Decoupling of stomatal conductance from net assimilation at high temperature as a mechanism to increase transpiration

Decoupling of stomatal conductance from net assimilation at high temperature as a mechanism to increase transpiration | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Decoupling of stomatal conductance from net assimilation at high temperature as a mechanism to increase transpiration View ORCID Profile Philipp Schuler , View ORCID Profile Margaux Didion-Gency , View ORCID Profile Giovanni Bortolami , View ORCID Profile Thibaut Julliard , View ORCID Profile Günter Hoch , View ORCID Profile Christoph Bachofen , View ORCID Profile Ansgar Kahmen , Charlotte Grossiord doi: https://doi.org/10.1101/2025.11.03.686201 Philipp Schuler 1 Plant Ecology Research Laboratory PERL, School of Architecture, Civil and Environmental Engineering, EPFL , CH-1015 Lausanne, Switzerland 2 Community Ecology Unit, Swiss Federal Institute for Forest, Snow and Landscape WSL , CH- 1015 Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Philipp Schuler For correspondence: philipp.schuler{at}wsl.ch Margaux Didion-Gency 3 Ecological and Forestry Applications Research Center (CREAF) , E-08193 Cerdanyola del Valley, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Margaux Didion-Gency Giovanni Bortolami 1 Plant Ecology Research Laboratory PERL, School of Architecture, Civil and Environmental Engineering, EPFL , CH-1015 Lausanne, Switzerland 2 Community Ecology Unit, Swiss Federal Institute for Forest, Snow and Landscape WSL , CH- 1015 Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giovanni Bortolami Thibaut Julliard 1 Plant Ecology Research Laboratory PERL, School of Architecture, Civil and Environmental Engineering, EPFL , CH-1015 Lausanne, Switzerland 2 Community Ecology Unit, Swiss Federal Institute for Forest, Snow and Landscape WSL , CH- 1015 Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thibaut Julliard Günter Hoch 4 Physiological Plant Ecology, Department of Environmental Sciences, University of Basel , Schönbeinstrasse 6, CH-4056 Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Günter Hoch Christoph Bachofen 1 Plant Ecology Research Laboratory PERL, School of Architecture, Civil and Environmental Engineering, EPFL , CH-1015 Lausanne, Switzerland 2 Community Ecology Unit, Swiss Federal Institute for Forest, Snow and Landscape WSL , CH- 1015 Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christoph Bachofen Ansgar Kahmen 4 Physiological Plant Ecology, Department of Environmental Sciences, University of Basel , Schönbeinstrasse 6, CH-4056 Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ansgar Kahmen Charlotte Grossiord 1 Plant Ecology Research Laboratory PERL, School of Architecture, Civil and Environmental Engineering, EPFL , CH-1015 Lausanne, Switzerland 2 Community Ecology Unit, Swiss Federal Institute for Forest, Snow and Landscape WSL , CH- 1015 Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary Photosynthetic assimilation (A net ) and stomatal conductance (g s ) are usually strongly coupled, but this relationship is decreased or even lost at high temperatures (T air ). The contributions of environmental drivers (T air , vapour pressure deficit (VPD), and soil moisture) in interaction with the physiological mechanisms behind this process are still unclear. We exposed saplings of three temperate and tropical species to rising T air (20 to 40°C) at low (1.2 to 1.9 kPa) and increasing VPD (1.1 to 5.6 kPa), and at stable T air (35°C) to increasing VPD (1.4 to 4.3 kPa) under well-watered or chronic soil drought conditions (≤10 %). A net, g s , and transpiration (E) in the light and the dark and leaf thermoregulation were tracked throughout the experiment. When VPD remained low, g s continued to increase while A net decreased at T air > 35°C, leading to stomatal decoupling. In contrast, under rising VPD, trees maintained the coupling between A net and g s at high T air. While a decoupling of A net and g s only occurred when VPD was low, A net and E decoupled under both VPD regimes at high T air . Our results indicate that, since g s and VPD collectively drive E, stomatal decoupling is needed to increase E when VPD is not sufficiently high. Introduction It is widely assumed that net photosynthesis (A net ) and stomatal conductance (g s ) are optimally coupled to maximize carbon gain while minimizing water loss ( Wong et al., 1979 ; Lawson et al., 2010 ; Duursma et al., 2014 ). Hence, when A net declines at high air temperatures (T air ) due to biochemical limitations (Sage & Kubien, 2007), g s should decrease simultaneously ( Wong et al., 1979 ; Lawson et al., 2010 ; Duursma et al., 2014 ). Most leaf-level stomatal models assume this strong coupling (e.g., Farquhar & Wong, 1984; Medlyn et al., 2011 ). Yet, more and more studies are challenging this understanding, both in natural and controlled conditions, showing a decoupling (i.e., the loss of the strong relationship) between A net and g s under high T air ( Schulze et al., 1973 ; Aphalo and Jarvis, 1991 ; Eamus et al., 2008 ; Urban et al., 2017 ; Schönbeck et al., 2022 ; Marchin et al., 2023 ; Diao et al., 2024a ; Gauthey et al., 2024 ) – a process referred to as “stomatal decoupling”. In most studies, while A net tends to drop at T air above 30-35°C (i.e., reflecting the optimum Tair for photosynthesis), g s either remains stable (e.g., Schönbeck et al., 2022 ; Gauthey et al., 2024 ) or rises (e.g., Schulze et al., 1973 ; Urban et al., 2017 ; Diao et al., 2024a ). Keeping high g s (and hence transpiration, E) could be a mechanism that serves to avoid lethal thermal damage during heatwaves through continued evaporative cooling (e.g., Drake et al., 2018; Gauthey et al., 2024 , Bachofen et al., 2025 ), even though it increases the risk of embolism (e.g., Schoenbeck et al., 2022). However, how high T air , high vapor pressure deficit (VPD), and low soil moisture - factors that usually co-occur during heatwaves - contribute to the decoupling of A net and g s remains unclear. Similarly, the species-specific variability and the physiological mechanisms behind stomatal decoupling at high T air remain elusive ( Mills et al., 2024 ), leading to significant uncertainties in leaf-level stomatal models and our understanding of tree thermal tolerance. Potential drivers of the stomatal decoupling at high T air can be separated into purely physical and physiological components (see Mills et al., 2024 , for a detailed review of these processes). Physical drivers include the T air -driven increase in the diffusive rate of water vapour in the air ( Massman, 1998 ), which leads to increasing g s at increasing T air even if the opening of the stomatal aperture remains constant. This is considered the baseline for the observed stomatal decoupling. In addition, higher T air will increase the xylem hydraulic conductivity (K leaf ) because water viscosity declines sharply with increasing T air ( Matzner and Comstock, 2001 ), leading to less negative leaf water potential (Ψ) for a given transpiration rate, and hence, higher g s ( Mills et al., 2024 ). Higher T air will also raise the minimum epidermal conductance (i.e., residual water loss once stomata are closed, g min ) ( Duursma et al., 2014 ), which could further contribute to the higher measured g s with warmer air ( Gauthey et al., 2024 ). However, a strong T air -driven increase of g min likely only occurs at high T air > 40°C ( Wang et al., 2024 ). Moreover, while photosynthesis, to our current understanding, does not directly impact the stomatal apparatus ( Von Caemmerer et al., 2004 ; Rogers et al., 1980 ; Urban et al., 2017 ), it might have an indirect influence via T air -driven shifts in photosystem (PS) I and PS II activity ( Messinger et al., 2006 ). High T air alters the balance between the proton generation in PS II and the proton consumption in PS I, causing changes in the redox state inside the cells ( Busch, 2014 ). Increasing photorespiration at high T air ( Dusenge et al., 2019 ) or degradation of Rubisco above 40°C ( Bose et al., 1999 ) might be reasons for an altered ratio between PS I and PS II. Such changes in the redox state could be sensed by guard cells ( Lawson et al., 2010 ), therefore influencing the stomatal aperture and increasing g s. Together, these physical and physiological processes establish a foundational framework through which rising T air alone could drive increases in g s , independent of changes in stomatal aperture or carbon assimilation. Still, most mechanisms have never been tested experimentally. In addition, stomatal decoupling should depend on the environmental conditions experienced by the plants, particularly the evaporative demand and soil moisture ( Schulze et al., 1973 ; Mott and Peak, 2010). An increase in VPD (e.g., Diao et al., 2024b ) and a reduction in soil moisture (e.g., Brodribb and Holbrook, 2003) trigger a decline in leaf Ψ, lowering the vapour content in the stomatal pore, which in turn leads to stomatal closure ( Peak and Mott, 2011 ), reduced A net and g s (Grossiord et al., 2020), and may prevent stomatal decoupling. Yet, multiple studies have reported stomatal decoupling during natural heatwaves where elevated T air co-occurs with high VPD and low soil moisture (e.g., Drake et al., 2018; Marchin et al., 2023 ; Gauthey et al., 2024 ), challenging these observations and our understanding of gas exchange regulation. Moreover, while drought typically suppresses E through strong stomatal closure ( Hall and Schulze, 1980 ; Bachofen et al., 2023 ), increasing VPD can enhance E due to a greater evaporative demand ( Massmann et al., 2019 ), until stomatal limitations dominate under severe stress. Further complicating our understanding of g s and E responses to high T air , the decoupling appears highly species-specific. Among the 22 species reviewed by Mills et al. (2024) , only half exhibited consistent stomatal decoupling, with varying intensity—on average, gₛ doubled between 10°C and 40°C. Nevertheless, given the limited number of species studied, the extent to which this pattern applies across taxa remains uncertain. Whether or above which T air threshold stomatal decoupling occurs probably differs between plants with different climatic adaptations ( Mills et al., 2024 ). Species adapted to low soil moisture and relatively high VPD, such as in many temperate regions, likely respond differently than species growing in high soil moisture and low VPD environments, such as in the wet tropics ( Cunningham, 2004 ; Middleby et al., 2024 ; Slot et al., 2024 ). However, no study has explored the variation in stomatal decoupling across species originating from contrasting climatic types, the relative influence of T air , VPD, and soil moisture in regulating this process. We hypothesized that (1) stomatal regulation at high T air will be strongly impacted by VPD and soil moisture availability, with reductions in g s at high VPD and in dry soil, while it would increase with T air at stable VPD and moist soil conditions (i.e., leading to stomatal decoupling and increased E). Further, (2) stomatal regulation of tree species from temperate regions should be more sensitive to high T air , thereby increasing decoupling at high T air , low VPD, and moist conditions. In contrast, tropical species, which evolve at higher T air and VPD environments, may show a lower magnitude of decoupling under the same conditions. Therefore, (3) we expect stomatal decoupling to contribute to leaf temperature regulation, with higher E leading to stronger evaporative leaf cooling at higher T air for temperate and tropical tree species. Materials and methods Plant handling prior to the experiment In January 2024, approximately 50-100 cm tall saplings of three tree species from the European temperate climates (Köppen climate classification: Alnus cordata (Loisel.) Duby (Cfa-Cfb), Acer platanoides L. (Cfa), Phillyrea angustifolia L. (Csa) – 30 replicates per species) and three tree species from tropical wet to monsoonal climates ( Terminalia microcarpa Decne. – 30 replicates, Trema tomentosa (Roxb.) H. Hara, Syzygium jambos L. (Alston) – 24 replicates each (all three species from Am-Af climates)) were planted in 5L pots, using Oekohum “Container CLASSIC – ohne Torf” (ökohum gmbh, 8585 Herrenhof, Switzerland), a mixture of bark humus, bark compost, wood fiber, hemp fiber, coconut, miscanthus, expanded clay, perlite. The species were selected to represent different phylogenetic lineages ( Table S1 ). Prior to the experiment, the tropical tree species were grown in a greenhouse with a T air of 20-25°C and a relative humidity (RH) between 50-60%. All trees were transferred to a greenhouse with additional overhead lights (10 hours a day), where the T air was kept at 25°C and the RH at 50% (stable VPD of 1.6 kPa) until they were exposed to the experimental conditions. The flushing of the deciduous temperate tree species started within 2 weeks, and for P. angustifolia , after about one month, and their leaves were fully mature when they were used for the experiment. The six species were exposed sequentially to the experimental conditions, which lasted 12 days. One week before the experiment, we reduced watering to half of the randomly selected saplings for the drought treatment to ensure low soil moisture. Soil moisture was measured every second day using a handheld soil moisture probe (TDR100 Soil Moisture Meter, FieldScout, Hoskin Scientific, Burnaby, Canada). Two days before the experimental conditions started, each tree species was transferred into the three climate chambers (see experimental details below). During these two days, the trees were acclimated to the conditions in the climate chamber at a T air of 25°C, an RH of 50%, a day/night cycle of 12/12 hours, and an average photosynthetic active radiation (PAR) of ∼700 µmol m -2 s -1 (no UV radiation). During the acclimation period, the T air was decreased by 5°C during the night, but VPD was held constant. Experimental design To disentangle the individual and additive effects of T air , VPD, and soil water availability on the measured parameters (see below), the plants were exposed to three different T air and VPD regimes, each with a subset of well-watered or drought conditions (n=5 trees per species, atmospheric regime, and soil moisture treatment; n=4 for T. tomentosa and S. jambos ). The setpoints for the three T air and VPD regimes were: “increasing T air , low VPD”: increasing T air setpoints from 20 to 40°C (20.0°C, 25.0°C, 30.0°C, 32.5°C, 35.0°C, 40.0°C) and maintaining a low VPD around 1.2 kPa by increasing setpoints RH (50%, 62%, 72%, 75%, 79%, 85%, respectively). “increasing T air and VPD”: rising T air from 20 to 40°C as in (1) but increasing VPD by decreasing RH setpoints (50%, 37%, 28%, 24%, 21%, 16%; VPD = 1.2 kPa, 2.00 kPa, 3.1 kPa, 3.7 kPa, 4.4 kPa, 6.2 kPa, respectively). “stable T air , increasing VPD”: constant T air at 35°C while increasing VPD by reducing RH setpoints (80%, 65%, 55%, 40%, 30%, 20%). The actual average T air and VPD of the chambers can be found in Table S3 . Each of the six T air /VPD steps was maintained for two days, with the measurements (detailed below) conducted systematically on the second day to ensure plants had been exposed to the corresponding conditions. As during the acclimation period, T air was decreased by 5°C during the night (relative to the previous daytime conditions), but VPD was held constant during day- and nighttime. The mean soil moisture (soil volumetric water content, VWC) was 32.6% ± 0.3% SE, 32.4% ± 0.4% SE, and 31.2% ± 0.4% SE (“increasing T air , low VPD”, “increasing Tair and VPD”, and “stable T air , increasing VPD”, respectively) for well-watered plants and 9.1% ± 0.3% SE, 9.2% ± 0.3% SE, and 7.4% ± 0.3% SE (“increasing T air , low VPD”, “increasing T air and VPD”, and “stable T air , increasing VPD”, respectively) for drought-exposed plants ( Table S2 ). While the soil moisture of the well-watered plants showed no significant trend during the experimental period, drought- exposed plants had a significant decrease in soil moisture from 12.2 ± 0.7% SE to 7.1% ± 0.5% SE, from 12.7 ± 0.9% to 6.6% ± 0.6%, and from 11.7% ± 0.8% SE to 5.6% ± 0.2% SE for “increasing T air , low VPD”, “increasing T air and VPD”, and “stable T air , increasing VPD”, respectively ( Table S2 ). Leaf-level measurements on light- and dark-adapted leaves To determine the importance of stomatal decoupling, as well as potential underlying drivers, leaf- level gas exchange in the light (A net , E, g s ) and dark (g s dark , E dark ), and T air (inside the Li-6800 measuring chamber) as well as leaf temperature (T leaf ) were measured every second day (i.e., at days 2, 4, 6, 8, 10, 12) with a Li-6800-01A (LI-COR Environmental, Lincoln, NE 68504, United States). In the morning, one light-exposed leaf from the upper part of the canopy was dark-adapted by covering it in aluminium foil for more than 20 minutes. A second leaf next to the one selected for the dark-adapted measurements was chosen for the light-adapted ones. The dark-adapted leaves were measured with the following settings: the cuvette Tair and RH were set to the current values of the corresponding climate chamber, a reference CO 2 concentration of 400 ppm, a PAR of 0 µmol m -2 s -1 , and a flow of 500 µmol s −1 . After every dark-adapted measurement, the light-adapted measurements had the same settings, except that PAR was set to 1500 µmol m -2 s -1 , ensuring light saturation for all species. All measurements were taken once the gas exchange stabilized, typically after 5-10 minutes. The average T air and VPD conditions during the measurements can be found in Table S3 . Data analysis The collected data was analyzed and displayed in R v.4.2.0 ( R Core Team, 2025 ) and base R. WUE i was calculated by dividing A net by g s , WUE was calculated by dividing A net by E, and the ratio between g s and g s dark (g s -ratio) as well as E and E dark (E-ratio) was calculated by dividing the former by the latter. Furthermore, we calculated the difference between T air and leaf T leaf (T offset ). To reduce “noise” due to species-specific offsets and display the general response of temperate and tropical species to T air and VPD, we normalized the A net , g s , g s dark , E, and E dark to their species- specific average values per treatment of day 2. However, not for the analysis of the relationship between any of these, and also not to calculate WUE i , WUE as well as the g s -ratio and E-ratio). Data from day 8 (T air = 32.5°C) were excluded from the analysis to maintain consistent 5°C steps. Overall responses of A net , g s , g s dark , g s -ratio, E, E dark , E-ratio, WUE i , WUE, T offset , and T offset dark to T air and VPD were displayed in simple ggplots plots with LOESS smooths without stats by using ggplot2 to display the data in plots (H Wickham, 2016 ). To statistically analyze the responses to T air and VPD for the subsets (“increasing T air , low VPD”, “increasing T air and VPD”, and “stable T air , increasing VPD”, split into temperate/tropical, wet/dry), we modeled the relationship between A net , g s , g s dark , E, E dark (all normalized), WUEi, WUE. Toffset, T offset dark , and T air with ordinary least squares (stats::lm) and then tested for a significant slope change (breakpoint) using the segmented ( Fasola et al., 2018 ) package’s Davies test; when indicated, we fit a one-breakpoint segmented regression, and extracted R², p-values, estimated breakpoint(s), and segment slopes. Figures were produced with ggplot2, ggpmisc ( Aphalo, 2025 ), ggpubr ( Kassambara, 2025 ), patchwork ( Pedersen, T., 2025 ) for layout/annotations. Furthermore, we tested the relationship between A net and g s , A net and E, E and g s , whether g s and E predicted T offset , both in the light and the dark, using a linear model with T air (°C) (for the two conditions with increasing T air ) or VPD (kPa) (for the condition with stable T air ) as a categorical factor and, for instance, g s x T air interactions (lm(A net ∼ g s * T air )). The simple slopes at each T air or VPD, respectively, were estimated with estimated marginal trends, pairwise-compared and compact-letter-displayed (cld) using the R package “emmeans” ( Lenth and Piaskowski, 2025 ), and we reported slope estimates with p-values. The impact of E and g s on T offset was done in the light and the dark to be able to observe other potential impacts of leaf traits, such as different absorbance or other leaf properties, on the efficiency of transpirational leaf cooling. Lastly, to assess the overall relationships between g s and E with T offset in the light and the dark, we fitted separate simple linear regressions per T air or VPD (T offset ∼ E, etc.) across the individual subsets to report R² and slope significance (with significance codes *, **, ***). Results T air and VPD response of net assimilation Across experimental conditions, net assimilation (A net ) responses to T air and VPD were highly variable between biogeographic groups and soil water availability ( Figs. 1 ; S1, S2). Download figure Open in new tab Figure 1: Response of the normalized net assimilation (A net , normalized to the species-specific average value at day 2) when temperature (T air ) increases while vapour pressure deficit (VPD) remains low (a, d), when T air and VPD simultaneously increase (b, e), and when VPD increases at a stable T air of 35°C (c, f) of well-watered plants (a, b, c) or drought-exposed (d, e, f) plants. Points show individual measurements, with color indicating biogeographic origin (temperate = blue, tropical = red) and shape denoting species. Thin, light species-level LOESS smooths (with shaded 95% confidence intervals) are overlaid by thicker biogeography-level (i.e., temperate and tropical) LOESS smooths (with shaded 95% confidence intervals). Under well-watered conditions and low VPD, temperate species showed a breaking point (BP) at 35°C (Davies p = 0.0203), with A net increasing significantly until this threshold ( R² = 0.18, p = 0.0041, slope = 0.032) and subsequently decreasing, however non-significantly, above it ( R² = 0.08, p = 0.13, slope = –0.054; Fig. S1a). By contrast, tropical species displayed no BP (Davies p = 0.132), and A net continuously increased up to 40°C ( R² = 0.38, p = 5.2 × 10⁻⁸, slope = 0.81; Fig. S1b). When T air and VPD increased simultaneously, no BP was detected in either group (Davies p = 0.629 and p = 0.162, respectively). A net of temperate species showed no significant response, whereas A net of tropical species still exhibited a significant positive trend ( R² = 0.12, p = 0.038, slope = 0.047; Figs. S1c, d). At constant 35°C, A net of temperate species showed no significant response (Fig. S1f), while tropical species exhibited a significant decrease of A net with rising VPD (Davies p = 0.492, R² = 0.13, p = 0.003, slope = –0.099; Fig. S1e). Under drought, responses shifted (Fig. S2). In temperate species, A net decreased steadily with increasing T air (Davies p = 0.199, R² = 0.07, p = 0.02, slope = –0.022; Fig. S2a). However, in tropical species, a BP occurred at 35°C (Davies p = 0.0021), where A net increased significantly until the BP ( R² = 0.21, p = 0.0034, slope = 0.08), but declined significantly above it ( R² = 0.19, p = 0.025, slope = –0.202; Fig. S2b). With increasing T air and VPD, temperate species showed no significant response (Fig. S2c), whereas tropical species decreased continuously (Davies p = 0.64, R² = 0.17, p = 6.4 × 10⁻⁴, slope = –0.029; Fig. S2d). At a stable T air of 35°C, A net decreased significantly with rising VPD in both groups ( R² = 0.23, p = 5.9 × 10⁻⁵, slope = –0.184; Fig. S2e in temperate and R² = 0.19, p = 9 × 10⁻⁵, slope = –0.151; Fig. S2f in tropical species). Overall, temperate species under well-watered conditions benefited from increasing T air only until 35°C when VPD remained low (Fig. S1a), but not when VPD simultaneously increased (Fig. S1c) or when drought-exposed (Fig. S2a, c). Their A net was unresponsive to rising VPD at 35°C when well-watered (Fig. S1e), but became VPD sensitive when drought-exposed (Fig. S1e). A net of tropical species, on the other hand, benefited more from increasing T air , even during soil drought, when VPD remained low (Figs. S1b, S2b), but only when well-watered when T air and VPD increased simultaneously (Figs. S1d, S2d), and not when VPD increased at a stable T air , independent of soil moisture availability (Figs. S1f, S2f). T air and VPD response of stomatal conductance in light- and dark-adapted leaves Responses of stomatal conductance in the light (g s ) partly mirrored those of photosynthesis but often revealed an independent response of the two processes, again with distinct differences between temperate and tropical species ( Fig. 2 ; Figs. S3, S4). Download figure Open in new tab Figure 2: Response of the normalized stomatal conductance (g s , normalized to the species-specific average value at day 2) when temperature (T air ) increases while vapour pressure deficit (VPD) remains low (a, d), when T air and VPD simultaneously increase (b, e), and when VPD increases at a stable T air of 35°C (c, f) of well-watered plants (a, b, c) or drought-exposed (d, e, f) plants. Points show individual measurements, with color indicating biogeographic origin (temperate = blue, tropical = red) and shape denoting species. Thin, light species-level LOESS smooths (with shaded 95% confidence intervals) are overlaid by thicker biogeography-level (i.e., temperate and tropical) LOESS smooths (with shaded 95% confidence intervals). Download figure Open in new tab Figure 3: Response of the normalized transpiration (E, normalized to the species-specific average value at day 2) when temperature (T air ) increases while vapour pressure deficit (VPD) remains low (a, d), when T air and VPD simultaneously increase (b, e), and when VPD increases at a stable T air of 35°C (c, f) of well-watered plants (a, b, c) or drought-exposed (d, e, f) plants. Points show individual measurements, with color indicating biogeographic origin (temperate = blue, tropical = red) and shape denoting species. Thin, light species-level LOESS smooths (with shaded 95% confidence intervals) are overlaid by thicker biogeography-level (i.e., temperate and tropical) LOESS smooths (with shaded 95% confidence intervals). Under well-watered and low VPD conditions, temperate species exhibited a continuous increase of g s with increasing T air (no BP, Davies p = 0.0876; R² = 0.43, p = 2.7 × 10⁻¹⁰, slope = 0.119; Fig. S3a). In tropical species, a BP was detected at 33.65°C (Davies p = 8.3 × 10⁻⁷), with stable g s below this threshold ( R² = 0.01, p = 0.58, slope = 0.014) and a strong increase thereafter ( R² = 0.44, p = 2.2 × 10⁻⁴, slope = 0.87; Fig. S3b). When T air and VPD rose simultaneously, a significant increase in g s occurred only in tropical species ( R 2 = 0.15, p = 0.0015, slope = 0.04; Figs. S3c, d). At constant 35°C, temperate species showed no significant VPD effect (Fig. S3e), while tropical species exhibited a strong continuous decline in g s ( R² = 0.26, p = 2.6 × 10⁻⁵, slope = –0.163; Fig. S3f). When drought-exposed, temperate species showed no significant changes in g s with increasing T air (Fig. S4a), while tropical species displayed a continuous increase ( R² = 0.26, p = 1.6 × 10⁻⁵, slope = 0.219; Fig. S4b). When T air and VPD increased together, g s of temperate species remained unresponsive (Fig. S4c), whereas g s of tropical species steadily declined ( R² = 0.17, p = 6 × 10⁻⁴, slope = –0.026; Fig. S4d). At constant 35°C, g s steadily decreased with rising VPD in both groups ( R² = 0.11, p = 0.0037, slope = –0.159; Fig. S4e in temperate, and R² = 0.20, p = 2.2 × 10⁻⁴, slope = –0.189; Fig. S4f in tropical species). Responses of stomatal conductance of dark-adapted leaves (g s dark ) to T air and VPD showed similar overall trends as during the light. However, the general sensitivity of g s dark differed from the observed response in the light, and between temperate and tropical species (Figs. S5–S7). Under well-watered conditions, temperate species exhibited a BP at 34.9°C (Davies p = 0.0122). Below this threshold, g s dark increased only slightly with rising T air ( R² = 0.13, p = 0.017, slope = 0.165), above the BP, the increase in g s was stronger ( R² = 0.21, p = 0.013, slope = 0.926; Fig. S6a). Similar to the light-adapted leaf, g s of tropical species displayed a BP at 32.7°C (Davies p = 0.00145), with no significant change below it ( R² = 0.01, p = 0.64, slope = 0.013) and a strong increase above ( R² = 0.20, p = 0.021, slope = 0.63; Fig. S6b). When T air and VPD increased simultaneously, g s dark remained unchanged in both groups (Fig. S6c, d). At constant 35°C, temperate and tropical species exhibited a significant constant decline of g s dark with increasing VPD ( R² = 0.07, p = 0.025, slope = –0.144; Fig. S6e, and R² = 0.30, p = 3.7 × 10⁻⁶, slope = –0.233; Fig. S6f, respectively). Under drought, g s dark of temperate and tropical species increased with rising T air ( R² = 0.06, p = 0.047, slope = 0.032; Fig. S7a and R² = 0.14, p = 0.0026, slope = 0.127; Fig. S7b, respectively). When T air and VPD rose simultaneously, temperate species exhibited a significant constant decline in g s dark ( R² = 0.29, p = 1.2 × 10⁻⁶, slope = –0.036; Fig. S7c), whereas tropical species showed no significant change (Fig. S7d). At constant 35°C, g s dark of temperate and tropical species displayed a BP at 2.31 kPa VPD (Davies p = 0.00113) and 2.53 kPa (Davies p = 9.8 × 10⁻ 4 ), respectively. Below this threshold, g s dark decreased strongly ( R² = 0.19, p = 0.016, slope = –0.559Fig. S7e and R² = 0.20, p = 0.034, slope = –0.588, Fig. S7e, respectively), while no further decrease was observed beyond it ( R² = 0.00, p = 0.89, slope = –0.004; Fig. S7e and R² = 0.02, p = 0.42, slope = –0.020; Fig. S7f, respectively). The ratio of stomatal conductance in the light to that in the dark (g s -ratio, g s divided by g s dark ) revealed additional contrasts between temperate and tropical species and between water treatments (Figs. S8–S10), further revealing different responses of stomatal regulation in light- and dark- adapted leaves. Under wet conditions and low VPD, temperate species showed exhibited a significant steady decline in the ratio from 3.36 ± 0.69 (SE) at 20°C to 1.30 ± 0.14 at 40°C ( R² = 0.16, p = 0.00071, slope = –0.107; Fig. S9a), meaning that g s was initially substantially higher in the light than in the dark, but this difference vanished and they became very similar at 40°C. In tropical species, no significant response occurred (Fig. S9b), meaning that g s in light- and dark-adapted leaves increased to a similar extent. When T air and VPD increased simultaneously, the ratio of neither group showed significant responses (Figs. S9c, d). At constant 35°C, temperate species exhibited a BP at 1.97 kPa (Davies p = 0.0456). Before this threshold, the g s -ratio increased steeply from 1.97 ± 0.32 to 4.27 ± 0.88 ( R² = 0.18, p = 0.021, slope = 2.707), whereas above it no significant VPD response was detected ( R² = 0.00, p = 0.94, slope = –0.025; Fig. S9e). Tropical species, on the other hand, had a BP at a higher VPD of 3.94 kPa (Davies p = 0.0453). Below the BP, the g s - ratio showed no significant trend ( R² = 0.09, p = 0.086, slope = 0.664), but above it, the g s -ratio increased strongly ( R² = 0.19, p = 0.029, slope = 4.722; Fig. S9f), showing a much stronger reduction of g s dark and gs during the light at the highest VPD. Overall, this shows a different regulation of stomatal conductance in the light and the dark in response to increasing T air and VPD among temperate species at a low VPD (Fig. S9a), but not if they increase simultaneously (Fig. S9c). The response of g s and g s dark in tropical species, however, responded very similarly in all but the most extreme VPD conditions at a stable T air of 35°C. Under drought and a low VPD, the g s -ratio of temperate species displayed a BP at 25.65°C (Davies p = 0.0307), with an increase between 20°C to 25°C ( R² = 0.09, p = 0.12, slope = 0.26), and a significant steady decrease until 40°C ( R² = 0.12, p = 0.027, slope = –0.184; Fig. S10a), driven by the steady increase of g s dark (Fig. S7a) while g s did not change (Fig. S4a). In tropical species, the g s -ratio increased steadily with T air ( R² = 0.12, p = 0.0057, slope = 0.071; Fig. S10b), caused by the stronger increase of g s in the light (Fig. S4b) than in the dark (Fig. S7b). With simultaneous increases in Tair and VPD, the g s -ratio of neither temperate nor tropical species showed significant responses (Figs. S10c, d). At constant 35°C, the g s -ratio of temperate species showed no significant response to rising VPD (Fig. S10e), whereas tropical species exhibited a steady increase ( R² = 0.08, p = 0.0024, slope = 0.395, Fig. S10f). T air and VPD response of transpiration in light- and dark-adapted leaves Unlike g s , transpiration (E) showed a much more consistent increase with T air under low and rising VPD, rather constant with rising VPD at a constant T air , and less differentiated response between temperate and tropical species within watering regimes (Figs. S11, S12). Under well-watered conditions and low VPD, temperate species exhibited a BP at 31.6°C (Davies p = 0.00114). Below this threshold, E increased slightly ( R² = 0.24, p = 0.0008, slope = 0.077), while above it the increase was much stronger ( R² = 0.24, p = 0.006, slope = 0.41; Fig. S11a). Tropical species showed a BP at 33.5°C (Davies p = 2.7 × 10⁻¹⁰). Below this threshold, E did not respond to rising T air ( R² = 0.07, p = 0.11, slope = 0.05), while above the BP, E increased sharply ( R² = 0.61, p = 2.3 × 10⁻⁶, slope = 1.169; Fig. S11b). When T air and VPD increased simultaneously, E of temperate species rose steadily ( R² = 0.31, p = 3.5 × 10⁻⁷, slope = 0.200; Fig. S11c), while tropical species exhibited a BP at 28.2°C (Davies p = 0.0292). Below this threshold, E did not respond significantly ( R² = 0.13, p = 0.073, slope = 0.070), while we observed a strong increase above ( R² = 0.38, p = 3.3 × 10⁻⁵, slope = 0.457; Fig. S11d). At constant 35°C, temperate species showed a significant steady increase with VPD ( p = 0.0021, slope = 0.394; Fig. S11e), while E of tropical species exhibited no significant response (Fig. S11f). Under drought, E of temperate species at a constant low VPD did not respond significantly to rising T air (Fig. S12a), while it slightly increased under simultaneous increasing VPD ( R² = 0.09, p = 0.0084, slope = 0.124, Fig. S12c). In tropical species, E increased significantly with T air , both under low ( R² = 0.37, p = 8.8 × 10⁻⁸, slope = 0.342; Fig. S12b) and simultaneous increasing VPD ( R² = 0.20, p = 2.3 × 10⁻⁴, slope = 0.342; Fig. S12d). At constant 35°C, E of neither group showed significant VPD responses (Figs. S12e, f). Overall, this demonstrates the importance of the interplay between g s and VPD in regulating E, with g s needing to be upregulated when T air increases to increase E when VPD remains low, but can remain unchanged when VPD simultaneously rises. Similar to light-adapted leaves, transpiration of dark-adapted leaves (E dark ) increased with T air in both temperate and tropical species, with distinct BPs under well-watered conditions and low VPD but largely linear responses under increasing VPD or when drought-exposed (Figs. S13-S15). Under wet conditions and low VPD, temperate species showed a BP at 33.9°C (Davies p = 0.00104). Below this threshold, E dark increased modestly ( R² = 0.12, p = 0.018, slope = 0.137), while above it the increase was much steeper ( R² = 0.24, p = 0.0076, slope = 1.70; Fig. S14a). Tropical species exhibited a BP at 32.5°C (Davies p = 0.00232). Below this point, no response to T air occurred ( R² = 0.00, p = 0.97, slope = 0.001), but above it, E dark increased strongly ( R² = 0.19, p = 0.027, slope = 0.932; Fig. S14b). When T air and VPD increased simultaneously, E dark of temperate and tropical species showed significant steady increases ( R² = 0.25, p = 6.4 × 10⁻⁶, slope = 0.304; and R² = 0.26, p = 1.5 × 10⁻⁵, slope = 0.394; respectively, Fig. S14c, d). At constant 35°C, temperate species showed no VPD response (Fig. S14e), whereas tropical species exhibited a significant decrease of E dark with increasing VPD ( R² = 0.07, p = 0.037, slope = –0.126; Fig. S14f). Under drought, E dark of temperate and tropical species increased steadily ( R² = 0.16, p = 0.00049, slope = 0.098; Fig. S15a, and R² = 0.21, p = 1.3 × 10⁻⁴, slope = 0.244; Fig. S15b, respectively). With simultaneous increases in T air and VPD, only temperate species showed a significant steady increase ( R² = 0.14, p = 0.0012, slope = 0.053; Fig. S15c, d). At constant 35°C, temperate species exhibited a BP at 2.4 kPa (Davies p = 0.0431). Below this threshold, E dark decreased slightly but not significantly ( R² = 0.06, p = 0.18, slope = –0.332), whereas above it a significant increase occurred ( R² = 0.09, p = 0.046, slope = 0.135; Fig. S15e). In tropical species, E dark decreased steadily in response to rising VPD ( R² = 0.17, p = 0.00091, slope = –0.176; Fig. S15f). This indicates that E of dark-adapted leaves is more conservatively regulated than transpiration in light- adapted leaves at high T air when water availability is restricted, but still remain responsive to changes in T air and VPD. The ratio of transpiration in the light to that in the dark (E-ratio; E divided by E dark ) showed less response compared to absolute fluxes, further verifying mostly similar responses of E independent of light availability (Figs. S16-18). However, under wet conditions and low VPD, temperate species exhibited a significant steady decrease in the ratio with increasing T air ( R² = 0.16, p = 0.00057, slope = –0.128; Fig. S17a), showing that the E and E dark become more and more similar. No significant change of the E-ratio was observed in tropical species when T air increased, both at a low VPD (Fig. S17b) and increasing VPD (Fig. S17d), and in temperate species when T air and VPD increased simultaneously (Fig. S17c) or VPD increased at constant 35°C (Fig. S17e). In contrast, the E-ratio of tropical species exhibited a strong linear increase with rising VPD at 35°C ( R² = 0.21, p = 0.00027, slope = 0.902; Fig. S17f), showing their stronger VPD sensitivity of E dark compared to E. Under drought, when T air increased while VPD remained low, temperate species displayed a BP at 25°C (Davies p = 0.033). Below this point, the E-ratio did increase but not overall significantly, whereas above it the E-ratio decreased strongly, but again not significantly (Fig. S18a). The E- ratio of tropical species increased steadily with rising T air ( R² = 0.11, p = 0.011, slope = 0.081; Fig. S18b). When T air and VPD increased simultaneously or VPD increased at a constant 35°C, the E- ratio of neither group showed a significant response (Figs. S18c, d, e, f). These findings indicate that E, both in the light and the dark, is mostly regulated in a similar range in response to rising T air , especially when water is not limited, while their ratio remains largely constant in response to rising VPD at a constant T air . T air and VPD response of the intrinsic water use efficiency The intrinsic water use efficiency (WUE i , A net divided by g s ) showed contrasting T air and VPD responses between biogeographic groups and water regimes, with distinct BPs under both wet and dry conditions ( Fig. 4 ; Figs. S19, S20). Download figure Open in new tab Figure 4: Response of the intrinsic water use efficiency (WUE i (A net divided by g s ), when Tair increases while vapour pressure deficit (VPD) remains low (a, d), when Tair and VPD simultaneously increase (b, e), and when VPD increases at a stable Tair of 35°C (c, f) of well- watered plants (a, b, c) or drought-exposed (d, e, f) plants. Points show individual measurements, with color indicating biogeographic origin (temperate = blue, tropical = red) and shape denoting species. Thin, light species-level LOESS smooths (with shaded 95% confidence intervals) are overlaid by thicker biogeography-level (i.e., temperate and tropical) LOESS smooths (with shaded 95% confidence intervals). Under well-watered conditions and low VPD, WUE i of temperate species exhibited a BP at 25°C (Davies p = 0.00878). Below this threshold, WUE i remained stable ( R² = 0.11, p = 0.079, slope = 5.568), while above it WUE i decreased significantly with increasing T air ( R² = 0.20, p = 0.002, slope = –4.185; Fig. S19a). In tropical species, a BP occurred at 31.9°C (Davies p = 0.00226), with no significant response below ( R² = 0.06, p = 0.13, slope = 3.645), but with WUE i declining sharply above ( R² = 0.28, p = 0.005, slope = –12.120; Fig. S19b). When T air and VPD increased simultaneously, WUE i remained unchanged in temperate species (Fig. S19c), but exhibited a BP at 35°C in tropical species (Davies p = 0.00649). Below the BP, WUE i remained constant ( R² = 0.00, p = 0.76, slope = 0.331), while above it, WUE i decreased steeply ( R² = 0.50, p = 7.1 × 10⁻⁵, slope = –6.715; Fig. S19d). At constant 35°C, WUE i remained stable in temperate species (Fig. S19e), whereas tropical species showed a steady increase with rising VPD ( R² = 0.14, p = 0.0024, slope = 8.277; Fig. S19f). Under drought, WUE i responses to T air further differentiated among temperate and tropical species (Fig. S20). Temperate species showed a BP with low VPD at 30°C (Davies p = 0.00354): below this point, WUE i did not change ( R² = 0.04, p = 0.32, slope = 3.101), but above it declined markedly ( R² = 0.29, p = 2.4 × 10⁻⁴, slope = –8.605; Fig. S20a). With simultaneously rising VPD, they showed a BP at 33.14°C (Davies p = 0.000706). Below the BP, a slight but significant increase was observed ( R² = 0.10, p = 0.039, slope = 2.05), while above it, WUE i decreased strongly ( R² = 0.27, p = 0.0036, slope = –6.619; Fig. S20c). On the other hand, WUE i of tropical species decreased continuously with T air independent of VPD ( R² = 0.20, p = 2.1 × 10⁻⁴, slope = –2.764; Fig. S20b and R² = 0.12, p = 0.0061, slope = –1.935; Fig. S20d, respectively). At constant T air , WUE i of neither group showed significant VPD responses (Fig. S20e, f). T air and VPD response of the water use efficiency Unlike WUE i , the actual water-use efficiency (WUE, A net divided by E) declined more consistently with rising T air , independent of VPD and soil moisture availability ( Fig. 5 ). We found distinct breaking points (BPs) in response to increasing T air under a low and rising VPD, both in temperate and tropical species, and a consistent decrease in response to rising VPD at a constant 35°C (Figs. S21, S22). Download figure Open in new tab Figure 5: Response of the normalized water use efficiency (WUE (A net divided by E), normalized to the species-specific average value at day 2) when Tair increases while vapour pressure deficit (VPD) remains low (a, d), when Tair and VPD simultaneously increase (b, e), and when VPD increases at a stable Tair of 35°C (c, f) of well-watered plants (a, b, c) or drought-exposed (d, e, f) plants. Points show individual measurements, with color indicating biogeographic origin (temperate = blue, tropical = red) and shape denoting species. Thin, light species-level LOESS smooths (with shaded 95% confidence intervals) are overlaid by thicker biogeography-level (i.e., temperate and tropical) LOESS smooths (with shaded 95% confidence intervals). Under well-watered conditions and low VPD, temperate species showed a BP at 25°C (Davies p = 0.00291). Below this threshold, WUE remained constant ( R² = 0.03, p = 0.33, slope = 0.199), while above it WUE declined sharply ( R² = 0.35, p = 2.15 × 10⁻⁵, slope = –0.409; Fig. S21a). In tropical species, a BP occurred at 30.8°C (Davies p = 0.02), where WUE did not respond below ( R² = 0.00, p = 0.78, slope = 0.039), but above it a strong decline was observed ( R² = 0.40, p = 5.3 × 10⁻⁴, slope = –0.564; Fig. S21b). When T air and VPD increased simultaneously, temperate species showed a BP at 25.6°C (Davies p = 0.003). Before this point, WUE decreased strongly ( R² = 0.33, p = 0.002, slope = –0.549), and after it continued to decline more gradually ( R² = 0.57, p = 3.8 × 10⁻⁹, slope = –0.158; Fig. S21c). Similarly, WUE of tropical species displayed a BP at 26.7°C (Davies p = 0.0193). Below this threshold, WUE decreased more steeply ( R² = 0.30, p = 0.0039, slope = –0.435), while above it the decline was less pronounced ( R² = 0.67, p = 2.9 × 10⁻¹⁰, slope = –0.159; Fig. S21d). At constant T air , temperate and tropical species showed steady decreases with rising VPD ( R² = 0.25, p = 5.9 × 10⁻⁶, slope = –0.573 and R² = 0.37, p = 1.2 × 10⁻⁷, slope = – 0.527, respectively; Fig. S21e, f). Under drought, the pattern remained largely remained (Fig. S22). Temperate species showed a BP at 29.9°C (Davies p = 0.00158). Below this T air , WUE remained unchanged ( R² = 0.00, p = 0.95, slope = 0.015), whereas above it, a pronounced decrease occurred (Fig. S22a). In tropical species, WUE decreased steadily ( R² = 0.36, p = 2.5 × 10⁻⁷, slope = –0.264; Fig. S22b). When both T air and VPD increased, temperate species exhibited a BP at 25°C (Davies p = 2.1 × 10⁻⁵). Below this point, WUE declined steeply ( R² = 0.57, p = 5.9 × 10⁻⁶, slope = –0.710), whereas above it the decline was weaker ( R² = 0.83, p = 5.6 × 10⁻¹⁸, slope = –0.252; Fig. S22c). Similar again in tropical species, a BP was detected at 26.1°C (Davies p = 0.0193). Before this threshold, WUE decreased strongly ( R² = 0.50, p = 7.2 × 10⁻⁵, slope = –0.831), and after it the decline flattened ( R² = 0.32, p = 3.8 × 10⁻⁴, slope = –0.135; Fig. S22d). At constant 35°C, both temperate and tropical species showed steady decreases in WUE with rising VPD ( R² = 0.46, p = 3.1 × 10⁻¹¹, slope = –0.762 and R² = 0.22, p = 1.2 × 10⁻⁴, slope = –1.310, respectively; Figs. S22e, f). The relationship between A net and g s in response to rising T air and VPD Across all treatments, A net and g s were strongly correlated, but the strength and slope of their relationship varied with T air , VPD, and biogeographic group ( Fig. 6 ; Fig. S23). Download figure Open in new tab Figure 6: Changes of the relationship between net assimilation (A net ) and stomatal conductance (g s ) when temperature (T air ) increases while vapour pressure deficit (VPD) remains low (a, d), Tair and VPD are simultaneously increasing (b, e), and VPD increases at a stable T air of 35°C (c, f) in temperate (a, b, c) and tropical (d, e, f) tree species of well-watered plants. Points show individual measurements; colored lines are linear fits plotted only when slopes are significant (p < 0.05) from global interaction models (A net ∼ g s × Temp or A net ∼ g s × VPD), with shaded 95% confidence intervals. Legends report per-level R² and slope m (A net /g s ) with significance asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001), and compact letter displays summarize pairwise slope differences. Under well-watered conditions, temperate species showed consistently high correlations between A net and g s ( Fig. 6a–c ). At low VPD, R² values ranged from 0.79 to 0.95 across temperatures. Slopes ( m ) declined from 56.3 (b) at 20°C ( R² = 0.85***) to 20.5 (a) at 40°C ( R² = 0.79***), indicating a progressive reduction in photosynthetic gain per unit stomatal conductance. When Tair and VPD increased simultaneously, slopes varied from 34 (a) at 20°C ( R² = 0.65***) to 63.7 (b) at 40°C ( R² = 0.95***; Fig. 6b ), but no clear trend in their relationship was visible. At constant 35°C and rising VPD, the A net –g s relationship steepened, with m = 23.3 (a) at 1.12 kPa ( R² = 0.73***) and m = 48.1 (b) at 4.5 kPa ( R² = 0.95***; Fig. 6c ). In tropical species, coupling between A net and g s was weaker and less consistent under wet conditions when VPD was low ( Fig. 6d ), where regressions were only significant at 35°C ( R² = 0.35, m = 42.7***). When Tair and VPD rose together, slopes remained relatively constant and did not significantly differ from each other ( m = 66.8 (a) to 76.1 (a); R² = 0.45***–0.93***; Fig. 6e ). At stable Tair, increasing VPD produced a progressive steepening of the A net -g s relationship: m = 27 (a) at 1.12 kPa ( R² = 0.89***) to m = 64.2 (b) at 4.5 kPa ( R² = 0.91***; Fig. 6f ). Under drought, A net –g s coupling was more consistent, with mostly no significant variation, though slope magnitudes differed (Fig. S23). In temperate species, slopes were relatively stable across Tair treatments at a constant low VPD ( m = 66 (a) to 84.8 (a); R² = 0.68***–0.82***; Fig. S23a) and increased when T air and VPD rose simultaneously ( m = 98.4 (a) at 20°C to 125 (a) at 35°C; R² = 0.48***–0.99***; Fig. S23b). Under constant T air , m rose from 44 (a) at 1.12 kPa ( R² = 0.84***) to 75.8 (a) at 4.5 kPa ( R² = 0.80***; Fig. S23c). In tropical species, when drought-exposed under constant low VPD, A net –g s correlations remained absent at 20–30°C but became significant at higher T air ( R² = 0.78***, m = 39.3 (b) at 35°C; R² = 0.43**, m = 13.9 (a) at 40°C; Fig. S23d). When T air and VPD increased together, slopes remained steep and uniform ( m = 83.4 (a) to 113.3 (a); R² = 0.72***–0.89***; Fig. S23e). At constant 35°C, rising VPD further strengthened the A net –g s coupling, but the regressions did not significantly differ from each other: m = 68.4 (a) at 1.12 kPa ( R² = 0.89***) to m = 132.3 (a) at 4.5 kPa ( R² = 0.87***; Fig. S23f). Overall, the A net –g s relationship weakened with increasing T air when VPD remained low in temperate species, but remained strong under rising VPD and increased under drought exposure. In tropical species, coupling was largely absent under low VPD but strengthened under high VPD, particularly when soils were dry. The relationship between A net and E in response to rising T air and VPD Coupling between A net and E (slopes m ) decreased with rising T air , both with a low and a simultaneously rising VPD, and was nearly absent in tropical species under low VPD ( Fig. 7 ; Fig. S24). Download figure Open in new tab Figure 7: Changes of the relationship between net assimilation (A net ) and transpiration (E) when temperature (T air ) increases while vapour pressure deficit (VPD) remains low (a, d), Tair and VPD are simultaneously increasing (b, e), and VPD increases at a stable Tair of 35°C (c, f) in temperate (a, b, c) and tropical (d, e, f) tree species of well-watered plants. Points show individual measurements; colored lines are linear fits plotted only when slopes are significant (p < 0.05) from global interaction models (A net ∼ g s × Temp or A net ∼ g s × VPD), with shaded 95% confidence intervals. Legends report per-level R² and slope m (A net /g s ) with significance asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001), and compact letter displays summarize pairwise slope differences. Under well-watered conditions, temperate species showed highly significant relationships at all T air ( Fig. 7a–c ). At low VPD, R² ranged from 0.81–0.95, with slopes declining from 4.3 (c) at 20°C ( R² = 0.85***) to 1.3 (a) at 40°C ( R² = 0.81***), reflecting reduced photosynthetic gain per unit transpiration. When T air and VPD rose simultaneously, relationships fell from 3.0 (b) at 20°C ( R² = 0.69***) to 1.2 (a) at 40°C ( R² = 0.97***; Fig. 7b ). At constant 35°C, increasing VPD did not impact the A net -E relationship ( m = 1.1–1.2 (a); R² = 0.60***–0.96***; Fig. 7c ). In tropical species, A net -E coupling was nearly absent at low VPD but strengthened under higher VPD ( Fig. 7d–f ). Relationships under rising T air and low VPD were only significant at 35°C ( R² = 0.44**, m = 2.4, Fig. 7d ). When T air and VPD increased together, R² values ranged 0.54***– 0.94*** and slopes decreased from 5.2 (c) at 20°C to 1.0 (a) at 40°C ( Fig. 7e ). At constant T air , increasing VPD did not significantly impact the relationships between Anet and E (1.5–2.1 (a), R² = 0.48***–0.96***; Fig. 7f ). Under drought, the general trends of the A net -E coupling remained unchanged (Fig. S24). In temperate species, slopes declined from 4.8 (b) at 20°C ( R² = 0.78***) to 1.8 (a) at 40°C ( R² = 0.77***; Fig. S24a). When T air and VPD increased simultaneously, slopes decreased systematically from 7.5 (c) at 20°C ( R² = 0.48***) to 1.5 (a) at 40°C ( R² = 0.99***; Fig. S24b). At constant T air , increasing VPD yielded steady slopes of 1.6–2.6, which did not differ significantly ((a), R² = 0.49***–0.86***; Fig. S24c). In drought-exposed tropical species, at constant low VPD and rising T air , no significant relationships occurred up to 30°C, but relationships became significant at 35°C ( R² = 0.70***, m = 2.6 (b)) and 40°C ( R² = 0.46**, m = 0.8 (a); Fig. S24d). When T air and VPD rose together, relationships were consistent ( m = 2.9–5.7 (a); R² = 0.55***–0.91***; Fig. S24e). At constant T air , the A net -E relationship weakened slightly with rising VPD, with m declining from 4.1*** (b) at 1.12 kPa ( R² = 0.89) to 2.3*** (a) at 3.94 kPa ( R² = 0.91; Fig. S24f). Overall, A net -E coupling expressed a consistent decline with increasing T air , independent of VPD, in both climatic groups. The relationship between E and g s in response to rising T air and VPD Under well-watered conditions, temperate species showed highly significant E – g s coupling across the full T air range ( Fig. 8a–c ). At low VPD, R² remained ≥ 0.97. Slopes increased slightly with T air from 13*** (ab) at 20°C to 16.1*** (a) at 40°C ( Fig. 8a ). When both T air and VPD increased, the relationship steepened strongly, with m rising from 11.7*** (a) at 20°C to 52.2*** (e) at 40°C ( R² = 0.99–1; Fig. 8b ). At constant T air , increasing VPD produced a similar pattern: m = 13.9*** (a) at 1.12 kPa ( R² = 0.92) and m = 41.1*** (e) at 4.5 kPa ( R² = 0.99; Fig. 8c ). Download figure Open in new tab Figure 8: Changes in the relationship between transpiration (E) and stomatal conductance (g s ) when temperature (T air ) increases while vapour pressure deficit (VPD) remains low (a, d), T air and VPD are simultaneously increasing (b, e), and VPD increases at a stable T air of 35°C (c, f) in temperate (a, b, c) and tropical (d, e, f) tree species of well-watered plants. Points show individual measurements; colored lines are linear fits plotted only when slopes are significant (p < 0.05) from global interaction models (A net ∼ g s × Temp or A net ∼ g s × VPD), with shaded 95% confidence intervals. Legends report per-level R² and slope m (A net /g s ) with significance asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001), and compact letter displays summarize pairwise slope differences. In tropical species, E – g s correlations were equally strong but displayed slightly lower R² values at the highest T air under low VPD ( Fig. 8d–f ). Under low VPD, slopes varied between 11.6*** (ab) at 20°C and 9.9*** (a) at 40°C ( R² = 0.83–0.98; Fig. 8d ). When T air and VPD increased together, m rose steadily from 13.8*** (a) at 20°C ( R² = 0.97) to 50.7*** (d) at 40°C ( R² = 0.99; Fig. 8e ). At constant T air , increasing VPD again resulted in steeper slopes, with m = 12.5*** (a) at 1.12 kPa ( R² = 0.99) and m = 41.2*** (d) at 4.5 kPa ( R² = 1; Fig. 8f ). Under drought, the E – g s relationship remained nearly linear but shifted toward steeper slopes, indicating higher transpirational output per unit stomatal conductance (Fig. S25). In temperate species, R² values stayed ≥ 0.89 across all T air . Slopes increased from 13.7*** (a) at 20°C to 25.6*** (a) at 40°C (Fig. S25a). When T air and VPD rose simultaneously, m increased strongly from 13.1*** (a) at 20°C to 63*** (e) at 40°C ( R² = 0.99–1; Fig. S25b). At constant T air , VPD caused a similar monotonic steepening: m = 16.4*** (a) at 1.12 kPa to 47.5*** (e) at 4.5 kPa ( R² = 0.96–1; Fig. S25c). Drought-exposed tropical species also exhibited near-perfect E – g s coupling (Fig. S25d–f). At low VPD, slopes increased modestly with T air , from 18.1*** (a) at 25°C ( R² = 0.98) to 16.2*** (a) at 40°C ( R² = 0.90; Fig. S25d). When T air and VPD rose together, slopes steepened sharply, from 13.9*** (a) at 20°C to 65.6*** (d) at 40°C ( R² = 0.94–1; Fig. S25e). Under constant 35°C, the relationship again strengthened with VPD, with m = 16.2*** (a) at 1.12 kPa ( R² = 0.98) and 44.8*** (e) at 4.5 kPa ( R² = 1; Fig. S25f). Overall, E – g s coupling remained almost perfectly linear across treatments, confirming the dominant control of stomatal conductance on transpirational water loss. The progressively steeper slopes with an increasing VPD, both when T air increased or remained stable at 35°C, demonstrate the enhanced water flux per unit stomatal conductance. In conclusion, when T air increases under a constant low VPD, E can only be increased by increasing g s ( Figs. 8a , 8d , S25a, S25d), while under simultaneous increasing VPD, g s can remain low ( Figs. 8b , 8e , S25b, S25e), or decrease when VPD increased at a stable T air ( Figs. 8c , 8f , S25c, S25f) to maintain E. T air and VPD effects on leaf temperature The temperature difference between leaves and air (T offset ; T leaf – T air ) decreased with rising T air and VPD in both biogeographic groups, indicating enhanced evaporative cooling under high thermal load (Figs. S26–S28). Under well-watered conditions and low VPD, temperate species exhibited no BP (Davies p = 0.285) but showed a steady decline in T offset from +0.13 ± 0.06°C at 20°C to –0.16 ± 0.09°C at 40°C ( R² = 0.11, p = 0.0042, slope = –0.014; Fig. S27a). Tropical species displayed a BP at 31.29°C (Davies p = 0.012): below this threshold, T offset remained stable (+0.55 ± 0.19°C at 20°C to +0.79 ± 0.19°C at 30°C), whereas above it, T offset declined ( R² = 0.28, p = 0.0074, slope = –0.108), reaching –0.11 ± 0.11°C at 40°C (Fig. S27b). When T air and VPD increased simultaneously, both groups exhibited continuous decreases (Davies p = 0.543, R² = 0.21, p = 6.2 × 10⁻⁵, slope = –0.056, and Davies p = 0.0815, in temperate and tropical species, respectively. Fig. S27c, d). At constant 35°C, T offset declined with increasing VPD in temperate species ( R² = 0.29, p = 6.8 × 10⁻⁷, slope = –0.224; Fig. S27e) from +0.28 ± 0.11°C at 1.12 kPa to –0.50 ± 0.15°C at 4.5 kPa, whereas tropical species exhibited a BP at 3.22 kPa (Davies p = 0.0354): below this threshold, T offset increased slightly ( R² = 0.14, p = 0.033, slope = 0.282), while above it, no further change was detected ( R² = 0.06, p = 0.23, slope = –0.550; Fig. S27f). Under drought, T offset responses were largely attenuated (Fig. S28). No BPs were detected for either species group when T air increased (Davies p > 0.05; Fig. S28a–d). At constant T air , temperate species showed a gradual decrease from +0.60 ± 0.10°C at 1.12 kPa to +0.33 ± 0.08°C at 4.5 kPa (Davies p = 0.532; Fig. S28e). In tropical species, a BP occurred at 3.41 kPa (Davies p = 0.00097): below this point, T offset increased ( R² = 0.27, p = 0.0015, slope = 0.401), reaching +0.94 ± 0.16°C at 2.53 kPa, whereas above it, T offset decreased ( R² = 0.19, p = 0.028, slope = –0.719), falling to +0.43 ± 0.09°C at 4.5 kPa (Fig. S28f). T air and VPD effects on leaf temperature in the dark While T leaf in the dark was consistently below T air , the trends in leaf–to-air temperature offsets in darkness (T offset dark , Figs. S29–S31) were comparable to those observed in the light. Under wet conditions, T offset dark of temperate species decreased steadily from –0.67 ± 0.07°C at 20°C to –1.27 ± 0.16°C at 40°C (Fig. S30a). Tropical species displayed a BP at 33.2°C (Davies p = 0.0139): below this point, no response was detected ( R² = 0.00, p = 0.71, slope = 0.003), whereas above it, T offset dark decreased significantly ( R² = 0.22, p = 0.022, slope = –0.053), from –0.65 ± 0.04°C at 30°C to –0.99 ± 0.12°C at 40°C (Fig. S30b). When T air and VPD rose together, both species groups exhibited continuous cooling ( R² = 0.13, p = 0.0021, slope = –0.038, and Davies p = 0.00217, R² = 0.58, p = 5.9 × 10⁻⁶, slope = –0.148 in temperate and tropical species, respectively. Fig. S30c, d). At constant 35°C, T offset dark showed small VPD effects, declining from –0.86 ± 0.11°C at 1.12 kPa to –1.16 ± 0.21°C at 4.5 kPa in temperate species ( R² = 0.06, p = 0.032, slope = –0.128; Fig. S30e), while tropical species remained stable (Davies p > 0.05; Fig. S30f). Under drought, T offset dark remained constant across all treatments (Fig. S31a, b, c, d, f), except in temperate species at constant 35°C with rising VPD, with a slight significant increase in cooling (–0.60 ± 0.08°C at 1.12 kPa to –0.82 ± 0.11°C at 4.5 kPa; R² = 0.08, p = 0.015, slope = –0.085; Fig. S31e). Overall, under a sufficient water supply, T offset and T offset dark decreased with increasing T air , but much more efficiently when VPD increased simultaneously (Figs. S26–S31). Despite this general trend, clear biogeographic contrasts emerged. Temperate species exhibited a stronger cooling response and larger absolute decreases in T offset with increasing T air and VPD than tropical species when light-exposed (Fig. S27). In contrast, T leaf of tropical species was largely higher relative to T air , with even delayed declines in T offset , indicating stronger radiative energy uptake. Under drought, evaporative cooling weakened in both groups, and no effective cooling of T leaf below T air remained. The relationship between leaf temperature offsets and stomatal conductance, and transpiration, with and without light Both with and without light, T offset became increasingly smaller (cooler leaves) with rising g s and E (Figs. S32–S39), confirming overall that evaporative fluxes exerted a strong physical influence on T leaf . However, the strength and consistency of this coupling were much stronger in temperate than in tropical species and weakened under drought. Under well-watered conditions, temperate species exhibited tight and mostly linear T offset –g s relationships, particularly when T air and VPD rose simultaneously. Slopes became increasingly negative with rising T air , reaching m = –8.11*** at 35°C and m = –7.72*** at 40°C ( R ² = 0.78 and 0.71; Fig. S32b), with cooling apparent once g s exceeded 0.05 mol m⁻² s⁻¹. At constant 35°C, rising VPD further strengthened this relation ( R ² = 0.62, m = –3.36***; Fig. S32c). In tropical species T offset –g s correlations were much weaker and less consistent, with significant cooling only at the highest T air or VPD (e.g., R ² = 0.73, m = –13.07**, Fig. S32e; R ² = 0.57, m = –8.45***, Fig. S32f), and cooling occurring at higher g s thresholds (> 0.18 mol m⁻² s⁻¹). Under drought, T offset –g s coupling weakened substantially (Fig. S33). Significant relations persisted only in a few cases, such as temperate leaves at 35°C ( R ² = 0.34, m = –12.85**, Fig. S33a) and tropical leaves under combined heat and VPD ( R ² = 0.46, m = –5.96*, Fig. S33d). Overall slopes across all T air were non-significant for most treatments, suggesting that the link between stomatal opening and leaf cooling was strongly limited once water supply was limited. In darkness, a consistent T offset – g s relationship within the treatments was restricted to the temperate species under high T air under well-watered conditions (Fig. S34). When T air increased, temperate species displayed overall a consistent linear decrease in T offset dark with increasing g s dark (across all species: R ² = 0.47, m = –2.75, p = 1 × 10⁻¹⁰ and R 2 = 0.44, m = -6.64, p = 2.9 × 10⁻¹⁰, respectively. Fig. S34a, b). However, this relationship was only significant at 30°C and above. In contrast, while the overall relationship across all T air or VPD remained in tropical species (overall R ² = 0.43, m = –2.45, p = 7.9 × 10⁻ 9 ; and R ² = 0.24, m = –7.2, p = 4.1 × 10⁻ 5 . Fig. S34d, e), it was inconsistent and often absent within a given T air , especially when VPD increased (Fig. S34e). At constant T air , increasing VPD accentuated cooling responses in temperate species (e.g., R ² = 0.84, m = –11.08*** at 4.5 kPa, Fig. S34c), but again, this relationship was much less consistent among the tropical species (Fig. S34f). Under drought (Fig. S35), however, these relations became sporadic and inconsistent across T air steps, with significant trends confined to tropical species when T air increased at a low VPD ( R ² = 0.52, m = –2.97, p = 1.5 × 10⁻¹⁰; Fig. S35d). The decline of T offset with increasing E was mainly visible across all T air and VPD (Figs. S36–S39). In well-watered plants, the T offset –E relationship was particularly strong in temperate species when T air and VPD increased together ( R ² = 0.76, m = –0.18, p = 2.9 × 10⁻²³; Fig. S36b), indicating that an increase in E yielded in actual leaf cooling (T offset 1.9 mmol m⁻² s⁻¹). Tropical species showed weaker associations ( R ² = 0.45, m = –0.22, p = 7.9 × 10⁻¹⁰; Fig. S36e), requiring nearly doubled transpiration rates (> 3.7 mmol m⁻² s⁻¹) to achieve comparable cooling. In darkness, and therefore in the absence of interferences by light-energy inputs of the leaf energy balance, the relationships between T offset dark and E dark were again only more consistently significant when all measurements were included across all T air and VPD treatments ( R ² = 0.33–0.48, m ≈ – 0.14 to –0.20, p ≤ 1.26 10⁻6; Figs. S38). A significant relationship between T offset dark and E dark within the different T air or VPD steps was again inconsistent, and largely restricted to temperate species at high T air . The slopes flattened or reversed under drought (Figs. S39), further revealing a strong reduction in the efficiency of evaporative cooling once hydraulic limitation set in. Taken together, T offset declined only overall across all T air by increasing g s and E, but not consistently within the separate T air steps, where E effectively contributes to leaf cooling mostly at high T air ≥ 30°C in temperate but not in tropical species when well-watered (Figs. S32, S34, S36, S38). Interestingly, leaf T leaf of tropical species was consistently higher than that of temperate species. This difference cannot be attributed to reduced evaporative cooling efficiency, as the overall relationship (slopes) between T offset and E (Fig. S36, S38) was similar. Instead, tropical leaves appear to operate at a higher thermal equilibrium when exposed to light, but not during darkness (Figs. S26, S29), implicating greater radiative heat absorption (e.g., higher short-wave absorptance or differences in wax/roughness affecting absorptance/emissivity and the near-leaf boundary layer) as the primary driver. Discussion The decoupling of photosynthesis from stomatal conductance at high T air Across treatments, we observed a T air -driven divergence and a VPD-driven convergence between photosynthetic assimilation (Aₙₑₜ) and stomatal conductance (gₛ), arising from the joint influence of T air and atmospheric demand, and further modulated by soil moisture availability ( Figs. 1 , 2 , 6 , S1 , S2, S3, S4, S23). Within the tested T air range (20–40°C), a T air -driven stomatal decoupling, here defined as a constantly decrease or loss of the relationship between A net and g s with rising T air , could only be observed in temperate species when VPD was kept low ( Fig. 6 ). As T air increased at low VPD, A net rose only up to approximately 35°C and then levelled or declined ( Fig. 1a ; S1a), whereas g s continued to increase ( Fig. 2a ; S3a), resulting in a progressive flattening of the A net -g s relationship (i.e., the slope (m) from 56.3 at 20°C to 20.5 at 40°C (R² = 0.85–0.79; Fig. 6a ). Since the leaf-to- air vapor gradient remained too small to increase E efficiently ( Fig. 8a ), plants had to actively raise g s to increase transpirational flux, especially at high T air above 30°C (E, Figs. 3a , S11), similar to what has been observed by Diao et al., 2024a . Furthermore, the strong and highly linear relationships between E and g s ( Figs. 8 , S25) confirm that stomatal regulation remained fully operational even at extreme conditions. Thus, it is implausible that the observed decoupling results from a breakdown of stomatal control but rather from an active upregulation of g s and therefore E, despite thermal constraints on photosynthesis. This agrees with the findings of Wang et al. 2024 , where the temperature-driven breakdown of the minimum leaf conductance occurred at T air > 40°C. In contrast, when T air and VPD increased together, we could not observe a decoupling between A net and g s , since the relationship between A net and g s remained largely unchanged ( Fig. 6b ; S23b), further demonstrating an effective stomatal control within the tested temperature range. The E increased, even if g s changed little or declined ( Fig. 2b ; S3d; S4d; S11c, d), since the rising VPD directly amplifies transpiration, as further reflected in the steepening E-g s relationships with increasing VPD ( Fig. 8b ). A similar response was observed at constant 35°C with rising VPD, similar to Diao et al., 2024b , where g s remained stable or decreased ( Fig. 2c ; S3e; S4e), while E increased (S11e), again showing that rising VPD maintained a constant E while g s decreased ( Fig. 8c ). Interestingly, in tropical species, the coupling between A net and g s (and hence between A net and E) was weak or even absent at low VPD across much of the T air range ( Fig. 6d ; Figs. 7d ; S23d; S24d), but re-emerged when VPD increased, either alongside T air or at a constant high Tair ( Figs. 6e , f; Fig. 7e, f ; S23e, f; S24e, f). Drought further increased the coupling between A net and g s , reflecting overall what has been seen in trees in the Amazon rain forest ( Janssen et al., 2020 ). The largely absence of coordination between stomatal conductance and photosynthesis at low VPD might derive from their adaptation to monsoonal climates, where low VPD and abundant water availability during the rainy season do not require a conservative, water-saving stomatal regulation, whereas VPD increases along with decreased water availability during the dry season, therefore fostering a more water-conservative stomatal control. A stronger sensitivity of g s to VPD than drought ( Figs. 2d, e, S4b, d ) has been observed in a tropical rainforest in Panama during an El Niño event ( Wu et al., 2020 ). Earlier studies reported an Aₙₑₜ-gₛ decoupling when both Tₐᵢᵣ and VPD increased ( Marchin et al., 2023 ; Gauthey et al., 2024 ), but those cases typically involved leaf T air exceeding 40–45°C, where photosynthetic biochemistry collapses ( Medlyn et al., 2002 ; Didion-Gency et al., 2025 ) while stomata remain partially open or leaf physical properties are no longer able to prevent a runaway water loss ( Riederer and Schreiber, 2001 ; Wang et al., 2024 ). In contrast, in our tested range (≤ 40°C), photosynthesis persisted, and gₛ adjustments remained functional, indicating that the observed decoupling arises from a true physiological mechanism for fluxes regulations rather than from thermal damage of the stomatal apparatus. The observed rise in E under high VPD, despite partial stomatal closure, underscores the dominant role of evaporative demand in driving water loss. Because E ≈ gₛ × ΔVPD (where ΔVPD is the leaf–air vapor gradient), E may increase either through higher gₛ or through a larger ΔVPD. When VPD remains low, gₛ must rise to elevate E; when VPD increases, ΔVPD dominates, allowing gₛ to remain constant or decline while E still rises ( Figs. 1 – 2 ; S11–S12). Thus, the Aₙₑₜ-gₛ decoupling at constant low VPD is the hydraulic counterpart of an attempt to sustain transpirational flux under thermal stress. This mechanistic interpretation is consistent with the contrasting behaviors of intrinsic (WUEᵢ = Aₙₑₜ/gₛ) and instantaneous (WUE = Aₙₑₜ/E) water-use efficiencies. At constant VPD, increasing Tₐᵢᵣ caused WUEᵢ and WUE to decline sharply (Figs. S19–S22), reflecting the steep rise in gₛ (and E) relative to the modest gain or decline in Aₙₑₜ. In contrast, when VPD increased with T air , WUE continued to decline even though gₛ remained stable, because E rose through the enhanced VPD gradient. Together, these responses show that the Aₙₑₜ–gₛ decoupling is a consequence of maintaining transpirational flux under high T air . Lastly, the near lack of changes in the ratio between gₛ and gs dark , as well as E and E dark (Figs. S8- S10, S16-S18), indicates a common control. Interestingly, their decrease in temperate species under a constant low VPD and rising T air was driven by the much stronger increase in g s dark than the observed increase in g s , leading them to be very similar at 40°C (Fig. S9a). This finding challenges the assumption that stomatal regulation is primarily light-driven, at least at high T air ≥ 30°C. Hence, the term “residual conductance” may underestimate the functional role of gₛ dark in maintaining continuous water flow, and therefore, other services such as nutrient and O 2 /CO 2 transport in heterotrophic tissues such as roots and stems ( Levy et al., 1999 ; Sorz and Hietz, 2006 ; Jensen et al., 2016 ). Leaf thermal regulation From a thermal perspective, T air -driven decoupling enhances transpiration and should therefore contribute to evaporative cooling ( Gauthey et al., 2024 ; Bachofen et al., 2025 ), and we found a consistent cooling of T leaf below T air only in temperate species (Figs. S26-S28). However, the relationships between T offset and g s and T offset and E are only consistently significant in temperate species in high temperatures (Figs. S26–S39). Furthermore, likely because the thermal conductivity of air decreases with increasing humidity ( Pernau et al., 2024 ), the relationship between E and leaf cooling was very low under the conditions we actually observed stomatal decoupling (Fig. S36). Across all T air and VPD steps, temperate and tropical species achieved a similar overall cooling efficiency per unit E (Figs. S36-S39). However, tropical species had leaves that were consistently warmer than leaves of temperate species and were rarely below T air , something that has been observed before ( Crous et al., 2023 ; Middleby et al., 2025a , 2025b ). Since the lower cooling ability with rising E of tropical species remains in the dark (Figs. S38), this points to a combination of radiative and structural differences (e.g., surface absorptance, boundary- layer conductance) as primary determinants of T leaf –T air offsets ( Kullberg and Feeley, 2024 ; Middleby et al., 2025a ). Overall, leaf transpirational cooling, at least in the investigated species, appears rather as a physical consequence of sustained transpiration rather than a targeted outcome of stomatal control and therefore, the observed decoupling. Conclusions and implications In conclusion, (1) we could confirm that stomatal regulation at high T air , and therefore stomatal decoupling, will be strongly impacted by VPD and soil moisture availability, with reductions in g s at high VPD and in dry soil, while rising T air at stable low VPD and moist soil conditions leads to stomatal decoupling and increased E. Interestingly, we found that (2) tree species from temperate regions had a stronger stomatal regulation across T air ranges, with a decoupling at high T air , low VPD, and moist conditions. In contrast, tropical species, which evolve at higher T air and VPD environments, only barely co-regulated A net and g s at low VPD. Furthermore, (3) we found that the increase of E by stomatal decoupling only barely contributed to T leaf regulation, while a VPD increase was much more efficient due to a better heat flux. Taken together, our results indicate that stomatal decoupling represents a biophysical strategy to sustain transpirational water flux under high T air , not a failure of regulation nor an active optimization for cooling. When Tₐᵢᵣ increases under constant VPD, gₛ must rise to elevate E ( Fig. 8a ), leading to a decrease in the relationship between A net and g s , and therefore a strong decline in WUE i (Fig. S19a) as well as WUE (Fig. S21a). When VPD increases, ΔVPD drives E ( Fig. 8b ), preserving Aₙₑₜ–gₛ coupling and therefore a stable WUE i (Fig. S19c) but reducing WUE (Fig. S21c) through atmospheric control. These findings could be important at the parameter level to model g s , as it points out that the g 0 parameter, for which g s dark can be taken as a proxy, representing the “residual” stomatal opening independent of photosynthetic activity ( Medlyn et al., 2011 ). In temperate species, g₀ (as approximated by g s dark ) slightly increased with Tair under well-watered, low-VPD conditions, and we found a clear breakpoint with a much steeper increase around 35°C (Fig. S6a). In tropical species, a similar change in the relationship occurred at slightly lower T air (∼33°C; Fig. S6b). When T air and VPD increased simultaneously, g s dark remained largely unchanged in both groups (Figs. S6c, d), and declined sharply when VPD increased at a constant 35°C (Figs. S6e, f). Under drought, however, gs dark increased only at a low rate with rising T air at a low VPD (Figs. S7a, b), and exhibited breakpoint-type accelerated decline with rising VPD (Figs. S7e, f), demonstrating a stronger regulation of the stomatal conductance during darkness in response to reduced water availability. Together, these trends suggest that g₀ is not a fixed trait but a dynamic quantity that increases with T air at a low evaporative demand independent of water limitations, yet is stable or decreases when VPD rises. Together, these findings demonstrate that g₀ should not be seen as a fixed residual conductance, especially not at high T air , but a dynamic physiological parameter actively modulated by T air , VPD, and water availability. This suggests that stomata at high T air remain more open than expected, even in the dark, to maintain a continuous water flow through the plant, supporting hydraulic and possibly other plant metabolic demands. Furthermore, similar to Middleby et al., 2024 , we found further support that the g 1 parameter in the Medlyn model ( Medlyn et al., 2011 ), which is inversely related to WUE i , changes with T air , VPD, and soil moisture availability (Figs. S19, S20), including distinct T air breaking points above which its trend reverses, both in temperate and tropical species. Other studies will need to investigate the behavior of stomatal regulation at more extreme T air above 40°C, where the loss of physiological control, for instance through increased cuticular permeability ( Riederer and Schreiber, 2001 ; Wang et al., 2024 ), and biochemical dysfunctionality ( Gauthey et al., 2024 ; Didion-Gency et al., 2025 ), will likely become more important. SNAcknowledgments PS, MD-G, TJ, CB, GB, and CG were supported by the Swiss National Science Foundation SNSF (310030_204697 and CRSK-3_220989) and the Sandoz Family Foundation. We thank Georges Grun for his support with the climate chamber control. Author contribution PS and CG planned and designed the research. PS, MD-G, TJ, and CG conducted the laboratory work. GH and AK maintained the climate chamber facility. PS analyzed the data and led the manuscript’s writing, and PS, CG, MD-G, TJ, GH, AK, CB, and GB contributed to the final version. Data availability Upon the manuscript’s acceptance, the data supporting the findings will be made openly available in a public repository (Envidat). Supplementary tables View this table: View inline View popup Download powerpoint Table S1: Characteristics of the studied tree species. View this table: View inline View popup Download powerpoint Table S2: Average soil moisture (SM, volume-%), the corresponding standard errors for the overall treatments, as well as for the single days of the experiment. The final trend analysis (trend LM) indicates if there was a significant change in SM during the experimental period. View this table: View inline View popup Download powerpoint Table S3: Mean measured Tair and VPD with the corresponding standard errors in the climate chambers and the measurement chamber of the Li-6800. Funder Information Declared Swiss National Science Foundation , 310030_204697 , CRSK-3_220989 References 1. ↵ Aphalo , P.J. , 2025 . ggpmisc: Miscellaneous Extensions to “ggplot2. ” 2. ↵ Aphalo , P.J. , Jarvis , P.G ., 1991 . Do stomata respond to relative humidity? Plant Cell & Environment 14 , 127 – 132 . doi: 10.1111/j.1365-3040.1991.tb01379.x OpenUrl CrossRef 3. ↵ Bachofen , C. , Peillon , M. , Meili , N. , Bourgeois , I. , Grossiord , C ., 2025 . High transpirational cooling by urban trees despite extreme summer heatwaves . Urban Forestry & Urban Greening 128819 . doi: 10.1016/j.ufug.2025.128819 OpenUrl CrossRef 4. ↵ Bachofen , C. , Poyatos , R. , Flo , V. , Martínez-Vilalta , J. , Mencuccini , M. , Granda , V. , Grossiord , C. , 2023 . Stand structure of Central European forests matters more than climate for transpiration sensitivity to VPD . Journal of Applied Ecology 60 , 886 – 897 . doi: 10.1111/1365-2664.14383 OpenUrl CrossRef 5. ↵ Bose , A. , Tiwari , B. , Chattopadhyay , M.K. , Gupta , S. , Ghosh , B ., 1999 . Thermal stress induces differential degradation of Rubisco in heat-sensitive and heat-tolerant rice . Physiologia Plantarum 105 , 89 – 94 . doi: 10.1034/j.1399-3054.1999.105114.x OpenUrl CrossRef 6. ↵ Busch , F.A ., 2014 . Opinion: The red-light response of stomatal movement is sensed by the redox state of the photosynthetic electron transport chain . Photosynth Res 119 , 131 – 140 . doi: 10.1007/s11120-013-9805-6 OpenUrl CrossRef PubMed 7. ↵ Crous , K.Y. , Cheesman , A.W. , Middleby , K. , Rogers , E.I.E. , Wujeska-Klause , A. , Bouet , A.Y.M. , Ellsworth , D.S. , Liddell , M.J. , Cernusak , L.A. , Barton , C.V.M ., 2023 . Similar patterns of leaf temperatures and thermal acclimation to warming in temperate and tropical tree canopies . Tree Physiology 43 , 1383 – 1399 . doi: 10.1093/treephys/tpad054 OpenUrl CrossRef PubMed 8. ↵ Cunningham , ShaunC ., 2004 . Stomatal sensitivity to vapour pressure deficit of temperate and tropical evergreen rainforest trees of Australia . Trees 18 . doi: 10.1007/s00468-004-0318-y OpenUrl CrossRef 9. ↵ Diao , H. , Cernusak , L.A. , Saurer , M. , Gessler , A. , Siegwolf , R.T.W. , Lehmann , M.M ., 2024a . Uncoupling of stomatal conductance and photosynthesis at high temperatures: mechanistic insights from online stable isotope techniques . New Phytologist 241 , 2366 – 2378 . doi: 10.1111/nph.19558 OpenUrl CrossRef PubMed 10. ↵ Diao , H. , Cernusak , L.A. , Saurer , M. , Gessler , A. , Siegwolf , R.T.W. , Lehmann , M.M ., 2024b . Dry inside: progressive unsaturation within leaves with increasing vapour pressure deficit affects estimation of key leaf gas exchange parameters . New Phytologist 244 , 1275 – 1287 . doi: 10.1111/nph.20078 OpenUrl CrossRef 11. ↵ Didion-Gency , M. , Gauthey , A. , Johnson , K.M. , Schuler , P. , Grossiord , C. , 2025 . Leaf Excision and Exposure Duration Alter the Estimates of the Irreversible Photosynthetic Thermal Thresholds . Plant Cell & Environment 48 , 5357 – 5368 . doi: 10.1111/pce.15521 OpenUrl CrossRef 12. ↵ Dusenge , M.E. , Duarte , A.G. , Way , D.A ., 2019 . Plant carbon metabolism and climate change: elevated CO 2 and temperature impacts on photosynthesis, photorespiration and respiration . New Phytologist 221 , 32 – 49 . doi: 10.1111/nph.15283 OpenUrl CrossRef PubMed 13. ↵ Duursma , R.A. , Barton , C.V.M. , Lin , Y.-S. , Medlyn , B.E. , Eamus , D. , Tissue , D.T. , Ellsworth , D.S. , McMurtrie , R.E ., 2014 . The peaked response of transpiration rate to vapour pressure deficit in field conditions can be explained by the temperature optimum of photosynthesis . Agricultural and Forest Meteorology 189–190 , 2 – 10 . doi: 10.1016/j.agrformet.2013.12.007 OpenUrl CrossRef 14. ↵ Eamus , D. , Taylor , D.T. , Macinnis-Ng , C.M.O. , Shanahan , S. , De Silva , L. , 2008 . Comparing model predictions and experimental data for the response of stomatal conductance and guard cell turgor to manipulations of cuticular conductance, leaf-to-air vapour pressure difference and temperature: feedback mechanisms are able to account for all observations . Plant Cell & Environment 31 , 269 – 277 . doi: 10.1111/j.1365-3040.2007.01771.x OpenUrl CrossRef PubMed Web of Science 15. ↵ Fasola , S. , Mueggo , V.M.R. , Kuchenhoff , H ., 2018 . A heuristic, iterative algorithm for change- point detection in abrupt change models . Computational Statistics 33 , 997 – 1015 . OpenUrl 16. ↵ Gauthey , A. , Kahmen , A. , Limousin , J. , Vilagrosa , A. , Didion-Gency , M. , Mas , E. , Milano , A. , Tunas , A. , Grossiord , C. , 2024 . High heat tolerance, evaporative cooling, and stomatal decoupling regulate canopy temperature and their safety margins in three European oak species . Global Change Biology 30 , e17439 . doi: 10.1111/gcb.17439 OpenUrl CrossRef PubMed 17. ↵ H Wickham , 2016 . ggplot2: Elegant Graphics for Data Analysis . 18. ↵ Hall , A. , Schulze , E ., 1980 . Drought Effects on Transpiration and Leaf Water Status of Cowpea in Controlled Environments . Functional Plant Biol . 7 , 141 . doi: 10.1071/PP9800141 OpenUrl CrossRef Web of Science 19. ↵ Janssen , T. , Fleischer , K. , Luyssaert , S. , Naudts , K. , Dolman , H. , 2020 . Drought resistance increases from the individual to the ecosystem level in highly diverse Neotropical rainforest: a meta-analysis of leaf, tree and ecosystem responses to drought . Biogeosciences 17 , 2621 – 2645 . doi: 10.5194/bg-17-2621-2020 OpenUrl CrossRef 20. ↵ Jensen , K.H. , Berg-Sørensen , K. , Bruus , H. , Holbrook , N.M. , Liesche , J. , Schulz , A. , Zwieniecki , M.A. , Bohr , T. , 2016 . Sap flow and sugar transport in plants . Rev. Mod. Phys. 88 , 035007 . doi: 10.1103/RevModPhys.88.035007 OpenUrl CrossRef 21. ↵ Kassambara , A. , 2025 . ggpubr: “ggplot2” Based Publication Ready Plots. 22. ↵ Kullberg , A.T. , Feeley , K.J ., 2024 . Seasonal acclimation of photosynthetic thermal tolerances in six woody tropical species along a thermal gradient . Functional Ecology 38 , 2493 – 2505 . doi: 10.1111/1365-2435.14657 OpenUrl CrossRef 23. ↵ Lawson , T. , Von Caemmerer , S. , Baroli , I ., 2010 . Photosynthesis and Stomatal Behaviour , in: Progress in Botany 72 . Springer , Berlin, Heidelberg , pp. 265 – 304 . 24. ↵ Lenth , R. , Piaskowski , J. , 2025 . emmeans: Estimated Marginal Means, aka Least-Squares Means . 25. ↵ Levy , P.E. , Meir , P. , Allen , S.J. , Jarvis , P.G ., 1999 . The effect of aqueous transport of CO2 in xylem sap on gas exchange in woody plants . Tree Physiology 19 , 53 – 58 . doi: 10.1093/treephys/19.1.53 OpenUrl CrossRef PubMed Web of Science 26. ↵ Marchin , R.M. , Medlyn , B.E. , Tjoelker , M.G. , Ellsworth , D.S ., 2023 . Decoupling between stomatal conductance and photosynthesis occurs under extreme heat in broadleaf tree species regardless of water access . Global Change Biology gcb . 16929 . doi: 10.1111/gcb.16929 OpenUrl CrossRef 27. ↵ Massman , W.J ., 1998 . A review of the molecular diffusivities of H2O, CO2, CH4, CO, O3, SO2, NH3, N2O, NO, and NO2 . Atmospheric Environment 32 , 1111 – 1127 . OpenUrl CrossRef Web of Science 28. ↵ Massmann , A. , Gentine , P. , Lin , C ., 2019 . When Does Vapor Pressure Deficit Drive or Reduce Evapotranspiration? J Adv Model Earth Syst 11 , 3305 – 3320 . doi: 10.1029/2019MS001790 OpenUrl CrossRef PubMed 29. ↵ Matzner , S. , Comstock , J ., 2001 . The temperature dependence of shoot hydraulic resistance: implications for stomatal behaviour and hydraulic limitation . Plant Cell & Environment 24 , 1299 – 1307 . doi: 10.1046/j.0016-8025.2001.00785.x OpenUrl CrossRef 30. ↵ Medlyn , B.E. , Dreyer , E. , Ellsworth , D. , Forstreuter , M. , Harley , P.C. , Kirschbaum , M.U.F. , Le Roux , X. , Montpied , P. , Strassemeyer , J. , Walcroft , A. , Wang , K. , Loustau , D. , 2002 . Temperature response of parameters of a biochemically based model of photosynthesis. II. A review of experimental data . Plant Cell & Environment 25 , 1167 – 1179 . doi: 10.1046/j.1365-3040.2002.00891.x OpenUrl CrossRef 31. ↵ Medlyn , B.E. , Duursma , R.A. , Eamus , D. , Ellsworth , D.S. , Prentice , I.C. , Barton , C.V.M. , Crous , K.Y. , De Angelis , P. , Freeman , M. , Wingate , L. , 2011 . Reconciling the optimal and empirical approaches to modelling stomatal conductance: RECONCILING OPTIMAL AND EMPIRICAL STOMATAL MODELS . Global Change Biology 17 , 2134 – 2144 . doi: 10.1111/j.1365-2486.2010.02375.x OpenUrl CrossRef 32. ↵ Messinger , S.M. , Buckley , T.N. , Mott , K.A ., 2006 . Evidence for Involvement of Photosynthetic Processes in the Stomatal Response to CO2 . Plant Physiology 140 , 771 – 778 . doi: 10.1104/pp.105.073676 OpenUrl Abstract / FREE Full Text 33. ↵ Middleby , K.B. , Cheesman , A.W. , Cernusak , L.A ., 2024 . Impacts of elevated temperature and vapour pressure deficit on leaf gas exchange and plant growth across six tropical rainforest tree species . New Phytologist 243 , 648 – 661 . doi: 10.1111/nph.19822 OpenUrl CrossRef PubMed 34. ↵ Middleby , K.B. , Cheesman , A.W. , Hopkinson , R. , Baker , L. , Ramirez Garavito , S. , Breed , M.F. , Cernusak , L.A ., 2025a . Ecotypic Variation in Leaf Thermoregulation and Heat Tolerance but Not Thermal Safety Margins in Tropical Trees . Plant Cell & Environment 48 , 649 – 663 . doi: 10.1111/pce.15141 OpenUrl CrossRef 35. ↵ Middleby , K.B. , Jordan , R. , Cheesman , A.W. , Rossetto , M. , Breed , M.F. , Crayn , D.M. , Cernusak , L.A ., 2025b . Local Adaptation Drives Leaf Thermoregulation in Tropical Rainforest Trees . Global Change Biology 31 , e70461 . doi: 10.1111/gcb.70461 OpenUrl CrossRef PubMed 36. ↵ Mills , C. , Bartlett , M.K. , Buckley , T.N ., 2024 . The poorly-explored stomatal response to temperature at constant evaporative.pdf . Plant, Cell & Environment . 37. ↵ Peak , D. , Mott , K.A ., 2011 . A new, vapour-phase mechanism for stomatal responses to humidity and temperature . Plant Cell & Environment 34 , 162 – 178 . doi: 10.1111/j.1365-3040.2010.02234.x OpenUrl CrossRef PubMed Web of Science 38. ↵ Pedersen , T. , T., 2025 . patchwork: The Composer of Plots . 39. ↵ Pernau , H.-F. , Benkendorf , M. , Jägle , M. , Heinrich , S. , Knittel , T. , Wöllenstein , J ., 2024 . Measuring the Thermal Conductivity of Humid Air over a Broad Temperature and Water Content Range , in: Eurosensors 2023 . Presented at the Eurosensors 2023, MDPI, p. 167 . doi: 10.3390/proceedings2024097167 OpenUrl CrossRef 40. ↵ R Core Team , 2025 . R: A language and environment for statistical computing . 41. ↵ Riederer , M. , Schreiber , L ., 2001 . Protecting against water loss: analysis of the barrier properties of plant cuticles . Journal of Experimental Botany 52 , 2023 – 2032 . doi: 10.1093/jexbot/52.363.2023 OpenUrl CrossRef PubMed Web of Science 42. ↵ Rogers , C. , Sharpe , P.J.H. , Powell , R.D ., 1980 . Dark Opening of Stomates of Vicia faba in CO 2 - free Air: EFFECT OF TEMPERATURE ON STOMATAL APERTURE AND POTASSIUM ACCUMULATION . Plant Physiol . 65 , 1036 – 1038 . doi: 10.1104/pp.65.6.1036 OpenUrl Abstract / FREE Full Text 43. ↵ Schönbeck , L.C. , Schuler , P. , Lehmann , M.M. , Mas , E. , Mekarni , L. , Pivovaroff , A.L. , Turberg , P. , Grossiord , C ., 2022 . Increasing temperature and vapour pressure deficit lead to hydraulic damages in the absence of soil drought . Plant Cell & Environment 45 , 3275 – 3289 . doi: 10.1111/pce.14425 OpenUrl CrossRef 44. ↵ Schulze , E.-D. , Lange , O.L. , Kappen , L. , Buschbom , U. , Evenari , M ., 1973 . Stomatal responses to changes in temperature at increasing water stress . Planta 110 , 29 – 42 . doi: 10.1007/BF00386920 OpenUrl CrossRef PubMed Web of Science 45. ↵ Slot , M. , Rifai , S.W. , Eze , C.E. , Winter , K ., 2024 . The stomatal response to vapor pressure deficit drives the apparent temperature response of photosynthesis in tropical forests . New Phytologist nph . 19806 . doi: 10.1111/nph.19806 OpenUrl CrossRef 46. ↵ Sorz , J. , Hietz , P ., 2006 . Gas diffusion through wood: implications for oxygen supply . Trees 20 , 34 – 41 . doi: 10.1007/s00468-005-0010-x OpenUrl CrossRef 47. ↵ Urban , J. , Ingwers , M.W. , McGuire , M.A. , Teskey , R.O ., 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 , 1757 – 1767 . doi: 10.1093/jxb/erx052 OpenUrl CrossRef PubMed 48. ↵ Von Caemmerer , S. , Lawson , T. , Oxborough , K. , Baker , N.R. , Andrews , T.J. , Raines , C.A. , 2004 . Stomatal conductance does not correlate with photosynthetic capacity in transgenic tobacco with reduced amounts of Rubisco . Journal of Experimental Botany 55 , 1157 – 1166 . doi: 10.1093/jxb/erh128 OpenUrl CrossRef PubMed Web of Science 49. ↵ Wang , S. , Hoch , G. , Grun , G. , Kahmen , A ., 2024 . Water loss after stomatal closure: quantifying leaf minimum conductance and minimal water use in nine temperate European tree species during a severe drought . Tree Physiology 44 , tpae027. doi: 10.1093/treephys/tpae027 OpenUrl CrossRef 50. ↵ Wong , S.C. , Cowan , I.R. , Farquhar , G.D ., 1979 . Stomatal conductance correlates with photosynthetic capacity . Nature 282 , 424 – 426 . doi: 10.1038/282424a0 OpenUrl CrossRef Web of Science 51. ↵ Wu , J. , Serbin , S.P. , Ely , K.S. , Wolfe , B.T. , Dickman , L.T. , Grossiord , C. , Michaletz , S.T. , Collins , A.D. , Detto , M. , McDowell , N.G. , Wright , S.J. , Rogers , A ., 2020 . The response of stomatal conductance to seasonal drought in tropical forests . Global Change Biology 26 , 823 – 839 . doi: 10.1111/gcb.14820 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted November 04, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Decoupling of stomatal conductance from net assimilation at high temperature as a mechanism to increase transpiration Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Decoupling of stomatal conductance from net assimilation at high temperature as a mechanism to increase transpiration Philipp Schuler , Margaux Didion-Gency , Giovanni Bortolami , Thibaut Julliard , Günter Hoch , Christoph Bachofen , Ansgar Kahmen , Charlotte Grossiord bioRxiv 2025.11.03.686201; doi: https://doi.org/10.1101/2025.11.03.686201 Share This Article: Copy Citation Tools Decoupling of stomatal conductance from net assimilation at high temperature as a mechanism to increase transpiration Philipp Schuler , Margaux Didion-Gency , Giovanni Bortolami , Thibaut Julliard , Günter Hoch , Christoph Bachofen , Ansgar Kahmen , Charlotte Grossiord bioRxiv 2025.11.03.686201; doi: https://doi.org/10.1101/2025.11.03.686201 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)