El Niño-Southern Oscillation forcing on carbon and water cycling in a Bornean tropical rainforest.

Carbon dioxide and water vapor exchanges between tropical forest canopies and the atmosphere through photosynthesis, respiration, and evapotranspiration (ET) influence carbon and water cycling at the regional and global scales. Their inter- and intra-annual variations are sensitive to seasonal rhythms and longer-timescale tropical climatic events. In the present study, we assessed the El Niño-Southern Oscillation (ENSO) influence on ET and on the net ecosystem exchange (NEE), using eddy-covariance flux observations in a Bornean rainforest over a 10-y period (2010-2019) that included several El Niño and La Niña events. From flux model inversions, we inferred ecophysiological properties, notably the canopy stomatal conductance and "big-leaf" maximum carboxylation rate (Vcmax25_BL). Mean ET values were similar between ENSO phases (El Niño, La Niña, and neutral conditions). Conversely, the mean net ecosystem productivity was highest during La Niña events and lowest during El Niño events. Combining Shapley additive explanation calculations for nine controlling factors with a machine-learning algorithm, we determined that the primary factors for ET and NEE in the La Niña and neutral phases were incoming shortwave solar radiation and Vcmax25_BL, respectively, but that canopy stomatal conductance was the most significant factor for both ET and NEE in the El Niño phase. A combined stomatal-photosynthesis model approach further indicated that Vcmax25_BL differences between ENSO phases were the most significant controlling factor for canopy photosynthesis, emphasizing the strong need to account for ENSO-induced ecophysiological factor variations in model projections of the long-term carbon balance in Southeast Asian tropical rainforests.

Constitutive expression of apple endo-POLYGALACTURONASE1 in fruit induces early maturation, alters skin structure and accelerates softening.

During fruit ripening, polygalacturonases (PGs) are key contributors to the softening process in many species. Apple is a crisp fruit that normally exhibits only minor changes to cell walls and limited fruit softening. Here, we explore the effects of PG overexpression during fruit development using transgenic apple lines overexpressing the ripening-related endo-POLYGALACTURONASE1 gene. MdPG1-overexpressing (PGox) fruit displayed early maturation/ripening with black seeds, conversion of starch to sugars and ethylene production occurring by 80 days after pollination (DAP). PGox fruit exhibited a striking, white-skinned phenotype that was evident from 60 DAP and most likely resulted from increased air spaces and separation of cells in the hypodermis due to degradation of the middle lamellae. Irregularities in the integrity of the epidermis and cuticle were also observed. By 120 DAP, PGox fruit cracked and showed lenticel-associated russeting. Increased cuticular permeability was associated with microcracks in the cuticle around lenticels and was correlated with reduced cortical firmness at all time points and extensive post-harvest water loss from the fruit, resulting in premature shrivelling. Transcriptomic analysis suggested that early maturation was associated with upregulation of genes involved in stress responses, and overexpression of MdPG1 also altered the expression of genes involved in cell wall metabolism (e.g. ß-galactosidase, MD15G1221000) and ethylene biosynthesis (e.g. ACC synthase, MD14G1111500). The results show that upregulation of PG not only has dramatic effects on the structure of the fruit outer cell layers, indirectly affecting water status and turgor, but also has unexpected consequences for fruit development.

A Solanum lycopersicum polyamine oxidase contributes to the control of plant growth, xylem differentiation, and drought stress tolerance.

Polyamines are involved in several plant physiological processes. In Arabidopsis thaliana, five FAD-dependent polyamine oxidases (AtPAO1 to AtPAO5) contribute to polyamine homeostasis. AtPAO5 catalyzes the back-conversion of thermospermine (T-Spm) to spermidine and plays a role in plant development, xylem differentiation, and abiotic stress tolerance. In the present study, to verify whether T-Spm metabolism can be exploited as a new route to improve stress tolerance in crops and to investigate the underlying mechanisms, tomato (Solanum lycopersicum) AtPAO5 homologs were identified (SlPAO2, SlPAO3, and SlPAO4) and CRISPR/Cas9-mediated loss-of-function slpao3 mutants were obtained. Morphological, molecular, and physiological analyses showed that slpao3 mutants display increased T-Spm levels and exhibit changes in growth parameters, number and size of xylem elements, and expression levels of auxin- and gibberellin-related genes compared to wild-type plants. The slpao3 mutants are also characterized by improved tolerance to drought stress, which can be attributed to a diminished xylem hydraulic conductivity that limits water loss, as well as to a reduced vulnerability to embolism. Altogether, this study evidences conservation, though with some significant variations, of the T-Spm-mediated regulatory mechanisms controlling plant growth and differentiation across different plant species and highlights the T-Spm role in improving stress tolerance while not constraining growth.

A meta-analysis of photosynthetic efficiency and stress mitigation by melatonin in enhancing wheat tolerance.

BACKGROUND: Our meta-analysis examines the effects of melatonin on wheat under varying abiotic stress conditions, focusing on photosynthetic parameters, chlorophyll fluorescence, leaf water status, and photosynthetic pigments. We initially collected 177 publications addressing the impact of melatonin on wheat. After meticulous screening, 31 published studies were selected, encompassing 170 observations on photosynthetic parameters, 73 on chlorophyll fluorescence, 65 on leaf water status, 240 on photosynthetic pigments. RESULTS: The analysis revealed significant heterogeneity across studies (I² > 99.90%) for the aforementioned parameters and evidence of publication bias, emphasizing the complex interaction between melatonin application and plant physiological responses. Melatonin enhanced the overall response ratio (lnRR) for photosynthetic rates, stomatal conductance, transpiration rates, and fluorescence yields by 20.49, 22.39, 30.96, and 1.09%, respectively, compared to the control (no melatonin). The most notable effects were under controlled environmental conditions. Moreover, melatonin significantly improved leaf water content and reduced water potential, particularly under hydroponic conditions and varied abiotic stresses, highlighting its role in mitigating water stress. The analysis also revealed increases in chlorophyll pigments with soil drenching and foliar spray, and these were considered the effective application methods. Furthermore, melatonin influenced chlorophyll SPAD and intercellular CO2 concentrations, suggesting its capacity to optimize photosynthetic efficiency. CONCLUSIONS: This synthesis of meta-analysis confirms that melatonin significantly enhances wheat's resilience to abiotic stress by improving photosynthetic parameters, chlorophyll fluorescence, leaf water status, and photosynthetic pigments. Despite observed heterogeneity and publication bias, the consistent beneficial effects of melatonin, particularly under controlled conditions with specific application methods e.g. soil drenching and foliar spray, demonstrate its utility as a plant growth regulator for stress management. These findings encourage focused research and application strategies to maximize the benefits of melatonin in wheat farming, and thus contributing to sustainable agricultural practices.

Highly Stretchable, Knittable, Wearable Fiberform Hydrovoltaic Generators Driven by Water Transpiration for Portable Self-Power Supply and Self-Powered Strain Sensor.

The development of excellently stretchable, highly mobile, and sustainable power supplies is of great importance for self-power wearable electronics. Transpiration-driven hydrovoltaic power generator (HPG) has been demonstrated to be a promising energy harvesting strategy with the advantages of negative heat and zero-carbon emissions. Herein, this work demonstrates a fiber-based stretchable HPG with the advantages of high output, portability, knittability, and sustainable power generation. Based on the functionalized micro-nano water diffusion channels constructed by the discarded mask straps (MSs) and oxidation-treated carbon nanomaterials, the applied water can continuously produce electricity during the spontaneous flow and diffusion. Experimentally, when a tiny 0.1 mL of water encounters one end of the proposed HPG, the centimeter-length device can yield a peak voltage of 0.43 V, peak current of 29.5 µA, and energy density of 5.833 mW h cm-3. By efficiently integrating multiple power generation units, sufficient output power can be provided to drive commercial electronic devices even in the stretched state. Furthermore, due to the reversibility of the electrical output during dynamic stretching-releasing, it can passively convert physiological activities and motion behaviors into quantifiable and processable current signals, opening up HPG's application in the field of self-powered wearable sensing.

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

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

The role of thermodiffusion in transpiration.

Plant leaf temperatures can differ from ambient air temperatures. A temperature gradient in a gas mixture gives rise to a phenomenon known as thermodiffusion, which operates in addition to ordinary diffusion. Whilst transpiration is generally understood to be driven solely by the ordinary diffusion of water vapour along a concentration gradient, we consider the implications of thermodiffusion for transpiration. We develop a new modelling framework that introduces the effects of thermodiffusion on the transpiration rate, E. By applying this framework, we quantify the proportion of E attributable to thermodiffusion for a set of physiological and environmental conditions, varied over a wide range. Thermodiffusion is found to be most significant (in some cases > 30% of E) when a leaf-to-air temperature difference coincides with a relatively small water vapour concentration difference across the boundary layer; a boundary layer conductance that is large as compared to the stomatal conductance; or a relatively low transpiration rate. Thermodiffusion also alters the conditions required for the onset of reverse transpiration, and the rate at which this water vapour uptake occurs.

Dynamic soil hydraulic resistance regulates stomata.

The onset of stomatal closure reduces transpiration during drought. In seed plants, drought causes declines in plant water status which increases leaf endogenous abscisic acid (ABA) levels required for stomatal closure. There are multiple possible points of increased belowground resistance in the soil-plant atmospheric continuum that could decrease leaf water potential enough to trigger ABA production and the subsequent decreases in transpiration. We investigate the dynamic patterns of leaf ABA levels, plant hydraulic conductance and the point of failure in the soil-plant conductance in the highly embolism-resistant species Callitris tuberculata using continuous dendrometer measurements of leaf water potential during drought. We show that decreases in transpiration and ABA biosynthesis begin before any permanent decreases in predawn water potential, collapse in soil-plant hydraulic pathway and xylem embolism spread. We find that a dynamic but recoverable increases in hydraulic resistance in the soil in close proximity to the roots is the most likely driver of declines in midday leaf water potential needed for ABA biosynthesis and the onset of decreases in transpiration.

Elevated aerosol enhances plant water-use efficiency by increasing carbon uptake while reducing water loss.

Aerosols could significantly influence ecosystem carbon and water fluxes, potentially altering their interconnected dynamics, typically characterized by water-use efficiency (WUE). However, our understanding of the underlying ecophysiological mechanisms remains limited due to insufficient field observations. We conducted 4-yr measurements of leaf photosynthesis and transpiration, as well as 3-yr measurements of stem growth (SG) and sap flow of poplar trees exposed to natural aerosol fluctuation, to elucidate aerosol's impact on plant WUE. We found that aerosol improved sun leaf WUE mainly because a sharp decline in photosynthetically active radiation (PAR) inhibited its transpiration, while photosynthesis was less affected, as the negative effect induced by declined PAR was offset by the positive effect induced by low leaf vapor pressure deficit (VPDleaf). Conversely, diffuse radiation fertilization (DRF) effect stimulated shade leaf photosynthesis with minimal impact on transpiration, leading to an improved WUE. The responses were further verified by a strong DRF on SG and a decrease in sap flow due to the suppresses in total radiation and VPD. Our field observations indicate that, contrary to the commonly assumed coupling response, carbon uptake and water use exhibited dissimilar reactions to aerosol pollution, ultimately enhancing WUE at the leaf and canopy level.

Back to the future for drought tolerance.

Global agriculture faces increasing pressure to produce more food with fewer resources. Drought, exacerbated by climate change, is a major agricultural constraint costing the industry an estimated US$80 billion per year in lost production. Wild relatives of domesticated crops, including wheat (Triticum spp.) and barley (Hordeum vulgare L.), are an underutilized source of drought tolerance genes. However, managing their undesirable characteristics, assessing drought responses, and selecting lines with heritable traits remains a significant challenge. Here, we propose a novel strategy of using multi-trait selection criteria based on high-throughput spectral images to facilitate the assessment and selection challenge. The importance of measuring plant capacity for sustained carbon fixation under drought stress is explored, and an image-based transpiration efficiency (iTE) index obtained via a combination of hyperspectral and thermal imaging, is proposed. Incorporating iTE along with other drought-related variables in selection criteria will allow the identification of accessions with diverse tolerance mechanisms. A comprehensive approach that merges high-throughput phenotyping and de novo domestication is proposed for developing drought-tolerant prebreeding material and providing breeders with access to gene pools containing unexplored drought tolerance mechanisms.

Elevated [CO2] and temperature augment gas exchange and shift the fitness landscape in a montane forb.

Climate change is simultaneously increasing carbon dioxide concentrations ([CO2]) and temperature. These factors could interact to influence plant physiology and performance. Alternatively, increased [CO2] may offset costs associated with elevated temperatures. Furthermore, the interaction between elevated temperature and [CO2] may differentially affect populations from along an elevational gradient and disrupt local adaptation. We conducted a multifactorial growth chamber experiment to examine the interactive effects of temperature and [CO2] on fitness and ecophysiology of diverse accessions of Boechera stricta (Brassicaceae) sourced from a broad elevational gradient in Colorado. We tested whether increased [CO2] would enhance photosynthesis across accessions, and whether warmer conditions would depress the fitness of high-elevation accessions owing to steep reductions in temperature with increasing elevation in this system. Elevational clines in [CO2] are not as evident, making it challenging to predict how locally adapted ecotypes will respond to elevated [CO2]. This experiment revealed that elevated [CO2] increased photosynthesis and intrinsic water use efficiency across all accessions. However, these instantaneous responses to treatments did not translate to changes in fitness. Instead, increased temperatures reduced the probability of reproduction for all accessions. Elevated [CO2] and increased temperatures interacted to shift the adaptive landscape, favoring lower elevation accessions for the probability of survival and fecundity. Our results suggest that elevated temperatures and [CO2] associated with climate change could have severe negative consequences, especially for high-elevation populations.

Long-term trend and interannual variation in evapotranspiration of a young temperate Douglas-fir stand over 2002-2022 reveals the impacts of climate change.

The shortage of decades-long continuous measurements of ecosystem processes limits our understanding of how changing climate impacts forest ecosystems. We used continuous eddy-covariance and hydrometeorological data over 2002-2022 from a young Douglas-fir stand on Vancouver Island, Canada to assess the long-term trend and interannual variability in evapotranspiration (ET) and transpiration (T). Collectively, annual T displayed a decreasing trend over the 21 years with a rate of 1% yr-1, which is attributed to the stomatal downregulation induced by rising atmospheric CO2 concentration. Similarly, annual ET also showed a decreasing trend since evaporation stayed relatively constant. Variability in detrended annual ET was mostly controlled by the average soil water storage during the growing season (May-October). Though the duration and intensity of the drought did not increase, the drought-induced decreases in T and ET showed an increasing trend. This pattern may reflect the changes in forest structure, related to the decline in the deciduous understory cover during the stand development. These results suggest that the water-saving effect of stomatal regulation and water-related factors mostly determined the trend and variability in ET, respectively. This may also imply an increase in the limitation of water availability on ET in young forests, associated with the structural and compositional changes related to forest growth.

Different roads, same destination: The shared future of plant ecophysiology and ecohydrology.

Terrestrial water fluxes are substantially mediated by vegetation, while the distribution, growth, health, and mortality of plants are strongly influenced by the availability of water. These interactions, playing out across multiple spatial and temporal scales, link the disciplines of plant ecophysiology and ecohydrology. Despite this connection, the disciplines have provided complementary, but largely independent, perspectives on the soil-plant-atmosphere continuum since their crystallization as modern scientific disciplines in the late 20th century. This review traces the development of the two disciplines, from their respective origins in engineering and ecology, their largely independent growth and maturation, and the eventual development of common conceptual and quantitative frameworks. This common ground has allowed explicit coupling of the disciplines to better understand plant function. Case studies both illuminate the limitations of the disciplines working in isolation, and reveal the exciting possibilities created by consilience between the disciplines. The histories of the two disciplines suggest opportunities for new advances will arise from sharing methodologies, working across multiple levels of complexity, and leveraging new observational technologies. Practically, these exchanges can be supported by creating shared scientific spaces. This review argues that consilience and collaboration are essential for robust and evidence-based predictions and policy responses under global change.

Unsaturation in the air spaces of leaves and its implications.

Modern plant physiological theory stipulates that the resistance to water movement from plants to the atmosphere is overwhelmingly dominated by stomata. This conception necessitates a corollary assumption-that the air spaces in leaves must be nearly saturated with water vapour; that is, with a relative humidity that does not decline materially below unity. As this idea became progressively engrained in scientific discourse and textbooks over the last century, observations inconsistent with this corollary assumption were occasionally reported. Yet, evidence of unsaturation gained little traction, with acceptance of the prevailing framework motivated by three considerations: (1) leaf water potentials measured by either thermocouple psychrometry or the Scholander pressure chamber are largely consistent with the framework; (2) being able to assume near saturation of intercellular air spaces was transformational to leaf gas exchange analysis; and (3) there has been no obvious mechanism to explain a variable, liquid-phase resistance in the leaf mesophyll. Here, we review the evidence that refutes the assumption of universal, near saturation of air spaces in leaves. Refining the prevailing paradigm with respect to this assumption provides opportunities for identifying and developing mechanisms for increased plant productivity in the face of increasing evaporative demand imposed by global climate change.

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

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

Unravelling the neglected role of ultraviolet radiation on stomata: A meta-analysis with implications for modelling ecosystem-climate interactions.

Stomata play a pivotal role in regulating gas exchange between plants and the atmosphere controlling water and carbon cycles. Accordingly, we investigated the impact of ultraviolet-B radiation, a neglected environmental factor varying with ongoing global change, on stomatal morphology and function by a Comprehensive Meta-Analysis. The overall UV effect at the leaf level is to decrease stomatal conductance, stomatal aperture and stomatal size, although stomatal density was increased. The significant decline in stomatal conductance is marked (6% in trees and >10% in grasses and herbs) in short-term experiments, with more modest decreases noted in long-term UV studies. Short-term experiments in growth chambers are not representative of long-term field UV effects on stomatal conductance. Important consequences of altered stomatal function are hypothesized. In the short term, UV-mediated stomatal closure may reduce carbon uptake but also water loss through transpiration, thereby alleviating deleterious effects of drought. However, in the long term, complex changes in stomatal aperture, size, and density may reduce the carbon sequestration capacity of plants and increase vegetation and land surface temperatures, potentially exacerbating negative effects of drought and/or heatwaves. Therefore, the expected future strength of carbon sink capacity in high-UV regions is likely overestimated.

Systemic stomatal responses in plants: Coordinating development, stress, and pathogen defense under a changing climate.

To successfully survive, develop, grow and reproduce, multicellular organisms must coordinate their molecular, physiological, developmental and metabolic responses among their different cells and tissues. This process is mediated by cell-to-cell, vascular and/or volatile communication, and involves electric, chemical and/or hydraulic signals. Within this context, stomata serve a dual role by coordinating their responses to the environment with their neighbouring cells at the epidermis, but also with other stomata present on other parts of the plant. As stomata represent one of the most important conduits between the plant and its above-ground environment, as well as directly affect photosynthesis, respiration and the hydraulic status of the plant by controlling its gas and vapour exchange with the atmosphere, coordinating the overall response of stomata within and between different leaves and tissues plays a cardinal role in plant growth, development and reproduction. Here, we discuss different examples of local and systemic stomatal coordination, the different signalling pathways that mediate them, and the importance of systemic stomatal coordination to our food supply, ecosystems and weather patterns, under our changing climate. We further discuss the potential biotechnological implications of regulating systemic stomatal responses for enhancing agricultural productivity in a warmer and CO2 -rich environment.

Restricted internal diffusion weakens transpiration-photosynthesis coupling during heatwaves: Evidence from leaf carbonyl sulphide exchange.

Increasingly frequent and intense heatwaves threaten ecosystem health in a warming climate. However, plant responses to heatwaves are poorly understood. A key uncertainty concerns the intensification of transpiration when heatwaves suppress photosynthesis, known as transpiration-photosynthesis decoupling. Field observations of such decoupling are scarce, and the underlying physiological mechanisms remain elusive. Here, we use carbonyl sulphide (COS) as a leaf gas exchange tracer to examine potential mechanisms leading to transpiration-photosynthesis decoupling on a coast live oak in a southern California woodland in spring 2013. We found that heatwaves suppressed both photosynthesis and leaf COS uptake but increased transpiration or sustained it at non-heatwave levels throughout the day. Despite statistically significant decoupling between transpiration and photosynthesis, stomatal sensitivity to environmental factors did not change during heatwaves. Instead, midday photosynthesis during heatwaves was restricted by internal diffusion, as indicated by the lower internal conductance to COS. Thus, increased evaporative demand and nonstomatal limitation to photosynthesis act jointly to decouple transpiration from photosynthesis without altering stomatal sensitivity. Decoupling offered limited potential cooling benefits, questioning its effectiveness for leaf thermoregulation in xeric ecosystems. We suggest that adding COS to leaf and ecosystem flux measurements helps elucidate diverse physiological mechanisms underlying transpiration-photosynthesis decoupling.

Hyperspectral imaging reveals small-scale water gradients in apple leaves due to minimal cuticle perforation by Venturia inaequalis conidiophores.

Effects of Venturia inaequalis on water relations of apple leaves were studied under controlled conditions without limitation of water supply to elucidate their impact on the non-haustorial biotrophy of this pathogen. Leaf water relations, namely leaf water content and transpiration, were spatially resolved by hyperspectral imaging and thermography; non-imaging techniques-gravimetry, a pressure chamber, and porometry-were used for calibration and validation. Reduced stomatal transpiration 3-4 d after inoculation coincided with a transient increase of water potential. Perforation of the plant cuticle by protruding conidiophores subsequently increased cuticular transpiration even before visible symptoms occurred. With sufficient water supply, cuticular transpiration remained at elevated levels for several weeks. Infections did not affect the leaf water content before scab lesions became visible. Only hyperspectral imaging was suitable to demonstrate that a decreased leaf water content was strictly limited to sites of emerging conidiophores and that cuticle porosity increased with sporulation. Microscopy confirmed marginal cuticle injury; although perforated, it tightly surrounded the base of conidiophores throughout sporulation and restricted water loss. The role of sustained redirection of water flow to the pathogen's hyphae in the subcuticular space above epidermal cells, to facilitate the acquisition and uptake of nutrients by V. inaequalis, is discussed.

Combined leaf gas-exchange system for model assessment.

Leaf gas-exchange measurements are useful in assessing plant environmental responses. However, uncertainties in the leaf gas-exchange model potentially limit its application. The main challenge in the model-dependent calculations is to detect violations of assumptions. Here, we developed a system that integrates into one instrument the direct measurement of leaf intercellular CO2 concentration and the standard open-flow (OF) and novel open-diffusion (OD) systems for flux measurement. In the OD system, a gas-permeable membrane between the leaf ambient air and outside air creates CO2 and H2O differentials, rather than the air flow in the OF chamber. We measured hypostomatous and amphistomatous leaves of several species with different photosynthetic capacities [sunflower (Helianthus annuus), grape (Vitis vinifera), lemon (Citrus limon), and cherry (Prunus avium)]. The CO2 and H2O differentials in the OD system strictly depend on the flux measured by the OF system. The lower permeability of the membrane resulted in a larger differential per flux, indicating that the OD system can increase the resolution for a small flux. An analysis of the conductance model along with observations suggested that cuticle and leaf intercellular conductances and the unsaturation of leaf humidity contributed to discrepancies between the direct measurement and standard calculation. The combined system developed here provides an opportunity to address these overlooked concepts in leaf gas exchange.