Summary: The strong covariation of temperature and vapour pressure deficit (VPD) in nature limits our understanding of the direct effects of temperature on leaf gas exchange. Stable isotopes in CO₂ and H₂O vapour provide mechanistic insight into physiological and biochemical processes during leaf gas exchange. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a leaf temperature range of 5–40°C, while maintaining a constant leaf‐to‐air VPD (0.8 kPa) without soil water limitation. Above the optimum temperature for photosynthesis (30°C) under the controlled environmental conditions, stomatal conductance (g_s) and net photosynthesis rate (A_n) decoupled across all tested species, with g_s increasing but A_n decreasing. During this decoupling, mesophyll conductance (cell wall, plasma membrane and chloroplast membrane conductance) consistently and significantly decreased among species; however, this reduction did not lead to reductions in CO₂ concentration at the chloroplast surface and stroma. We question the conventional understanding that diffusional limitations of CO₂ contribute to the reduction in photosynthesis at high temperatures. We suggest that stomata and mesophyll membranes could work strategically to facilitate transpiration cooling and CO₂ supply, thus alleviating heat stress on leaf photosynthetic function, albeit at the cost of reduced water‐use efficiency.

Recent studies have challenged the interpretation of plant water isotopes obtained through cryogenic vacuum distillation (CVD) based on observations of a large ²H fractionation. These studies have hypothesized the existence of an H-atom exchange between water and organic tissue during CVD extraction with the magnitude of H exchange related to relative water content of the sample; however, clear evidence is lacking. Here, we systematically tested the uncertainties in the isotopic composition of CVD-extracted water by conducting a series of incubation and rehydration experiments using isotopically depleted water, water at natural isotope abundance, woody materials with exchangeable H, and organic materials without exchangeable H (cellulose triacetate and caffeine). We show that the offsets between hydrogen and oxygen isotope ratios and expected reference values (Δ²H and Δ¹⁸O) have inversely proportional relationships with the absolute amount of water being extracted, i.e. the lower the water amount, the higher the Δ²H and Δ¹⁸O. However, neither Δ²H nor Δ¹⁸O values, were related to sample relative water content. The Δ²H pattern was more pronounced for materials with exchangeable H atoms than with non-exchangeable H atoms. This is caused by the combined effect of H exchange during the incubation of materials in water and isotopic enrichments during evaporation and sublimation that depend on absolute water amount. The H exchange during CVD extraction itself was negligible. Despite these technical issues, we observed that the water amount-dependent patterns were much less pronounced for samples at natural isotope abundance and particularly low when sufficiently high amounts of water were extracted (>600 µL). Our study provides new insights into the mechanisms causing isotope fractionation during CVD extraction of water. The methodological uncertainties can be controlled if large samples of natural isotope abundance are used in ecohydrological studies.

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

Forest evapotranspiration (ET) is one of the most important factors influencing the terrestrial hydrological cycle and is prone to being influenced by increasing drought events. This highlights the need to understand the interaction between global forest ET and drought. Consequently, we drew 710 sets of ecosystem-scale ET observations from 69 forest sites around the world and then systematically assessed the ET anomalies during droughts across the dominant forest and climate types. Overall, the response of forest ET to drought is non-monotonic. Under severe and extreme droughts with long durations, ET was reduced in most of forests in the world. We attributed the decreased forest ET to both restricted water supply and restricted water consumption by forests; however, lower magnitude and short-term droughts generally increased forest ET, and in some cases, ET even exceeded precipitation during droughts. We attributed this to the increased surface evaporation and the utilization of deep water by deep root systems. Specifically, we find that the positive anomaly of ET under droughts implied the drought paradox, which acts to accelerate the terrestrial hydrological cycle and further amplify the drought. This study as a global synthetic analysis of case studies on the site scale may have great potential for understanding the importance of the drought-modulated forest water cycle and the possibility of increasing drought stress with the effects of the drought paradox.

Carbon (C) isotope discrimination during photosynthetic CO₂ assimilation has been extensively studied, but the whole process of fractionation from leaf to soil has been less well investigated. In the present study, we investigated the δ¹³C signature along the C transfer pathway from air to soil in a coniferous and broad-leaved mixed forest in northeast China and examined the relationship between δ¹³C of respiratory fluxes (leaf, trunk, soil, and the entire ecosystem) and environmental factors over a full growing season. This study found that the δ¹³C signal of CO₂ from canopy air was strongly imprinted in the organic and respiratory pools throughout C transfer due to the effects of discrimination and isotopic mixing on C assimilation, allocation, and respiration processes. A significant difference in isotopic patterns was found between conifer and broadleaf species in terms of seasonal variations in leaf organic matter. This study also found that δ¹³C in trunk respiration, compared with that in leaf and soil respiration, was more sensitive to seasonal variations of environmental factors, especially soil temperature and soil moisture. Variation in the δ¹³C of ecosystem respiration was correlated with air temperature with no time lag and correlated with soil temperature and vapor pressure deficit with a lag time of 10 days, but this correlation was relatively weak, indicating a delayed linkage between above- and belowground processes. The isotopic linkage might be confounded by variations in atmospheric aerodynamic and soil diffusion conditions. These results will help with understanding species differences in isotopic patterns and promoting the incorporation of more influencing factors related to isotopic variation into process-based ecosystem models.