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At least 19 records

Hydraulic constraints to stomatal conductance in flooded trees

Stomatal closure is a pervasive response among trees exposed to flooded soil. We tested whether this response is caused by reduced hydraulic conductance in the soil-to-leaf hydraulic continuum (k total ), and particularly by reduced root hydraulic conductance (k root ), which has been widely hypothesized. We tracked stomatal conductance at the leaf level (g s ) and canopy scale (G s ) along with physiological conditions in two temperate tree species, Magnolia grandiflora and Quercus virginiana, that were subjected to flood and control conditions in a greenhouse experiment. Flooding reduced g s , G s , k root and k total . Path analysis showed strong support for direct effects of k total on g s and for flood duration on k total , but not k root on k total . A process-based model that accounted for the k total reduction predicted the timeseries of G s in flood and control treatment trees reasonably well (predicted versus observed G s R 2 = 0.80 and 0.51 for M. grandiflora and Q. virginiana, respectively). However, accounting only for k root reduction in flooded trees was insufficient for predicting observed G s reduction. Together, these results suggest that hydraulic constraints were not limited to roots and highlight the need to account for flooding effects on k total when projecting forest ecosystem function using process-based models.

Plant stress↗

Effects of Warming and Elevated CO2 on Stomatal Conductance and Chlorophyll Fluorescence of C3 and C4 Coastal Wetland Species

Abstract Coastal wetland communities provide valuable ecosystem services such as erosion prevention, soil accretion, and essential habitat for coastal wildlife, but are some of the most vulnerable to the threats of climate change. This work investigates the combined effects of two climate stressors, elevated temperature (ambient, + 1.7 °C, + 3.4 °C, and 5.1 °C) and elevated CO 2 ( e CO 2 ), on leaf physiological traits of dominant salt marsh plant species. The research took place at the Salt Marsh Accretion Response to Temperature eXperiment (SMARTX) at the Smithsonian Environmental Research Center, which includes two plant communities: a C 3 sedge community and a C 4 grass community. Here we present data collected over five years on rates of stomatal conductance (g s ), quantum efficiency of PSII photochemistry ( F v / F m ), and rates of electron transport (ETR max ). We found that both warming and e CO 2 caused declines in all traits, but the warming effects were greater for the C 3 sedge. This species showed a strong negative stomatal response to warming in 2017 and 2018 (28% and 17% reduction, respectively in + 5.1 °C). However, in later years the negative response to warming was dampened to < 7%, indicating that S. americanus was able to partially acclimate to the warming over time. In 2022, we found that sedges growing in the combined + 5.1 °C e CO 2 plots exhibited more significant declines in g s , F v /F m , and ETR max than in either treatment individually. These results are important for predicting future trends in growth of wetland species, which serve as a large carbon sink that may help mitigate the effects of climate change.

54 ENVIRONMENTAL SCIENCES↗

The effect of relative humidity and temperature on the response of stomatal conductance to vapor pressure deficit in tropical trees

Understanding how leaf gas exchange responds to changes in vapor pressure deficit (VPD) is key to predicting tropical forest resilience to climate change. Stomata regulate leaf water and CO2 diffusion, and respond to changes in temperature and relative humidity (RH), two drivers of VPD. At high temperatures, the cuticular pathway may also become significant and participate in the overall leaf conductance. Here, we measured gas exchange under light and dark conditions to investigate the stomatal and cuticular responses to temperature and RH on detached branches in five tropical tree species. Leaf conductance in the dark, when stomata are essentially closed, was not markedly impacted by temperature and RH, suggesting a minimal response of the cuticular pathway to these conditions. We compared six steady-state conductance models incorporating different effects of photosynthesis and evaporative demand on stomatal control. All models performed well (residual standard deviation, σ, < 0.025 mol m-2 s-1), but the best-fitting model (σ = 0.017 mol m-2 s-1) used a nonlinear relationship between photosynthesis and stomatal conductance and incorporated RH rather than vapor pressure difference. All models overestimated the steady-state conductance at high RH. Furthermore, leaf conductance immediately increased after a decrease in RH (wrong-way response), but not after an increase in leaf temperature. This suggests that the mechanisms underlying stomatal response to VPD need further investigation. The non-linear coupling between photosynthetic rate and stomatal conductance indicates a sharper physiological response than previously acknowledged.

Lamour, Julien↗

Evaluating the Influence of Plants on Hydrologic Cycling: Quantifying and Validating the Role of Plant Processes and Stomatal Conductance (Final Report)

Plants can exert strong controls on water cycling, both locally and across the globe. The impacts are driven by how plants control the exchange of water and energy between the land and the atmosphere, which in turn are influenced by how plants photosynthesize and grow (e.g. biogeochemical cycling). Our team has shown previously that plant responses to increasing CO 2 in the atmosphere can influence the water cycle through changes in rainfall and the amount of water on land with implications for drought. Plant responses can also alter both average and extreme flow of water in rivers, with implications for freshwater availability and flood frequency. Although the impact of plant responses on water cycling has been demonstrated to exist, significant uncertainty remains in our understanding of the magnitude and form of the plant responses themselves, as well as their ultimate impact on water availability for people and ecosystems. In this project we worked to quantify the role of plant processes in regulating water cycling (e.g., precipitation, evapotranspiration, runoff, droughts, etc.). We addressed the following objectives: quantify the control of physiological vs. radiative effects on water cycling across many coupled models in idealized and realistic scenarios, as well as to quantify how our assumptions about leaf-level processes (e.g. coupling between stomatal conductance and photosynthesis), and organism-level processes (e.g. leaf area response to high CO 2 ) contributed to uncertainties and biases in water cycling. We focused on the impact of these processes on water cycling to assess the sensitivities of precipitation, evapotranspiration, and runoff to model assumptions about plant processes at the leaf, organism, and community level. As part of this project we used measurements of stable carbon isotopes to generate observationally based estimates of plant functioning during the historical period. This work is directly relevant to DOE’s primary scientific research questions in the RGMA topic area (b) on Biogeochemical processes, feedbacks, and interactions in the Earth system.

59 BASIC BIOLOGICAL SCIENCES↗

Altered cell wall hydroxycinnamate composition impacts leaf- and canopy-level CO2 uptake and water use in rice

Abstract Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall (CW) properties might help improve photosynthesis and crop water use efficiency (WUE). We tested this using 2 independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a “BAHD” CoA acyltransferase), which alters CW hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, CW composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll CW thickness (−14%) and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic WUE (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in aboveground biomass (AGB) (+12.5%) and canopy-level WUE (+8.8%; ratio of AGB to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in CW composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic WUE will need to enhance mesophyll conductance and photosynthesis while maintaining or decreasing stomatal conductance.

59 BASIC BIOLOGICAL SCIENCES↗

Atmospheric Feedbacks Reverse the Sensitivity of Modeled Photosynthesis to Stomatal Function

Stomata mediate fluxes of carbon and water between terrestrial plants and the atmosphere. These fluxes are governed by stomatal function and can be modulated in many Earth system models by an empirical parameter within the calculation of stomatal conductance, the stomatal slope (𝑔 1⁢𝑀 ). Intuitively, 𝑔 1⁢𝑀 represents the marginal water cost of carbon, relating it to the emergent plant property of water use efficiency. Observations show that 𝑔 1⁢𝑀 can range widely across and within plant types in varying environments, and this distribution of 𝑔 1⁢𝑀 is not captured within Earth system models which represent each plant type with a single 𝑔 1⁢𝑀 value. Here we examine how 𝑔 1⁢𝑀 influences photosynthesis using coupled Earth system model simulations by perturbing 𝑔 1⁢𝑀 to observed 5⁢t⁢h and 95⁢t⁢h percentiles for each plant type. We find that high 𝑔 1⁢𝑀 reduces photosynthesis nearly everywhere, while low 𝑔 1⁢𝑀 has regionally dependent responses. Under fixed atmospheric conditions, low 𝑔 1⁢𝑀 increases photosynthesis in the Amazon and central North America but decreases photosynthesis in boreal Canada. These responses reverse when the atmosphere responds interactively due to spatially differing sensitivity to increases in temperature and vapor pressure deficit. Choice of 𝑔 1⁢𝑀 also influences photosynthetic response to changes in atmospheric carbon dioxide (CO 2 ), with lower and higher 𝑔1⁢𝑀 modifying total global response to elevated 2x preindustrial CO 2 by +6.4% and −9.6%, respectively. Our work demonstrates that atmospheric feedbacks are critical for determining the photosynthetic response to 𝑔 1⁢𝑀 assumptions and some regions are particularly sensitive to choice of 𝑔 1⁢𝑀 .

Liu, Amy X. [University of Washington, Seattle, WA↗

Endophyte mediated Populus trichocarpa water use efficiency is dependent on time of day and plant water status

Endophytes are potential partners for improving the resource use efficiency of bioenergy feedstock systems such as short rotation coppiced Populus. Endophytes isolated from members of the Salicaceae family have broad host compatibility and can improve water use efficiency (WUE) through decreases in stomatal conductance. However, the literature is inconsistent with regards to the environmental conditions and temporal patterns of these benefits. This study investigates how endophyte mediated changes in Populus trichocarpa (Torr. and Gray) ‘Nisqually-1’ stomatal conductance and WUE shift with time and scale in response to water-deficit stress. Leaf gas exchange and above-ground productivity were used to evaluate the carbon and water balances of glasshouse grown plants in response to endophyte inoculation and water-deficit. Differences in stomatal conductance between control and inoculated plants were more pronounced (39.7 % decrease, Welch’s two-sample t(14.34 dfadj) = -2.358, p-value = 0.033) under water-deficit conditions in the late morning during a period of higher light intensity. The decrease in stomatal conductance accompanied a substantial increase in intrinsic WUE (iWUE) for water-deficit inoculated plants. However, increases in iWUE did not result in improvements in aboveground productivity or shoot biomass WUE (WUEsb) for water-deficit inoculated plants. This decoupling between iWUE and aboveground productivity may be an indicator of assimilate allocation to microbial metabolism as an additional carbon sink or a shift in carbon allocation towards belowground biomass. Future work should take a whole-plant approach that accounts for diurnal patterns in incident irradiance to evaluate the impact of endophyte inoculation on host WUE and stress tolerance.

59 BASIC BIOLOGICAL SCIENCES↗

Save or spend? Diverging water‐use strategies of grasses and encroaching clonal shrubs

Abstract Shrub encroachment is one of the primary threats to mesic grasslands around the world. This dramatic shift in plant cover has the potential to alter ecosystem‐scale water budgets and responses to novel rainfall regimes. Understanding divergent water‐use strategies among encroaching shrubs and the grasses they replace is critical for predicting shifts in ecosystem‐scale water dynamics as a result of shrub encroachment, particularly if drought events become more frequent and/or severe in the future. In this study, we assessed how water‐use traits of a rapidly encroaching clonal shrub (Cornus drummondii) and a dominant C 4 grass (Andropogon gerardii) impact responses to changes in water availability in tallgrass prairie. We assessed intra‐annual change in depth of water uptake, turgor loss point and stomatal regulation in each species. Sampling took place at Konza Prairie Biological Station (northeastern KS, USA) during the 2021 and 2022 growing seasons. Cornus drummondiishifted from shallow to deep soil water sources across the 2021 and 2022 growing seasons. This plasticity in depth of water uptake facilitated a ‘wasteful’ water‐use strategy inC. drummondii, where stomatal conductance and transpiration rates continued to increase even when no further gain in photosynthetic rate occurred.A. gerardiiphotosynthetic rates and stomatal conductance were more variable through time and were more responsive to changes in leaf water potential thanC. drummondii. However, intra‐annual adjustment of turgor loss point was more pronounced inC. drummondii(Δπ TLP = −0.48 MPa ± 0.15 SD) than inA. gerardii(Δπ TLP = −0.29 MPa ± 0.19 SD). Synthesis. These results suggest thatC. drummondiiis highly resilient to changes in water availability in surface soils and will likely remain unaffected by future droughts unless they are severe enough to reduce the availability of deep soil water. Given that clonal shrubs are key invaders of grasslands world‐wide, increased leaf‐level water loss is expected to accelerate ecosystem‐level drying as clonal shrub encroachment proceeds in mesic grasslands.

Environmental Sciences & Ecology↗

Development of a multi-layer canopy model for E3SM Land Model with support for heterogeneous computing

The vertical structure of vegetation canopies creates micro-climates. However, the land components of most Earth System Models, including the Energy Exascale Earth System Model (E3SM), typically neglect vertical canopy structure by using a single layer big-leaf representation to simulate water, CO 2 , and energy exchanges between the land and the atmosphere. In this study, we developed a Multi-Layer Canopy Model for the E3SM Land Model to resolve the micro-climate created by vegetation canopies. The model developed in this study re-implements the CLM-ml_v1 to support heterogeneous computing architectures consisting of CPUs and GPUs and includes three additional optimization-based stomatal conductance models. The use of Portable, Extensible Toolkit for Scientific Computation provides a speedup of 25–50 times on a GPU relative to a CPU. The numerical implementation of the model was verified against CLM-ml_v1 for a month-long simulation using data from the Ameriflux US-University of Michigan Biological Station site. Model structural uncertainty was explored by performing control simulations for five stomatal conductance models that exclude and include the control of plant hydrodynamics (PHD) on photosynthesis. The bias in simulated sensible and latent heat fluxes was lower when PHD was accounted for in the model. Additionally, six idealized simulations were performed to study the impact of three environmental variables (i.e. air temperature, atmospheric CO 2 , and soil moisture) on canopy processes (i.e. net CO 2 assimilation, leaf temperature, and leaf water potential). Increasing air temperature reduced net CO 2 assimilation and increased air temperature. Net CO 2 assimilation increased at higher atmospheric CO 2 , while decreasing soil moisture resulted in lower leaf water potential.

54 ENVIRONMENTAL SCIENCES↗

Greater aperture counteracts effects of reduced stomatal density on WUE: a case study on sugarcane and meta-analysis

Stomata regulate CO 2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (g sw ). However, the quantitative relationship between reduced SD, g sw , and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEFP2) in sugarcane reduced SD by 26-38% but did not affect gsw compared to wildtype. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO 2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C 3 dicots, C 3 monocots, and C 4 monocots revealed engineered reductions in SD are strongly correlated with lower gsw (r 2 =0.60-0.98), but this response is damped relative to the change in anatomy.

59 BASIC BIOLOGICAL SCIENCES↗

Impacts of Pasture Conversion to Sugarcane on Water Fluxes and Water Use Efficiency in the Southeastern US

The expansion of sugarcane (cane), a high-yielding perennial crop, will likely reshape the bioenergy landscape in the Southeastern US. However, its ecohydrological implications, particularly following conversion from grazed pastures, a dominant land use in the region, remain highly uncertain. We investigated the impact of cane expansion on evapotranspiration (ET) and its partitioning, and the mechanisms influencing both ET components and water use efficiency (WUE) across multiple scales and growth cycles in subtropical Florida. We combined eddy covariance, biometric measurements, and process-based stomatal conductance (g s ) models. ET was 1.7% lower in cane than in improved pasture (IMP) but exceeded that in semi-native pasture (SN) by 21%. Transpiration (T) followed a similar pattern, consistent with lower g s in cane relative to IMP. Cane had more conservative water use and greater sensitivity of g s to vapor pressure deficit (VPD) compared to IMP pasture, suggesting cane may be more tolerant of increasing atmospheric water demand. In contrast, SN showed lower g s and weaker stomatal sensitivity to VPD compared to cane, resulting in lower T. In cane, stomatal regulation and T varied across growth cycles, with stomata becoming less water conservative as stands matured, highlighting the importance of incorporating stand age-dependent stomatal regulation into hydrological models. Evaporation (E) was higher in cane than pastures (19%–26%), partially offsetting WUE gains. Cane exhibited higher intrinsic WUE (GPP/g s ; Gross Primary Productivity), ecosystem WUE (GPP/ET), and harvest WUE (harvest/ET) than both pasture types. Large-scale pasture-to-cane conversion could produce widely contrasting hydrological outcomes. The net regional impact will depend on the proportion of each pasture type converted and on cane's high g s sensitivity to VPD, which triggers tight stomatal regulation and conservative water use, both of which will become increasingly consequential under intensifying atmospheric water demand.

bioenergy↗

Wood‐density has no effect on stomatal control of leaf‐level water use efficiency in an Amazonian forest

Forest disturbances increase the proportion of fast-growing tree species compared to slow-growing ones. To understand their relative capacity for carbon uptake and their vulnerability to climate change, and to represent those differences in Earth system models, it is necessary to characterise the physiological differences in their leaf-level control of water use efficiency and carbon assimilation. We used wood density as a proxy for the fast-slow growth spectrum and tested the assumption that trees with a low wood density (LWD) have a lower water-use efficiency than trees with a high wood density (HWD). We selected 5 LWD tree species and 5 HWD tree species growing in the same location in an Amazonian tropical forest and measured in situ steady-state gas exchange on top-of-canopy leaves with parallel sampling and measurement of leaf mass area and leaf nitrogen content. We found that LWD species invested more nitrogen in photosynthetic capacity than HWD species, had higher photosynthetic rates and higher stomatal conductance. Furthermore, contrary to expectations, we showed that the stomatal control of the balance between transpiration and carbon assimilation was similar in LWD and HWD species and that they had the same dark respiration rates.

54 ENVIRONMENTAL SCIENCES↗

Data for Regier et al. (2025), "Short-term experimental flooding impacts soil biogeochemistry but not aboveground vegetation in a coastal forest"

Rising sea levels and intensifying storms increase flooding pressure on coastal forests, but the mechanisms that drive coastal forest mortality remain unclear. This study used an ecosystem-scale manipulation (TEMPEST, Terrestrial Ecosystem Manipulation to Probe the Effects of Storm Treatments) to simulate hurricane-level flooding of a coastal forest and explore the individual and interactive impacts of inundation and salinity. This dataset comprises the results reported by Regier et al. (2025) in their paper "Short-term experimental flooding impacts soil biogeochemistry but not aboveground vegetation in a coastal forest." It consists of measurements of:- Belowground conductance- Soil dissolved oxygen and redox- Soil and tree greenhouse gas fluxes- Leaf photosynthesis, stomatal conductance, and intercellular carbon dioxide- Sap flux- Soil volumetric water content and electrical conductivityAll files are plain-text CSV (comma separated value) and no special software is required to read them.

54 ENVIRONMENTAL SCIENCES↗

Systematic Underestimation of Canopy Conductance Sensitivity to Drought by Earth System Models

The response of vegetation canopy conductance (g c ) to changes in moisture availability ($γ^{m}_{gc}$) is a major source of uncertainty in climate projections. While vegetation typically reduces stomatal conductance during drought, accurately modeling how and to what degree stomata respond to changes in moisture availability at global scales is particularly challenging, because no global scale g c observations exist. Here, we leverage a collection of satellite, reanalysis and station-based near-surface air and surface temperature estimates, which are physically and statistically linked to $γ^{m}_{gc}$ due to the local cooling effect of g c through transpiration, to develop a novel emergent constraint of $γ^{m}_{gc}$ in an ensemble of Earth System Models (ESMs). We find that ESMs systematically underestimate $γ^{m}_{gc}$ by ~33%, particularly in grasslands, croplands, and savannas in semi-arid and bordering regions of the Central United States, Central Europe, Southeastern South America, Southern Africa, Eastern Australia, and parts of East Asia. We show that this underestimation occurs because ESMs inadequately reduce g c when soil moisture decreases. As g c controls carbon, water and energy fluxes, the misrepresentation of modeled $γ^{m}_{gc}$ contributes to biases in ESM projections of gross primary production, transpiration, and temperature during droughts. Our results suggest that the severity and duration of droughts may be misrepresented in ESMs due to the impact of sustained g c on both soil moisture dynamics and the biosphere-atmosphere feedbacks that affect local temperatures and regional weather patterns.

54 ENVIRONMENTAL SCIENCES↗

Ecosystem Water‐Saving Timescale Varies Spatially With Typical Drydown Length

Abstract Stomatal optimization theory is a commonly used framework for modeling how plants regulate transpiration in response to the environment. Most stomatal optimization models assume that plants instantaneously optimize a reward function such as carbon gain. However, plants are expected to optimize over longer timescales given the rapid environmental variability they encounter. There are currently no observational constraints on these timescales. Here, a new stomatal model is developed and is used to analyze the timescales over which stomatal closure is optimized. The proposed model assumes plants maximize carbon gain subject to the constraint that they cannot draw down soil moisture below a critical value. The reward is integrated over time, after being weighted by a discount factor that represents the timescale ( τ ) that a plant considers when optimizing stomatal conductance to save water. The model is simple enough to be analytically solvable, which allows the value of τ to be inferred from observations of stomatal behavior under known environmental conditions. The model is fitted to eddy covariance data in a range of ecosystems, finding the value of τ that best predicts the dynamics of evapotranspiration at each site. Across 82 sites, the climate metrics with the strongest correlation to τ are measures of the average number of dry days between rainfall events. Values of τ are similar in magnitude to the longest such dry period encountered in an average year. The results here shed light on which climate characteristics shape spatial variations in ecosystem‐level water use strategy.

54 ENVIRONMENTAL SCIENCES↗

Patch-level CO2 and CH4 fluxes and porewater concentrations in experimental wetlands, 5 and 10 PPT saltwater intrusion simulations, Louisiana 2023-2024

This dataset containes carbon dioxide (CO2) and methane (CH4) flux measurements collected from wetland vegetation patches dominated by Typha domingensis and Panicum hemitomon to assess greenhouse gas flux responses to experimental saltwater intrusion (SWI) pulses. Measurements were conducted before, during, and after simulated SWI events at target salinities of approximately 5 parts per thousand (ppt) with durations of 6, 10, and 17 days and 10 ppt with a duration of 48 days, alongside a control wetland (with no salinity added, flood manipulation only). These data were generated to evaluate how the magnitude and duration of SWI alter wetland carbon exchange and related biogeochemical and plant responses. This data package includes flux measurements from the wetland surface (i.e, soil/water surface and enclosed vegetation) and from the soil/water surface only; porewater and surface water concentrations of CO2 and CH4; salinity, pH, electrical conductivity collected in porewater (at 5, 10, and 20 cm soil depths) and in surface water; soil redox potential; leaf spectral indices, leaf vapor pressure deficit, stomatal conductance; water level, salinity, and photosynthetically active radiation; and aboveground biomass.

EARTH SCIENCE > AGRICULTURE > SOILS > SOIL RESPIRA↗

Hysteresis area at the canopy level during and after a drought event in the Central Amazon

Understanding forest water limitation during droughts within a warming climate is essential for accurate predictions of forest-climate interactions. In hyperdiverse ecosystems like the Amazon forest, the mechanisms shaping hysteresis patterns in transpiration relative to environmental factors are not well understood. From this perspective, we investigated these dynamics by conducting in situ leaf-level measurements throughout and after the 2015 El Niño-Southern Oscillation (ENSO) drought. Our findings indicate a substantial increase in the hysteresis area (H area ) among transpiration (E), vapor pressure deficit (VPD), and stomatal conductance (g s ) at canopy level during the ENSO peak, attributed to both temporal lag and differences in magnitude between g s and VPD peaks. Specifically, the canopy species Pouteria anomala exhibited an increased H area , due to earlier maximum g s rates leading to a greater temporal lag with VPD compared to the post-drought period. Additionally, leaf water potential (ψ L ) and canopy temperature (T canopy ) showed larger H area during the ENSO peak compared to post-drought conditions across all studied species, suggesting that stomatal closure, particularly during the afternoon, acts to minimize water loss and may explain the counterclockwise hysteresis observed between ψ L and T canopy . Here, the pronounced H area during the drought points to a potential imbalance between water supply and demand, underlining the role of stomatal behavior of isohydric species in response to drought.

54 ENVIRONMENTAL SCIENCES↗

Engineering quantitative stomatal trait variation and local adaptation potential by cis‐regulatory editing

Summary Cis‐regulatory element editing can generate quantitative trait variation that mitigates extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed CRISPR/Cas9 approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter ofOsSTOMAGEN, a positive regulator of rice stomatal density. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild‐type stomatal density. This panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation and intrinsic water use efficiency in steady‐state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought‐responsive stomatal density reprogramming instomagenand two cis‐regulatory edited alleles was reduced. Collectively our data demonstrate that cis‐regulatory element editing can generate near‐isogenic trait variation that can be leveraged for establishing relationships between anatomy and physiology, providing a basis for optimizing traits across diverse environments.

Biotechnology & Applied Microbiology↗