement that warming below 5 °C does not affect g . The intention of L415-430 in the original version was to s highlight the interactive impacts of warming magnitude and duration. To better capture this variability, in the revision we re-analyzed the joint effects of warming amount and duration by fitting models for each leaf trait. For traits showing significant interaction (p < 0.05), we generated predictions across the observed duration range at three levels of warming (10th/50th/90th percentiles: Low/Mid/High) with 95% confidence intervals (Fig. 3). Corresponding text in the Results (lines 248-256) and Discussion (lines 459-467) has been revised.\nL430: Do you mean trade-offs between hydraulic safety and thermal regulation? Please rephrase.\nResponse: Yes. We meant the trade-off between hydraulic safety and leaf thermal regulation (via evaporative cooling). In the revision, we removed the original sentence because it was redundant with earlier text, and we rephrased the passage as follows: “In contrast, stronger warming initially stimulated gs, but this effect diminished and often reversed at longer durations (Fig. 3a), consistent with high gs triggering cavitation and hydraulic failure 52. To prevent such failure, plants likely prioritize water conservation at the expense of carbon gain and evaporative cooling 17 (lines 463-467)”.\nL449-455: I think enhanced photorespiration with warming also contributes significantly to the observed pattern described in this paragraph, in addition to enhanced dark respiration, right?\nResponse: Thanks. Enhanced photorespiration has been added as a factor contributing to decline in A under warming.\nnet\nL468-482: Nice and compelling results, see the extensive work by Brent Helliker and co-workers on this topic for further support to this interpretation.\nResponse: We appreciate the reviewer’s positive feedback and helpful suggestion. We have already cited the relevant work by Brent Helliker and co-workers in our manuscript, specifically referencing the Nature Plants paper by Michaletz, et al., 2016 and the PNAS article by Still et al. (2022), which includes Dr. Helliker as a co-author. These references are used to support our interpretation regarding leaf-air thermal coupling and the differential temperature responses plant growth forms. We believe these references aligns well with the reviewer’s comment and strengthens the conceptual basis of our discussion.\nReference\nMichaletz, S.T., Weiser, M.D., McDowell, N.G., Zhou, J., Kaspari, M., Helliker, B.R. & Enquist, B.J., (2016). The energetic and carbon economic origins of leaf thermoregulation. Nature Plants 2(9), 16129. https://doi.org/10.1038/nplants.2016.129\nStill, C.J., Page, G., Rastogi, B., Griffith, D.M., Aubrecht, D.M., Kim, Y., Burns, S.P., Hanson, C.V., Kwon, H., Hawkins, L., Meinzer, F.C., Sevanto, S., Roberts, D., Goulden, M., Pau, S., Detto, M., Helliker, B. & Richardson, A.D., (2022). No evidence of canopy- scale leaf thermoregulation to cool leaves below air temperature across a range of forest ecosystems. Proceedings of the National Academy of Sciences 119(38), e220568211\nL506-506-525: Very nice and compelling section, see work by Leon-Sanchez et al 2018-2020 and Aparecido et al 2020 for further support for your interpretation of the interactions between warming and drought.\nResponse: Thanks for the positive feedback on this section and the helpful suggestion to consider the work of León-Sánchez et al. (2018, 2020) and Aparecido et al. (2020) to further support our interpretation of the interactions between warming and drought. We have revised the discussion to incorporate key insights from these studies. Specifically, we now highlight that drought constrained the capacity for transpirational cooling despite a tendency for increased stomatal conductance, suggesting a limitation in the potential for sustained heat avoidance under low soil moisture availability, as discussed by Aparecido et al. (2020). We also reference the findings of León-Sánchez et al. (2020), who reported similarly strong reductions in photosynthesis under both warming and combined warming-drought conditions. These studies reinforce our discussion. Some of them were cited as:\n“For instance, the lower wind speeds typically occurring within OTCs can increase boundary layer resistance, thereby affecting whole-plant transpiration (León-Sánchez et al., 2020)” (lines 116-118).\n“This decoupling of carbon assimilation from water flux was further evidenced by pronounced reductions in WUE and WUE (León-Sánchez et al., 2020)” (lines 433- i t 434).\n“This aligns with the findings of León-Sánchez et al., (2020), who reported that reductions in A were similarly pronounced under warming alone and the combined net warming and rainfall reduction treatment” (lines 612-614).\n“This pattern suggests that drought constrained transpirational cooling despite a tendency for higher g , supporting that heat avoidance through increased water loss s may not be sustainable over extended periods in arid environments constrained by soil moisture availability (Aparecido et al., 2020)” (lines 599-602).\nL526-554: The Conclusions section is very nice, compelling and timely, and elegantly summarizes the main findings of the study. My only reservation is that I think the warming thresholds reported for significant enhancement of gs and E (5 and 3 degrees) appear to be unrealistically high in my own experience, as I have often encountered large increases in gs and E in the field with low/moderate experimental warming (2- 3ºC). I think that including WUEi and WUEt in the meta-analysis, and reanalysing field studies separately from pot/growth chamber studies might help clarify this (in my opinion) controversial and inconclusive aspect of the study (i.e. the high warming thresholds reported).\nResponse: We sincerely thank the reviewer for this thoughtful and constructive comment. A reanalysis of field-based experiments showed that g decreased linearly s with warming amount, but the threshold responses remained for E (3 °C) appeared high compared with observations from some field experiments, which also suggests the decoupling of g and E. We also included WUE and WUE, and compared the impacts s i t of experimental protocols.\nL166-167: I think there are some highly relevant studies with suitable and useful data that are currently missing from the 184 references list used for the meta-analysis (see Supp Material), and that could/should be also included to expand its scope and the strength of the conclusions. Some of them are even cited in the main text, but are missing from the 184 references list in the Supp Information section, such as:\nUrban (2017). Stomatal conductance increases with rising temperature. Plant signaling & behavior, 12(8), e1356534.\nUrban (2017). Increase in leaf temperature opens stomata and decouples net photosynthesis from stomatal conductance in Pinus taeda and Populus deltoides x nigra. Journal of experimental botany, 68(7), 1757-1767.\nMarchin (2016). Stomatal acclimation to vapour pressure deficit doubles transpiration of small tree seedlings with warming. Plant, Cell & Environment, 39(10), 2221-2234.\nLeón‐Sánchez et al (2020). Altered leaf elemental composition with climate change is linked to reductions in photosynthesis, growth and survival in a semi‐arid shrubland. Journal of Ecology, 108(1), 47-60.\nResponse: Thank you for suggesting these relevant studies. We agree that they provide valuable insights into plant physiological responses to warming. However, the main reason we did not include the two papers by Urban et al. (2017) and Marchin et al. (2016) in our meta-analysis is that the warming treatments in these studies were accompanied by simultaneous changes in VPD. According to our data selection criteria, control and warming treatments must differ only in temperature, while all other environmental conditions should remain comparable. This restriction ensures that the observed responses can be attributed specifically to temperature changes, thereby avoiding confounding effects from other factors. We acknowledge that León‐Sánchez et al. (2020) was inadvertently omitted, and it has now been included. In addition, after reviewing related literature, we have added 11 more relevant studies to the dataset.\nFinally, I would like to recommend the authors to have a look at a very recent paper that I believe is highly relevant to the topic of the present paper, and that should be useful for a more fine-tuned interpretation of the patterns and main findings encountered in the meta-analysis.\nEvans, M. E., Hu, J., & Michaletz, S. T. (2025). Scaling plant responses to heat: From molecules to the biosphere. Science, 388(6752), 1167-1173.\nResponse: Thank you for bringing this reference to our attention. It is highly insightful and inspired us to consider the physiological basis of heatwave definitions. Heatwaves are typically defined as periods of at least three to five consecutive days during which daily maximum temperature exceeds the climatological average by more than 5 °C. Accordingly, our finding that g exhibits a threshold at 5 °C, together with the sharp s exacerbation of A and WUE reductions beyond this level, may provide physiological net support for this definition. We have added the following statement in the Discussion: Furthermore, heatwaves are typically defined as periods of at least three or five consecutive days during which daily maximum temperature exceeds the climatological average by more than 5 °C 49. Consistent with this definition, we found that warming beyond 5 °C sharply exacerbated the reductions in A and WUE (Fig. S11), net highlighting the detrimental impacts of heatwaves 13, with the g threshold at 5 °C s providing physiological support for this benchmark (lines 452-458).\nReviewer #2 (Remarks to the Author):\nThis manuscript presents an exhaustive literature review of elevated temperature studies with terrestrial plants using meta-analysis. The analysis appears to be well done, and quantitatively summarizes the literature in a concise manner. A few major concerns are:\n1) The Introduction does not describe what other meta-analyses have been done concerning elevated temperature in terrestrial plants. Thus, it is not entirely clear what is novel about the current manuscript. Such information is critical. Some examples of previous meta analyses include\nZhang, J., Deng, L., Jiang, H. et al. The effects of elevated CO2, elevated O3, elevated temperature, and drought on plant leaf gas exchanges: a global meta-analysis of experimental studies. Environ Sci Pollut Res 28, 15274–15289 (2021).\nhttps://doi.org/10.1007/s11356-020-11728-6.\nLiang, J, Xia, J, Liu, L. Global patterns of the responses of leaf-level photosynthesis and respiration in terrestrial plants to experimental warming. Journal of Plant Ecology 6: 437 – 447 (2013). DOI 10.1093/jpe/rtt003\nWang, D., Heckathorn, S.A., Wang, X. et al. A meta-analysis of plant physiological and growth responses to temperature and elevated CO2 . Oecologia 169, 1–13 (2012). https://doi.org/10.1007/s00442-011-2172-0\nResponse: Thank you for this helpful suggestion. We have revised the Introduction to summarize previous meta-analyses on the effects of elevated temperature on terrestrial plants. These studies provide important syntheses of plant physiological responses to warming, but they differ from our work in scope and focus. The following texts have been added in the introduction “Previous meta-analyses have synthesized plant physiological responses to climate warming, including thermal acclimation of leaf respiration 36, single and combined global-change drivers on g 37, A 38 and leaf s net respiration 29,39-41. To the best of our knowledge, no study has yet comprehensively investigated the complex mechanisms underlying stomatal regulation and its interrelations with A and E under warming. (lines 155-159)”\nnet\n2) Broad statements are often made based on regressions that sometimes explain less than 10% of the variability between the values of a given gas exchange variable and the magnitude in temperature difference between treatment and control. Something similar occurs between gas exchange variables and the duration of the temperature treatment. It would be helpful for the reasons behind the very low r2 values and the implications of this to be mentioned in the Discussion section.\nResponse: We appreciate the reviewer’s insightful comment. We fully agree that low R2 values in single-variable regressions may raise concerns about the strength of the relationships. Since gas exchange responses to warming are driven by multiple interacting factors, and that low R2 values in univariate regressions likely reflect this complexity. To better address this issue, we implemented a random forest model that includes plant functional type (PFT), warming duration, warming facility, rooting condition, warming intensity, mean annual temperature (MAT), mean annual precipitation (MAP), and aridity index (AI). While each variable individually explains a limited amount of variation, together they significantly improve model performance (R2 = 0.52-0.82). This reinforces the need for multivariate approaches to understand plant physiological responses under climate change.\n3) The Conclusions section contains a lengthy summary of the Results. This could possibly be shortened and more emphasis on why the results are important could be mentioned. Currently, the text only indicates that the results would help with modeling.\nResponse: Thank you for this valuable suggestion. In the revised manuscript, we have shortened the summary of the Results and expanded the discussion of their broader implications.\nA list of minor comments is given below:\n1) The title appears to be missing a word? Change “associated” to “under” or “in response to”?\nResponse: Thanks. The title has been revised as “Decoupling of stomatal conductance, transpiration and photosynthesis in terrestrial plants under elevated temperature”.\n2) Acknowledgements (lines 21) – improve grammar “…..contributed to this in the present meta-analysis.” Change to “contributed to the present meta-analysis”\nResponse: Thanks. Revised.\n3) Abstract (lines 56-57) – is the increase in E under elevated temperature related to a concurrent increase in air VPD? If so, this should be clarified briefly in the text.\nThank you for the comment. Yes. Warming-induced increases in E reflect not only g s but also concurrent rises in VPD. Because of word-limit constraints and since we emphasized VPD later in the abstract (For warming < 3°C, E remained stable while g s declined for warming < 5°C; above these thresholds, both increased, with E having a lower threshold, indicating E is influenced not only by g but also by temperature-driven s increases in vapor pressure deficit (lines55-58)), we did not elaborate at that specific sentence. We have now clarified this in the Discussion (In contrast, E did not track the responses of g , but instead showed a positive response to warming (Fig. 1a), suggesting s that E was driven not only by g but also by additional factors such as VPD 4 (lines407- s 409)).\n4) Abstract (line 60) – again, is the E increase related to a concurrent increase in VPD?\nResponse: Thanks for your comment. This sentence has been revised to For warming < 3°C, E remained stable while g declined for warming < 5°C; above these thresholds, s both increased, with E having a lower threshold, indicating E is influenced not only by g but also by temperature-driven increases in vapor pressure deficit (lines55-58)”. s\n5) Abstract (lines 62-64) – Under elevated CO2, was the amplification of the Anet response to temperature due to decreasing gs under elevated CO2? If so, please rephrase the text.\nResponse: Thanks for your comment. Our results showed that despite the magnitude of the g response to warming differed between the two CO treatments (QM = 5.95, p = s 2 0.015; Fig. 7a; Table S21), g showed no consistent responses to warming under either s ambient or elevated CO conditions. g tended to decrease in both treatments and the 2 s decrease was smaller under elevated CO conditions. Thus, the greater decrease in A 2 net was not caused by g . We deduced that photosynthetic acclimation to eCO involves a s 2 downregulation of photosynthetic capacity, reflected in reductions in V and J . cmax max This downregulation is thought to occur because sustained exposure to high CO leads 2 to a reallocation of nitrogen away from photosynthetic proteins (e.g., Rubisco) toward other metabolic processes, thereby lowering the maximum capacity for carboxylation and electron transport. Under warming, this effect can be exacerbated, since higher temperatures accelerate photorespiration and respiratory carbon losses while simultaneously reducing Rubisco specificity for CO . As a result, the combination of 2 eCO and warming imposes a compounded limitation on photosynthetic capacity, 2 leading to a stronger decline in A .\nnet\n6) Intro – Not much information on what other meta-analyses of elevated termperature have been done are given. Please add relevant studies at some point in the Introduction. This would be important for understanding the novelty of the current manuscript.\nResponse: We thank the reviewer for this helpful suggestion. We have revised the Introduction to provide a brief overview of key meta-analyses addressing plant physiological responses to climate warming. Specifically, we now cite studies on the thermal acclimation of leaf respiration (Slot & Kitajima, 2015), the effects of single and combined global change drivers on stomatal conductance (Liang et al., 2023), as well as meta-analyses focusing on net photosynthesis (A ) and respiration (Wang et al., net 2012; Liang et al., 2013; Crous, Uddling & De Kauwe, 2022; Wu et al., 2025), and overall leaf gas exchange responses (Zhang et al., 2021). We also highlight that, to our knowledge, no previous study has comprehensively explored the coordinated responses among stomatal conductance, A , and transpiration under warming, nor investigated net the climatic and experimental factors underlying these relationships. These knowledge gaps limit our ability to predict ecosystem functioning under future climate warming and extreme heat events.\n7) Intro (line 73) – “stomata” is plural. So, the phrase should be “stomata play” rather than “stomata plays”?\nResponse: Thanks. Revised.\n8) Intro (line 77) – “this coupling can become variable…..” What does this mean?\nPlease revise the text.\nResponse: Thanks for pointing this out. This sentence has been revised as “This coordination, however, tends to weaken under elevated temperatures, which may compromise the balance between water loss and carbon gain 4,5, and violate predictions based on stomatal optimization assumptions (lines72-74)”.\n9) Intro (lines 85-86) – the authors state, “Elevated temperature typically leads to an increase in vapor pressure deficit (VPD), which drives stomatal closure to minimize water loss”. How much of an increase in temperature is the text referring to? It is awkward that the Abstract states that no increase in gs occurs with moderate elevated temperature increase, but here “stomatal closure” is referred to.\nResponse: We thank the reviewer for this valuable comment. We agree that the current wording in the Introduction may appear contradictory to our overall results. In fact, this reflects the very motivation of our study. As noted, temperature influences g through s two opposing mechanisms. On one hand, elevated temperature increases VPD, which typically induces stomatal closure to minimize water loss. On the other hand, further temperature increases can shift the priority toward leaf cooling via transpiration, which may promote stomatal opening. Our meta-analysis was specifically designed to test whether such a threshold exists. Indeed, we found that g tends to decrease with s moderate warming, but when temperature rises by 5 °C or more, stomata begin to open, suggesting a switch in regulatory strategy. Therefore, although the overall average response of g to warming was not significant, this does not imply insensitivity; rather, s it reflects variable and non-linear stomatal responses across different warming levels.\n10) Intro (line 148) – Does the word “global” mean “overall”? Please change if this interpretation is correct.\nResponse: Thank you for pointing this out. We confirm that “global” was intended to mean “overall,” and we have now revised the wording accordingly in the manuscript.\n11) Intro (last paragraph) – Would one or more hypotheses be helpful? Or, maybe it is felt that it is unnecessary because the study is a quantitative review of the existing literature?\nResponse: We thank the reviewer for the suggestion. Given that this study is a quantitative synthesis aimed at estimating overall effects and exploring heterogeneity across diverse studies and environments, we framed our approach around research questions rather than directional hypotheses. The existing literature shows substantial context dependence and likely non-linear responses, meaning that narrow a priori hypotheses could be misleading. We have revised the primary objectives of the present study in the revised manuscript.\n12) Intro (line 155) – fix the formatting of “{Blonder, 2023 #37641}”\nResponse: Thanks. Revised.\n13) M+M (lines 158-159) – Please give the web addresses of the sites that were used, and the dates that they were accessed.\nResponse: We have added the web addresses of all databases used, along with the dates they were last accessed, in the Materials and Methods section.\n14) M+M (line 164) – Please re-phrase the following “maintenance of the same initial conditions.” It is not entirely clear what this means.\nResponse: Thank you for pointing this out. We agree that the phrase “maintenance of the same initial conditions” could be clearer. We have revised the sentence to clarify that we refer to studies in which the elevated temperature and control treatments started with comparable environmental and biological baseline conditions (e.g., soil properties, plant age, species, and initial climate conditions). The revised sentence now reads: “comparable baseline environmental and biological conditions between the elevated temperature and control treatments”.\n15) Figure S1 – Is a “register” something different from the two databases used? If not, please delete “registers”.\nResponse: Thanks. Deleted.\n16) M+M (line 168) – change “parameters” to “variables”?\nResponse: Thanks. Revised.\n17) M+M (lines 205-206) – it is common that the number of replications is not given in many studies, or the authors sometimes incorrectly state the number of replications due to pseudoreplication issues. How were such difficulties handled? Were only studies used where the number of replicates was given?\nResponse: Yes. We only included studies where the number of replicates was explicitly reported.\n18) M+M (lines 226-230) – were possibly different effects in growth chambers, greenhouses, and field studies contemplated? If not, could this lead to a bias in the results obtained?\nResponse: Yes, we accounted for the potential differences among growth chambers, greenhouses, OTC, and IR heaters by including the warming facility type as a categorical variable in our random forest model. This allowed us to assess and control for the influence of different experimental setups. In addition, we conducted comparisons across facility types to evaluate whether they produced significantly different responses (Fig. 5).\n19) Results (line 267) – change “globally” to “overall”?\nResponse: Thank you for the suggestion. We agree that “overall” is a more appropriate term in this context, and we have revised the sentence accordingly.\n20) Results (lines 286-286)- The authors state, “, the impacts on A (p < 0.001; Figure 2c; Table S1) decreased significantly with ΔT,”. Maybe the text is not clear? If the response is linear, why would the “impact” decrease as deltaT increases?\nResponse: Thank you for pointing this out. Our original wording was indeed unclear. What we intended to convey is that the negative effects of warming on A increased net became stronger as warming amount increased. We have revised the sentence accordingly to “In contrast, A responses showed a negative correlation with net increasing temperatures (p < 0.05; Fig. 2c; Table S1)” lines (216-217).\n21) Supplementary material – A title of “Stomatal regulation at elevated temperature: Trade-offs between water use, transpirational cooling and photosynthesis” is given at the top of the document. What does this refer to?\nResponse: Sorry for the mistake. The title has been revised as “Decoupling of stomatal conductance, transpiration and photosynthesis in terrestrial plants under elevated temperature”.\n22) Figure 4 (legend) – the text states, “The dashed horizontal line marks the threshold for significant temperature effects.” This is unclear. The dashed line is at zero. Please change if necessary.\nResponse: Thanks. We have revised the legend to: “Fig. 4 Effects of plant functional types on leaf physiological responses to experimental warming. a-f, Mean effect sizes with 95% confidence intervals for paired PFT contrasts: herbs versus woody species (a), shrubs versus trees (b), broadleaf trees versus conifers (c), deciduous broadleaf versus evergreen broadleaf (d), C3 herbs versus C4 herbs (e), and annual herbs versus perennial herbs (f). Bars show mean effect sizes; error bars indicate 95% CIs. Effect sizes are considered significant when their 95% CI does not overlap zero. Numbers beneath bars denote sample sizes. Between-group heterogeneity was assessed using the omnibus test of meta-regressions with intercepts. Significance levels are denoted as †0.05 < p < 0.1, *p < 0.05, **p < 0.01, and ***p < 0.001. Variable abbreviations as in Fig. 1.”\n23) Figure 7c – move asterisk.\nResponse: Thank you for the suggestion. We have adjusted the placement of the asterisks in all figures to ensure that significance marks are displayed clearly and accurately.\n24) Discussion (lines 389-390) – the text states, “reported that decreased with rising temperatures until 42 °C,”. Is something missing here? E decreased?\nResponse: Thanks for pointing this out. The sentence has been corrected to “A comparable threshold pattern was observed in experimental warming up to 42 °C, where g decreased with temperature but rose again at higher values, even when A s net approached zero or became negative” (lines450-452).\n25) Discussion (line 406) – change “minimize” to “minimizing”?\nResponse: Thanks. Revised\n26) Discussion (line 417) – change “conflict” to “conflictive”?\nResponse: Thanks. This sentence has been revised as “Debate persists on whether stomatal acclimation to VPD under warming primarily maintains carbon balance or preserves water status” (lines459-460).\n27) Discussion (line 544) – spelling of “decupling”?\nResponse: Sorry for the mistake. Revised.\n28) Discussion (line 555) – uncategorized references?\nResponse: Sorry for the mistake. Revised.\n29) Conclusions – most of the text reads as a summary of the results. Please shorten this aspect of the Conclusions. More emphasis could be placed on the implications of the study. Just stating that the results are interesting for modeling purposes seems rather limited.\nResponse: We thank the reviewer for this helpful suggestion. We have shortened the summary of results and expanded the discussion on how the observed nonlinear and context-dependent responses of g , E, and A challenge the assumptions of stomatal s net optimization theory."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have now adequately and satisfactorily addressed all the major issues raised in my review report on the earlier version of the manuscript. Most notably, the explicit inclusion of WUEi and WUEt data in the meta-analysis significantly supports and strengthens the key message of the paper that photosynthesis and leaf water flux tend to decouple with warming, and I commend the authors for this. Overall, I am rather impressed by the quality and comprehensiveness of the revisions, which have improved the revised paper very significantly relative to the earlier version. The revised manuscript has been thoroughly revised and largely rewritten to address the concerns and suggestions of the reviewers. I really enjoyed reading the revised manuscript, which is very well written. I also appreciate the detailed, thoughtful and knowledgeable letter of response to the reviewers, in which all the arguments made are very compelling and convincing.\nIn my opinion, this work represents a very timely and strong contribution that successfully synthetizes and advances current knowledge on an important research topic that will be of interest to a wide readership.\nAt this stage, I can only offer a few more relatively minor comments and suggestions for further improvement.\na) Figure 1: Panel a is obviously the most important part of this compound figure, so I would suggest to enlarge it relative to the other panels, to better highlight its key relevance.\nb) I suggest to add a brief paragraph in the discussion to specifically address, summarize and clarify the impacts of warming duration on the assessed traits, as some of the figures (e.g. Fig 2) appear to suggest progressive acclimation of Anet, E and WUE to warming in longer lasting experiments, right? Warming duration (WD) appears to have sizeable effect sizes on the magnitude of response of most of the physiological parameters measured (Fig 1), and I think this is a very interesting, intriguing and important aspect of the study that could be better highlighted and clarified in the Discussion section. Please elaborate and expand on this aspect (possibly around L243-258?).\nc) In Figure 6, please clarify the interpretation of the Aridity Index for readers that are not familiar with this index, as the interpretation might appear counterintuitive to some readers (higher AI values mean less arid climatic conditions, right?). Alternatively, use the more intuitive “Aridity” (1-AI), which is positively related to climatic aridity (e.g. see Muñoz-Gálvez 2025 and references therein as an example). Also, please consider changing the colours selected for the HW and LW categories (blue seems more appropriate and intuitive for the HW category, right?).\nd) L120-122: this is an important consideration, so it might be useful to briefly mention here that topsoil desiccation with warming may lead to significant vertical decoupling of water and nutrient availability to plants along the soil profile, which may exert strong negative impacts on Anet and WUE due to nutrients becoming unavailable to plants in the dry topsoil layer (which is is usually the most fertile and nutrient-rich by far. See work by Ryel et al, Cramer et al and Querejeta et al.).\ne) L149: heat stress (or damage) avoidance?\nf) Please consider defining WUEi and WUEt in the Introduction section, for the sake of clarity and readability to readers unfamiliar with these parameters. L161: interrelations with Anet, E and WUE? I think an “Introduction” header is missing from L70, right?\ng) L165: and WUE also?\nh) L265-267: Please consider removing this sentence, since the difference is not statistically significant? Then mention E in the next sentence as well?\ni) L606: resulting in a LARGER rise in Tleaf? Is this an error?\nj) L614-616: See also León-Sánchez et al 2018 (included in the meta-analysis) which shows very similar patterns in another long-term field experiment. Explicitly include also the large decreases in WUE (in addition to Anet) in this sentence?\nk) L449-453: Wertin et al 2017 (not included in the meta-analysis) provides a nice example for the claims made in L449-453, L464 and L482-483.\nl) I think Figures S11 and S18 are very nice and could be moved to the main text, if the authors agree (just a suggestion, feel free to ignore it).\nm) Figure S2: what are the implications of the strong publication bias detected for Anet in this nice figure? Could you briefly mention and explain this in the Discussion section (or at least in the figure caption)?\nn) I recommend revising the quality, grammar and style of English in the Supporting Material, as some important sentences are extremely succinct and sometimes ambiguous, which may interfere with straightforward understanding of key points by readers.\nSuggested references:\nRyel, R. J., Ivans, C. Y., Peek, M. S., & Leffler, A. J. (2008). Functional differences in soil water pools: a new perspective on plant water use in water-limited ecosystems. In Progress in botany (pp. 397-422). Berlin, Heidelberg: Springer Berlin Heidelberg.\nQuerejeta, J. I., Ren, W., & Prieto, I. (2021). Vertical decoupling of soil nutrients and water under climate warming reduces plant cumulative nutrient uptake, water‐use efficiency and productivity. New Phytologist, 230(4), 1378-1393.\nCramer, M. D., Hawkins, H. J., & Verboom, G. A. (2009). The importance of nutritional regulation of plant water flux. Oecologia, 161(1), 15-24.\nWertin, T.M., Belnap, J. and Reed, S.C. (2017), Experimental warming in a dryland community reduced plant photosynthesis and soil CO2 efflux although the relationship between the fluxes remained unchanged. Funct Ecol, 31: 297-305.", "reviewer_2": "The revisions undertaken are comprehensive and increase the overall breadth of the study. I feel that the revisions have been well done. However, the revised results in the Abstract could be expressed more clearly. Some suggestions in this regard are given below:\n1. The Abstract heading is missing as are the Keywords.\n2. Abstract (Lines 57-60) – This sentence is not well constructed and is difficult to follow. Please re-write by separating it into two sentences.\n3. Abstract (Lines 60-63) – “Trees experienced smaller decreases in water-use efficiency (WUE) than shrubs, conifers showed stronger declines in gs and Anet than broadleaf species, and C3 herbs were more negatively affected for Anet and WUE than C4 herbs.” Going into the specifics of a wide number of responses for different growth forms, photosynthetic pathways, etc., may be of limited importance here in the Abstract. Maybe something general about WUE response to elevated temperature, and then how WUE in different growth forms responded to warming?\n4. Abstract (Lines 63-64) – “Warmer temperature promoted positive gs and E responses, whereas higher precipitation buffered warming-induced declines in Anet.” Doesn´t the text already say that gs and E increased at warmer temperatures in lines 57-60,? Or, are you referring to something else such as mean annual temperature (MAT)? Also, what does “higher precipitation” mean? Please clarify these points in the text.\n5. Discussion (Line 405) – It is unclear why the text is in bold."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have now adequately and satisfactorily addressed all the major issues raised in my review report on the earlier version of the manuscript. Most notably, the explicit inclusion of WUEi and WUEt data in the meta-analysis significantly supports and strengthens the key message of the paper that photosynthesis and leaf water flux tend to decouple with warming, and I commend the authors for this. Overall, I am rather impressed by the quality and comprehensiveness of the revisions, which have improved the revised paper very significantly relative to the earlier version. The revised manuscript has been thoroughly revised and largely rewritten to address the concerns and suggestions of the reviewers. I really enjoyed reading the revised manuscript, which is very well written. I also appreciate the detailed, thoughtful and knowledgeable letter of response to the reviewers, in which all the arguments made are very compelling and convincing.\nIn my opinion, this work represents a very timely and strong contribution that successfully synthetizes and advances current knowledge on an important research topic that will be of interest to a wide readership.\nAt this stage, I can only offer a few more relatively minor comments and suggestions for further improvement.\na) Figure 1: Panel a is obviously the most important part of this compound figure, so I would suggest to enlarge it relative to the other panels, to better highlight its key relevance.\nResponse: Thank you for your valuable suggestion. We agree that Panel a is indeed the most important component of this figure. In the revised version, we have retained only Panel a in the main figure, while moving the other panels to the Supplementary Information.\nb) I suggest to add a brief paragraph in the discussion to specifically address, summarize and clarify the impacts of warming duration on the assessed traits, as some of the figures (e.g. Fig 2) appear to suggest progressive acclimation of Anet, E and WUE to warming in longer lasting experiments, right? Warming duration (WD) appears to have sizeable effect sizes on the magnitude of response of most of the physiological parameters measured (Fig 1), and I think this is a very interesting, intriguing and important aspect of the study that could be better highlighted and clarified in the Discussion section. Please elaborate and expand on this aspect (possibly around L243-258?).\nResponse: Thanks for your advice. A paragraph was added in Discussion as: “Our analysis shows that warming duration plays a crucial role in shaping plant physiological responses. In short-term experiments, warming tended to increase g and s E, while reducing A and both WUE and WUE; however, these initial responses net i t progressively weakened with longer warming durations (Fig. 2h-l). This pattern indicates partial acclimation of both carbon and water fluxes as warming persisted, likely driven by adjustments in biomass allocation. For instance, a previous meta- analysis reported that warming increases biomass allocation to roots, thereby enhancing access to deeper soil water 21.”\nc) In Figure 6, please clarify the interpretation of the Aridity Index for readers that are not familiar with this index, as the interpretation might appear counterintuitive to some readers (higher AI values mean less arid climatic conditions, right?). Alternatively, use the more intuitive “Aridity” (1-AI), which is positively related to climatic aridity (e.g. see Muñoz-Gálvez 2025 and references therein as an example). Also, please consider changing the colours selected for the HW and LW categories (blue seems more appropriate and intuitive for the HW category, right?).\nResponse: Thank you for pointing this out. We agree that the directionality of the Aridity Index may be counterintuitive to some readers. Accordingly, we have added a clearer explanation in the Methods section: “The AI, defined as the ratio of MAP to mean annual potential evapotranspiration, was extracted for each study site from CHELSA version 2.1 raster layers based on geographic coordinates 71. Lower AI values indicate drier, more arid conditions, whereas higher values represent wetter, more humid climates, respectively”.\nThe colours selected for the HW and LW categories in Figure 7b has been changed.\nd) L120-122: this is an important consideration, so it might be useful to briefly mention here that topsoil desiccation with warming may lead to significant vertical decoupling of water and nutrient availability to plants along the soil profile, which may exert strong negative impacts on Anet and WUE due to nutrients becoming unavailable to plants in the dry topsoil layer (which is is usually the most fertile and nutrient-rich by far. See work by Ryel et al, Cramer et al and Querejeta et al.).\nResponse: Thanks for your advice. This part has been revised as “In addition, warming exacerbates surface soil water stress, driving plants to rely more heavily on deeper water sources 27. Warming-induced topsoil desiccation can lead to pronounced vertical decoupling of water and nutrient availability within the soil profile, which may reduce A and water use efficiency (WUE) because nutrients in the dried topsoil become net inaccessible to plants 28. Consequently, rooting conditions, including soil depth and rooting volume, further constrain shape plant physiological responses to warming.”\ne) L149: heat stress (or damage) avoidance?\nResponse: Thanks. Heat stress avoidance has been used now.\nf) Please consider defining WUEi and WUEt in the Introduction section, for the sake of clarity and readability to readers unfamiliar with these parameters. L161: interrelations with Anet, E and WUE? I think an “Introduction” header is missing from L70, right?\nResponse: Thanks for your advice. WUEi and WUEt have been explained as “To the best of our knowledge, no study has yet comprehensively investigated the complex mechanisms underlying stomatal regulation and its interrelations with A and E under net warming. Changes in g , A and E inevitably lead to variations in intrinsic (WUE = s net i A /g ) and instantaneous water use efficiency (WUE = A /E)”. Additionally, net s i net Introduction title has been added.\ng) L165: and WUE also?\nResponse: Thanks for your advice. This sentence has been revised as “Here, we conducted a comprehensive meta-analysis to evaluate the effects of warming on stomatal behavior and its consequences for water loss, thermal regulation, carbon assimilation and WUE.”\nh) L265-267: Please consider removing this sentence, since the difference is not statistically significant? Then mention E in the next sentence as well?\nResponse: Thanks for this helpful suggestion. We agree that the difference between herbs and woody species in g is not statistically significant at the conventional α = 0.05 s level. However, we would prefer to retain the sentence for two reasons.\nFirst, the between‐group test shows a marginal effect (QM = 2.76, p = 0.097), which we report cautiously without overstating its significance.\nSecond, another related comparison shows a similar near-significant trend (p = 0.051), and presenting these patterns together provides context for the direction and magnitude of the functional-type differences even if they do not meet strict significance thresholds.\nTo avoid any misinterpretation, we have revised the wording to clearly indicate that these represent weak or suggestive trends, rather than significant effects.\nRegarding E, we now describe this response as statistically insignificant, consistent with your suggestion. The revised text reads: “For g , the 95% CIs of both groups s overlapped with zero, indicating no consistent responses. The difference between herbs and woody species was marginally significant (Q = 2.76, p = 0.097), with herbaceous M species showing a slight positive effect, whereas woody species exhibited a negative effect. E increased in herbs but remained unchanged in woody plants; however, this difference was not statistically significant (Q = 2.08, p = 0.15; Fig. 4a; Table S10). M Herbs and woody plants showed similar directional responses in A (Q = 1.03, p = net M 0.31), WUE (Q = 1.32, p = 0.25), WUE (Q = 0.06, p = 0.81), T (Q = 0.0016, p i M t M leaf M = 0.97) and Ψ (Q = 0.71, p = 0.4; Fig. 4a; Table S10).”\nleaf M\ni) L606: resulting in a LARGER rise in Tleaf? Is this an error?\nResponse: Sorry for this mistake. Yes, it is an error. This sentence has been revised as “resulting in a larger rise in T ”.\nleaf\nj) L614-616: See also León-Sánchez et al 2018 (included in the meta-analysis) which shows very similar patterns in another long-term field experiment. Explicitly include also the large decreases in WUE (in addition to Anet) in this sentence?\nResponse: Thanks for your advice. WUE and WUE have been added as “In contrast, i t A , WUE and WUE declined progressively with temperature.”\nnet i t\nk) L449-453: Wertin et al 2017 (not included in the meta-analysis) provides a nice example for the claims made in L449-453, L464 and L482-483.\nResponse: Thanks for you recommendation. This reference has been properly cited as “Notably, g exhibited a threshold response to warming: under modest warming (< s 5 °C), g declined (Fig. 2a), consistent with findings that a 2 °C warming can s significantly reduce g (Wertin, Belnap & Reed, 2017), likely driven by higher VPD s and evaporative demand (De Boeck et al., 2012; Grossiord et al., 2020). In contrast, g s increased when warming exceeded 5 °C, reflecting a shift toward transpirational cooling (Marchin et al., 2023).”\nl) I think Figures S11 and S18 are very nice and could be moved to the main text, if the authors agree (just a suggestion, feel free to ignore it).\nResponse: Thank you very much for your thoughtful suggestion. We agree that Figures S11 and S18 are informative; however, given the space limitations and the overall structure of the main text, we feel that it is more appropriate to keep these figures in the Supplementary Information.\nm) Figure S2: what are the implications of the strong publication bias detected for Anet in this nice figure? Could you briefly mention and explain this in the Discussion section (or at least in the figure caption)?\nResponse: Thank you for this helpful comment. We agree that the funnel plot for A net shows substantial asymmetry and Egger’s test indicates a significant publication bias. Given that our meta-analysis shows an overall negative response of A to warming, net this bias likely arose because studies reporting stronger or significant decreases in net photosynthesis were more likely to be published than those showing weak or non- significant responses. This would lead to an overestimation of the mean effect size.\nWe have added the following explanation to the Discussion: “Notably, the funnel plot and Egger’s test indicate strong publication bias for A . Because our overall result net shows that warming suppressed A , such bias likely reflects selective reporting of net studies with stronger or statistically significant declines in A . As a consequence, the net magnitude of the negative warming effect on A may be inflated, and this result should net be interpreted with caution.”\nn) I recommend revising the quality, grammar and style of English in the Supporting Material, as some important sentences are extremely succinct and sometimes ambiguous, which may interfere with straightforward understanding of key points by readers.\nResponse: Thank you for this helpful comment. According to the journal’s guidelines, all Supplementary Notes must be explicitly referenced in the main text. Because many of the results originally included in the Supplementary Information were not mentioned in the manuscript, we have moved all key results and descriptions that were central to the narrative from the Supplementary Information into the main text. Consequently, the former supplementary notes have been fully integrated into the revised manuscript and deleted from the supplementary file. The accompanying text has been carefully polished for grammar, clarity, and style. The remaining Supplementary Information now consists primarily of extended figures, tables and Supplementary References.\nSuggested references:\nRyel, R. J., Ivans, C. Y., Peek, M. S., & Leffler, A. J. (2008). Functional differences in soil water pools: a new perspective on plant water use in water-limited ecosystems. In Progress in botany (pp. 397-422). Berlin, Heidelberg: Springer Berlin Heidelberg.\nQuerejeta, J. I., Ren, W., & Prieto, I. (2021). Vertical decoupling of soil nutrients and water under climate warming reduces plant cumulative nutrient uptake, water‐use efficiency and productivity. New Phytologist, 230(4), 1378-1393.\nCramer, M. D., Hawkins, H. J., & Verboom, G. A. (2009). The importance of nutritional regulation of plant water flux. Oecologia, 161(1), 15-24.\nWertin, T.M., Belnap, J. and Reed, S.C. (2017), Experimental warming in a dryland community reduced plant photosynthesis and soil CO2 efflux although the relationship between the fluxes remained unchanged. Funct Ecol, 31: 297-305.\nReviewer #2 (Remarks to the Author):\nThe revisions undertaken are comprehensive and increase the overall breadth of the study. I feel that the revisions have been well done. However, the revised results in the Abstract could be expressed more clearly. Some suggestions in this regard are given below:\n1. The Abstract heading is missing as are the Keywords.\nResponse: The Abstract heading has been added.\n2. Abstract (Lines 57-60) – This sentence is not well constructed and is difficult to follow. Please re-write by separating it into two sentences.\nResponse: Thanks. This sentence has been revised as “g declined until temperatures s exceeded 5°C above ambient, whereas E remained stable under warming < 3°C. Beyond these two thresholds, both g and E increased with further warming.” s\n3. Abstract (Lines 60-63) – “Trees experienced smaller decreases in water-use efficiency (WUE) than shrubs, conifers showed stronger declines in gs and Anet than broadleaf species, and C3 herbs were more negatively affected for Anet and WUE than C4 herbs.” Going into the specifics of a wide number of responses for different growth forms, photosynthetic pathways, etc., may be of limited importance here in the Abstract. Maybe something general about WUE response to elevated temperature, and then how WUE in different growth forms responded to warming?\nResponse: Thanks for your advice. This sentence has been revised as “The sensitivity of g , A , and water-use efficiency to warming varied substantially among plant s net functional types, with distinct responses across, phylogenetic groups, and photosynthetic pathways.”\n4. Abstract (Lines 63-64) – “Warmer temperature promoted positive gs and E responses, whereas higher precipitation buffered warming-induced declines in Anet.” Doesn´t the text already say that gs and E increased at warmer temperatures in lines 57-60,? Or, are you referring to something else such as mean annual temperature (MAT)? Also, what does “higher precipitation” mean? Please clarify these points in the text.\nResponse: We thank the reviewer for this helpful comment. We agree that the wording in the original sentence was ambiguous. Our intention was to refer to background climatic conditions, specifically mean annual temperature (MAT) and mean annual precipitation (MAP), which modulated the magnitude of plant physiological responses to experimental warming. To avoid confusion, we have revised the sentence to explicitly state MAT and MAP. The revised text in the Abstract now reads: “Higher mean annual temperature amplified the positive warming responses of g and E, s whereas greater mean annual precipitation buffered the warming-induced declines in Anet.”\n5. Discussion (Line 405) – It is unclear why the text is in bold.\nResponse: Thank you for your observation. The bold formatting in Line 405 is intentional, as this sentence serves as the subsection heading for that part of the Discussion."}]

Decision letter