Reconciling water-use efficiency estimates from carbon isotope discrimination of leaf biomass and tree rings: nonphotosynthetic fractionation matters.

Carbon isotope discrimination (∆) in leaf biomass (∆BL) and tree rings (∆TR) provides important proxies for plant responses to climate change, specifically in terms of intrinsic water-use efficiency (iWUE). However, the nonphotosynthetic 12C/13C fractionation in plant tissues has rarely been quantified and its influence on iWUE estimation remains uncertain. We derived a comprehensive, ∆ based iWUE model (iWUEcom) which includes nonphotosynthetic fractionations (d) and characterized tissue-specific d-values based on global compilations of data of ∆BL, ∆TR and real-time ∆ in leaf photosynthesis (∆online). iWUEcom was further validated with independent datasets. ∆BL was larger than ∆online by 2.53‰, while ∆BL and ∆TR showed a mean offset of 2.76‰, indicating that ∆TR is quantitatively very similar to ∆online. Applying the tissue-specific d-values (dBL = 2.5‰, dTR = 0‰), iWUE estimated from ∆BL aligned well with those estimated from ∆TR or gas exchange. ∆BL and ∆TR showed a consistent iWUE trend with an average CO2 sensitivity of 0.15 ppm ppm-1 during 1975-2015. Accounting for nonphotosynthetic fractionations improves the estimation of iWUE based on isotope records in leaf biomass and tree rings, which is ultimate for inferring changes in carbon and water cycles under historical and future climate.

The intricacies of vegetation responses to changing moisture conditions.

A long-standing debate looks at whether air or soil dryness is more limiting to vegetation water use and productivity. The answer has large implications for future ecosystem functioning, as atmospheric dryness is predicted to increase globally while changes in soil moisture are predicted to be far more variable. Here, I review the complexities that contribute to this debate, including the strong coupling between atmospheric and soil dryness, and the widespread heterogeneity in vegetation hydraulic traits, acclimations, and adaptations to water stress. I discuss solutions to improve understanding and modeling of vegetation sensitivity to dryness, including how different types of observational data can be used together to gain insight into vegetation response to water stress across spatial and temporal scales.

High water use efficiency due to maintenance of photosynthetic capacity in sorghum under water stress.

Environmental change requires more crop production per water use to meet the rising global food demands. However, improving crop intrinsic water use efficiency (iWUE) usually comes at the expense of carbon assimilation. Sorghum is a key crop in many vulnerable agricultural systems with higher tolerance to water stress (WS) than most widely planted crops. To investigate physiological controls on iWUE and its inheritance in sorghum we screened 89 genotypes selected based on inherited haplotypes from an elite or five exotics lines, containing a mix of geographical origins and dry vs. milder climates, which included different aquaporin (AQP) alleles. We found significant variation among key highly heritable gas exchange and hydraulic traits, with some being significantly affected by variation in haplotypes among parental lines. Plants with a higher proportion of the non-stomatal component of iWUE still maintained iWUE under WS by maintaining photosynthetic capacity, independently of reduction in leaf hydraulic conductance. Haplotypes associated with two AQPs (SbPIP1.1 and SbTIP3.2) influenced iWUE and related traits. These findings expand the range of traits that bridge the trade-off between iWUE and productivity in C4 crops, and provide possible genetic regions that can be targeted for breeding.

Transcriptomic dynamics of ABA response in Brassica napus guard cells.

Drought has a significant, negative impact on crop production; and these effects are poised to increase with climate change. Plants acclimate to drought and water stress through diverse physiological responses, primarily mediated by the hormone abscisic acid (ABA). Because plants lose the majority of their water through stomatal pores on aerial surfaces of plants, stomatal closure is one of the rapid responses mediated by ABA to reduce transpirational water loss. The dynamic changes in the transcriptome of stomatal guard cells in response to ABA have been investigated in the model plant Arabidopsis thaliana. However, guard cell transcriptomes have not been analyzed in agronomically valuable crops such as a major oilseed crop, rapeseed. In this study, we investigated the dynamics of ABA-regulated transcriptomes in stomatal guard cells of Brassica napus and conducted comparison analysis with the transcriptomes of A. thaliana. We discovered changes in gene expression indicating alterations in a host of physiological processes, including stomatal movement, metabolic reprogramming, and light responses. Our results suggest the existence of both immediate and delayed responses to ABA in Brassica guard cells. Furthermore, the transcription factors and regulatory networks mediating these responses are compared to those identified in Arabidopsis. Our results imply the continuing evolution of ABA responses in Brassica since its divergence from a common ancestor, involving both protein-coding and non-coding nucleotide sequences. Together, our results will provide a basis for developing strategies for molecular manipulation of drought tolerance in crop plants.

Molecular Mechanisms for Regulating Stomatal Formation across Diverse Plant Species.

Plant stomata play a crucial role in photosynthesis by regulating transpiration and gas exchange. Meanwhile, environmental cues can also affect the formation of stomata. Stomatal formation, therefore, is optimized for the survival and growth of the plant despite variable environmental conditions. To adapt to environmental conditions, plants open and close stomatal pores and even regulate the number of stomata that develop on the epidermis. There are great differences in the leaf structure and developmental origin of the cell in the leaf between Arabidopsis and grass plants. These differences affect the fine regulation of stomatal formation due to different plant species. In this paper, a comprehensive overview of stomatal formation and the molecular networks and genetic mechanisms regulating the polar division and cell fate of stomatal progenitor cells in dicotyledonous plants such as Arabidopsis and Poaceae plants such as Oryza sativa and Zea mays is provided. The processes of stomatal formation mediated by plant hormones and environmental factors are summarized, and a model of stomatal formation in plants based on the regulation of multiple signaling pathways is outlined. These results contribute to a better understanding of the mechanisms of stomatal formation and epidermal morphogenesis in plants and provide a valuable theoretical basis and gene resources for improving crop resilience and yield traits.

StEPF2 and StEPFL9 Play Opposing Roles in Regulating Stomatal Development and Drought Tolerance in Potato (Solanum tuberosum L.).

Stomata are essential for photosynthesis and water-use efficiency in plants. When expressed in transgenic Arabidopsis thaliana plants, the potato (Solanum tuberosum) proteins EPIDERMAL PATTERNING FACTOR 2 (StEPF2) and StEPF-LIKE9 (StEPFL9) play antagonistic roles in regulating stomatal density. Little is known, however, about how these proteins regulate stomatal development, growth, and response to water deficit in potato. Transgenic potato plants overexpressing StEPF2 (E2 plants) or StEPFL9 (ST plants) were generated, and RT-PCR and Western blot analyses were used to select two lines overexpressing each gene. E2 plants showed reduced stomatal density, whereas ST plants produced excessive stomata. Under well-watered conditions, ST plants displayed vigorous growth with improved leaf gas exchange and also showed increased biomass/yields compared with non-transgenic and E2 plants. E2 plants maintained lower H2O2 content and higher levels of stomatal conductance and photosynthetic capacity than non-transgenic and ST plants, which resulted in higher water-use efficiency and biomass/yields during water restriction. These results suggest that StEPF2 and StEPFL9 functioned in pathways regulating stomatal development. These genes are thus promising candidates for use in future breeding programs aimed at increasing potato water-use efficiency and yield under climate change scenarios.

Effects of Salicylic Acid on Physiological Responses of Pepper Plants Pre-Subjected to Drought under Rehydration Conditions.

Capsicum annuum L. has worldwide distribution, but drought has limited its production. There is a lack of research to better understand how this species copes with drought stress, whether it is reversible, and the effects of mitigating agents such as salicylic acid (SA). Therefore, this study aimed to understand the mechanisms of action of SA and rehydration on the physiology of pepper plants grown under drought conditions. The factorial scheme adopted was 3 × 4, with three water regimes (irrigation, drought, and rehydration) and four SA concentrations, namely: 0 (control), 0.5, 1, and 1.5 mM. This study evaluated leaf water percentage, water potential of shoots, chlorophylls (a and b), carotenoids, stomatal conductance, chlorophyll a fluorescence, and hydrogen peroxide (H2O2) concentration at different times of day, water conditions (irrigation, drought, and rehydration), and SA applications (without the addition of a regulator (0) and with the addition of SA at concentrations equal to 0.5, 1, and 1.5 mM). In general, exogenous SA application increased stomatal conductance (gs) responses and modified the fluorescence parameters (ΦPSII, qP, ETR, NPQ, D, and E) of sweet pepper plants subjected to drought followed by rehydration. It was found that the use of SA, especially at concentrations of 1 mM in combination with rehydration, modulates gs, which is reflected in a higher electron transport rate. This, along with the production of photosynthetic pigments, suggests that H2O2 did not cause membrane damage, thereby mitigating the water deficit in pepper plants. Plants under drought conditions and rehydration with foliar SA application at concentrations of 1 mM demonstrated protection against damage resulting from water stress. Focusing on sustainable productivity, foliar SA application of 1 mM could be recommended as a technique to overcome the adverse effects of water stress on pepper plants cultivated in arid and semi-arid regions.

Genome-Wide Identification of the Phospholipase D (PLD) Gene Family in Chinese White Pear (Pyrus bretschneideri) and the Role of PbrPLD2 in Drought Resistance.

The Chinese white pear (Pyrus bretschneideri), a vital fruit crop, is highly susceptible to abiotic stresses, especially drought, which poses a major threat to its growth and productivity. Phospholipase D (PLD) genes are pivotal in orchestrating plant responses to abiotic stresses, acting as key regulators in stress adaptation mechanisms. This study systematically identified and functionally characterized the entire PLD gene family in P. bretschneideri through a comprehensive genome-wide analysis. A total of 20 PbrPLD genes were identified, and they were categorized into five subfamilies based on phylogenetic analysis. chromosome localization, gene structure, and conserved motif analyses revealed that these genes have diverse evolutionary histories. Cis-acting element analysis and expression profiling under drought stress indicated that several PbrPLD genes, particularly PbrPLD2, are strongly induced by drought. Overexpression of PbrPLD2 in both Arabidopsis thaliana and pear demonstrated enhanced drought tolerance through improved stomatal closure and increased expression of drought-responsive genes. These findings highlight the critical role of PbrPLD2 in drought resistance and provide a theoretical and experimental foundation for molecular breeding in pear and other fruit crops.

Exogenous application of serotonin, with the modulation of redox homeostasis and photosynthetic characteristics, enhances the drought resistance of the saffron plant.

Water stress is one of the most significant abiotic stresses that disrupts the osmotic balance of plants and consequently reduces their growth and performance. In recent years, it has been found that serotonin, as a signaling and regulatory molecule, can play important roles in the growth and development of plants and enhance their tolerance to abiotic stresses. Saffron is a plant known for its medicinal and culinary properties. Its distinct flavor, aroma, and vibrant color make it a sought-after ingredient in various cuisines and traditional medicines. The aim of this study is to investigate the possible effect of serotonin growth regulator on some morphophysiological and biochemical characteristics of saffron plant under water stress conditions. Water stress was applied using polyethylene glycol 6000 at a level of 30%, w/v. Serotonin was also applied exogenously at a concentration of 100 µM in both foliar and root applications. The experimental findings demonstrated that water stress had a detrimental impact on various growth and photosynthetic parameters including FW, DW, SH, RWC, photosynthetic pigments content, Pn, Fv/Fm, C and Ci. Under these conditions, H2O2 content and ion leakage increased. The increase in the content of proline and sugars also confirmed that the saffron plant was placed in unfavorable growth conditions. Serotonin application in both foliar and root applications and especially root treatment under stressful conditions improved plant growth by activating enzymatic and non-enzymatic antioxidant systems. Overall, the exogenous application of serotonin increased the resistance of saffron plants to water stress.

Machine learning-enabled computer vision for plant phenotyping: a primer on AI/ML and case study on stomatal patterning.

Artificial intelligence and machine learning (AI/ML) can be used to automatically analyze large image datasets. One valuable application of this approach is estimation of plant trait data contained within images. Here we review 39 papers that describe the development and/or application of such models for estimation of stomatal traits from epidermal micrographs. In doing so, we hope to provide plant biologists with a foundational understanding of AI/ML and summarize the current capabilities and limitations of published tools. While most models show human-level performance for stomatal density (SD) quantification at superhuman speed, they are often likely to be limited in how broadly they can be applied across phenotypic diversity associated with genetic, environmental or developmental variation. Other models can make predictions across greater phenotypic diversity and/or additional stomatal/epidermal traits, but require significantly greater time investment to generate ground-truth data. We discuss the challenges and opportunities presented by AI/ML-enabled computer vision analysis, and make recommendations for future work to advance accelerated stomatal phenotyping.

Intensive leaf cooling promotes tree survival during a record heatwave.

Increasing heatwaves are threatening forest ecosystems globally. Leaf thermal regulation and tolerance are important for plant survival during heatwaves, though the interaction between these processes and water availability is unclear. Genotypes of the widely distributed foundation tree species Populus fremontii were studied in a controlled common garden during a record summer heatwave-where air temperature exceeded 48 °C. When water was not limiting, all genotypes cooled leaves 2 to 5 °C below air temperatures. Homeothermic cooling was disrupted for weeks following a 72-h reduction in soil water, resulting in leaf temperatures rising 3 °C above air temperature and 1.3 °C above leaf thresholds for physiological damage, despite the water stress having little effect on leaf water potentials. Tradeoffs between leaf thermal safety and hydraulic safety emerged but, regardless of water use strategy, all genotypes experienced significant leaf mortality following water stress. Genotypes from warmer climates showed greater leaf cooling and less leaf mortality after water stress in comparison with genotypes from cooler climates. These results illustrate how brief soil water limitation disrupts leaf thermal regulation and potentially compromises plant survival during extreme heatwaves, thus providing insight into future scenarios in which ecosystems will be challenged with extreme heat and unreliable soil water access.

Overexpression of StERECTA enhances drought tolerance in Arabidopsis thaliana.

Drought is a major abiotic stresses that severely hinder plant growth and agricultural productivity. The receptor-like kinase gene, ERECTA, has been proved to play important role in promoting the response to abiotic stress in crops. However, the specific molecular mechanisms underlying the drought resistance mediated by ERECTA in potato (Solanum tuberosum L.) are not well understood. In this study, sequence analysis confirmed that the StERECTA gene contains eight leucine-rich repeat (LRR) domains and an S_TKc domain, and these domains were highly conserved in Solanaceae family. Under drought stress, Arabidopsis thaliana strains overexpressing StERECTA showed increased biomass, proline (PRO) content, and antioxidant enzyme activities compared to the wild-type strains while the mutant ERECTA strain (er105) exhibited opposite phenotype. Additionally, StERECTA overexpression upregulated the expression of drought response marker genes (LEA3, DREB2A and P5CS1), improved levels of ABA and auxin, reduced stomatal density and relative expression level of stomatal development related genes (SPCH, FAMA and MUTE). Furthermore, Co-immunoprecipitation (Co-IP) assays demonstrated that StERECTA physically interacted with the YODA protein. In conclusion, our study provides new insights into the role and regulatory mechanism of StERECTA in response to drought stress. These findings may serve as a basis for genetic improvement of potato to enhance their tolerance to abiotic stress.

Stomata Are Driving the Direction of CO2-Induced Water-Use Efficiency Gain in Selected Tropical Trees in Fiji.

Understanding plant physiological response to a rising atmospheric CO2 concentration (ca) is key in predicting Earth system plant-climate feedbacks; however, the effects of long-term rising ca on plant gas-exchange characteristics in the tropics are largely unknown. Studying this long-term trend using herbarium records is challenging due to specimen trait variation. We assessed the impact of a ca rise of ~95 ppm (1927-2015) on the intrinsic water-use efficiency (iWUE) and maximum stomatal conductance (gsmax) of five tropical tree species in Fiji using the isotopic composition and stomatal traits of herbarium leaves. Empirical results were compared with simulated values using models that uniquely incorporated the variation in the empirical gsmax responses and species-specific parameterisation. The magnitude of the empirical iWUE and gsmax response was species-specific, ranging from strong to negligible. Stomatal density was more influential than the pore size in determining the gsmax response to ca. While our simulation results indicated that photosynthesis is the main factor contributing to the iWUE gain, stomata were driving the iWUE trend across the tree species. Generally, a stronger increase in the iWUE was accompanied by a stronger decline in stomatal response. This study demonstrates that the incorporation of variation in the gsmax in simulations is necessary for assessing an individual species' iWUE response to changing ca.

Synergistic Interaction Between PbbZIP88 and PbSRK2E Enhances Drought Resistance in Pear Through Regulation of PbATL18 Expression and Stomatal Closure.

Drought poses significant challenges to agricultural production, ecological stability and global food security. While wild pear trees exhibit strong drought resistance, cultivated varieties show weaker drought tolerance. This study aims to elucidate the molecular mechanisms underlying pear trees' response to drought stress. We identified a drought resistance-related transcription factor, PbbZIP88, which binds to and activates the expression of the drought-responsive gene PbATL18. Overexpression of PbbZIP88 in Arabidopsis and pear seedlings resulted in enhanced drought resistance and significantly improved physiological parameters under drought stress. We discovered that PbbZIP88 interacts with the key protein PbSRK2E in the ABA signalling pathway. This interaction enhances PbbZIP88's ability to activate PbATL18 expression, leading to higher levels of PbATL18. Furthermore, the PbbZIP88 and PbSRK2E interaction accelerates the regulation of stomatal closure under ABA treatment conditions, reducing water loss more effectively. Experimental evidence showed that silencing PbbZIP88 and PbSRK2E genes significantly decreased drought resistance in pear seedlings. In conclusion, this study reveals the synergistic role of PbbZIP88 and PbSRK2E in enhancing drought resistance in pear trees, particularly in the upregulation of PbATL18 expression, and the accelerated promotion of stomatal closure. These findings provide new candidate genes for breeding drought-resistant varieties and offer a theoretical foundation and technical support for achieving sustainable agriculture.

The Transcription Factor ZmMYB56 Regulates High CO2-Induced Stomatal Closure in Maize Seedlings by Regulating ZmHLT1 Expression.

Stomata are channels through which plants exchange H2O and CO2 with the external environment. The regulation of stomatal movement has significant impacts on plant growth, development and stress adaptation. Here, we present a maize R2R3 -MYB transcription factor, ZmMYB56, which regulates high CO2-induced stomatal closure in maize seedlings. ZmMYB56 is highly expressed in stomatal guard cells and is negatively regulated by CO2 and HCO3 -. Loss of ZmMYB56 function leads to insensitivity to high CO2. As a transcription factor, ZmMYB56 binds to a cis-acting element in the ZmHLT1 promoter and regulates its expression. ZmHLT1 plays a key role in CO2- induced maize stomatal movement, and its absence causes a severe weakening of maize's response to ambient CO2. ZmHLT1 expression is negatively regulated by bicarbonate, which does not occur in Zmmyb56 mutants, highlighting the significance of this regulatory relationship in plant responses to CO2 and HCO3 -. Taken together, these results show that ZmMYB56-regulated ZmHLT1 expression is important for high CO2-induced stomatal closure in maize. Our findings provide insight into genetic pathways that could be manipulated to improve maize growth and stress tolerance, especially under increasing atmospheric CO2 concentrations.

The Small Auxin-Up RNA 50 (SAUR50) Gene from Ammopiptanthus nanus Negatively Regulates Drought Tolerance.

Drought stress is a primary abiotic stress that causes significant losses to forestry and agricultural production. Therefore, exploring drought-responsive genes and their regulatory mechanism is crucial for plant molecular breeding for forestry and agriculture production safety. Small auxin-up RNA (SAUR) proteins are essential in plant growth and development but show functional diversity in stress response. In this study, the transcriptome sequencing data of Ammopiptanthus nanus seedlings revealed that the expression of AnSAUR50 was continuously downregulated under drought stress. Hence, the AnSAUR50 gene was cloned and functionally analyzed in drought response. The results showed that the coding sequence of AnSAUR50 was 315 bp in length and encoded 104 amino acids. The AnSAUR50 protein showed high conservation, possessed a SAUR-specific domain, and localized in the nucleus and cell membrane. The heterologous expression of the AnSAUR50 gene enhanced the drought sensitivity of the transgenic Arabidopsis with a lower survival rate, biomass, and higher malondialdehyde content and relative electrolyte leakage. Moreover, transgenic plants showed shorter root lengths and bigger stomatal apertures, resulting in facilitating water loss under drought stress. The study indicates that AnSAUR50 negatively regulates drought tolerance by inhibiting root growth and stomatal closure, which provides insights into the underlying function and regulatory mechanism of SAURs in plant stress response.

Contrasting regulation of leaf gas exchange of semi-arid tree species under repeated drought.

Predicting how plants respond to drought requires an understanding of how physiological mechanisms and drought response strategies occur, as these strategies underlie rates of gas exchange and productivity. We assessed the response of eleven plant traits to repeated experimental droughts in four co-occurring species of central Australia. The main goals of this study were to i) compare the response to drought between species; ii) evaluate whether plants acclimated to repeated drought; and iii) examine the degree of recovery in leaf gas exchange after cessation of drought. Our four species of study were two tree species and two shrub species, which field studies have shown to occupy different ecohydrological niches. The two tree species (Eucalyptus camaldulensis and Corymbia opaca) had large reductions in stomatal conductance (gs) values, declining by 90% in the second drought. By contrast, the shrub species (A. aptaneura and H. macrocarpa) had smaller reductions gs in the second drought of 52 and 65% respectively. Only, A. aptaneura showed a physiological acclimatation to drought due to small declines in gs vs á´ªpd (0.08 slope) during repeated droughts, meaning they maintained higher rates of gs compared with plants that only experienced one final drought (0.19 slope). All species in all treatments rapidly recovered leaf gas exchange and leaf mass per area following drought, displaying physiological plasticity to drought exposure. This research refines our understanding of plant physiological responses to recurrent water stress, which has implications for modelling of vegetation, carbon assimilation and water-use in semi-arid environments under drought.

Different model assumptions about plant hydraulics and photosynthetic temperature acclimation yield diverging implications for tropical forest gross primary production under warming.

Tropical forest photosynthesis can decline at high temperatures due to (1) biochemical responses to increasing temperature and (2) stomatal responses to increasing vapor pressure deficit (VPD), which is associated with increasing temperature. It is challenging to disentangle the influence of these two mechanisms on photosynthesis in observations, because temperature and VPD are tightly correlated in tropical forests. Nonetheless, quantifying the relative strength of these two mechanisms is essential for understanding how tropical gross primary production (GPP) will respond to climate change, because increasing atmospheric CO2 concentration may partially offset VPD-driven stomatal responses, but is not expected to mitigate the effects of temperature-driven biochemical responses. We used two terrestrial biosphere models to quantify how physiological process assumptions (photosynthetic temperature acclimation and plant hydraulic stress) and functional traits (e.g., maximum xylem conductivity) influence the relative strength of modeled temperature versus VPD effects on light-saturated GPP at an Amazonian forest site, a seasonally dry tropical forest site, and an experimental tropical forest mesocosm. By simulating idealized climate change scenarios, we quantified the divergence in GPP predictions under model configurations with stronger VPD effects compared with stronger direct temperature effects. Assumptions consistent with stronger direct temperature effects resulted in larger GPP declines under warming, while assumptions consistent with stronger VPD effects resulted in more resilient GPP under warming. Our findings underscore the importance of quantifying the role of direct temperature and indirect VPD effects for projecting the resilience of tropical forests in the future, and demonstrate that the relative strength of temperature versus VPD effects in models is highly sensitive to plant functional parameters and structural assumptions about photosynthetic temperature acclimation and plant hydraulics.

Growing at the arid edge: Anatomical variations in leaves are more extensive than in stems of five Mediterranean species across contrasting moisture regimes.

PREMISE: Increasing aridity in the Mediterranean region affects ecosystems and plant life. Various anatomical changes in plants help them cope with dry conditions. This study focused on anatomical differences in leaves and xylem of five co-occurring Mediterranean plant species namely Quercus calliprinos, Pistacia palaestina, Pistacia lentiscus, Rhamnus lycioides, and Phillyrea latifolia in wet and dry sites. METHODS: Stomatal density, stomatal length, leaf mass area, lamina composition, percentage of intercellular air spaces, and mesophyll cell area in leaves of plants in wet and dry sites were analyzed. Xylem anatomy was assessed through vessel length and area in branches. RESULTS: In the dry site, three species had increased stomatal density and decreased stomatal length. Four species had increased palisade mesophyll and reduced air space volume. In contrast, phenotypic changes in the xylem were less pronounced; vessel length was unaffected by site conditions, but vessel diameter decreased in two species. Intercellular air spaces proved to be the most dynamic anatomical feature. Quercus calliprinos had the most extensive anatomical changes; Rhamnus lycioides had only minor changes. All these changes were observed in comparison to the species in the wet site. CONCLUSIONS: This study elucidated variations in anatomical responses in leaves among co-occurring Mediterranean plant species and identified the most dynamic traits. Understanding these adaptations provides valuable insights into the ability of plants to thrive under changing climate conditions.

Shorting the metaphorical circuit: vascular partitioning and stomatal patchiness can create apparent unsaturation and CO2 gradient inversion in the Ohmic analogy for leaf gas exchange.

Analyses of leaf gas exchange rely on an Ohmic analogy that arrays single stomatal, internal air space, and mesophyll conductances in series. Such models underlie inferences of mesophyll conductance and the relative humidity of leaf airspaces, reported to fall as low as 80%. An unresolved question is whether such series models are biased with respect to real leaves, whose internal air spaces are chambered at various scales by vasculature. To test whether unsaturation could emerge from modeling artifacts, we compared series model estimates with true parameter values for a chambered leaf with varying distributions and magnitudes of leaf surface conductance ('patchiness'). Distributions of surface conductance can create large biases in gas exchange calculations. Both apparent unsaturation and internal CO2 gradient inversion can be produced by the evolution of broader distributions of stomatal apertures consistent with a decrease in surface conductance, as might occur under increasing vapor pressure deficit. In gas exchange experiments, the behaviors of derived quantities defined by simple series models are highly sensitive to the true partitioning of flux and stomatal apertures across leaf surfaces. New methods are needed to disentangle model artifacts from real biological responses.