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  1. Abstract Rising atmospheric CO2levels place terrestrial ecosystems under novel environmental conditions, and research in field settings is key to understanding how real plant communities will respond. Despite decades of progress in elevated CO2(eCO2) experiments, major gaps persist in our knowledge of plant responses to interacting influences of climate change, especially in areas outside North America and Western Europe.With a goal to expand access to field‐based eCO2research, we designed, built, and tested TinyCO2, a low‐cost field experiment for climate change research on plants. TinyCO2features sixteen 0.62‐m2plot areas, half with ambient and half with elevated (+200 ppm) CO2concentrations, and is suitable for short‐stature plants (≤0.5 m in height).Using a proportional‐integral control algorithm and constant sampling of air within the plots, TinyCO2achieves consistent elevation of [CO2] averaging +196.9 ppm. During testing, 95.1% of measured CO2concentrations fell within 20% of the setpoint (ambient CO2 + 200 ppm). A streamlined design and efficient use of instrumentation reduced the cost of the system to roughly one‐fifth of the cost of similar experiments from the past 30 years ($13.68 vs. $64.65 ppm−1 m−2, adjusted to 2024 USD).Our results demonstrate a system capable of precise and accurate field‐based CO2elevation for significantly reduced cost. We envision the TinyCO2design being implemented in a multitude of field‐based eCO2studies, perhaps as part of a globally distributed collaborative network experiment. 
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  2. Summary Mature leaf area (LA) is a showcase of diversity – varying enormously within and across species, and associated with the productivity and distribution of plants and ecosystems. Yet, it remains unclear how developmental processes determine variation in LA.We introduce a mathematical framework pinpointing the origin of variation in LA by quantifying six epidermal ‘developmental traits’: initial mean cell size and number (approximating values within the leaf primordium), and the maximum relative rates and durations of cell proliferation and expansion until leaf maturity. We analyzed a novel database of developmental trajectories of LA and epidermal anatomy, representing 12 eudicotyledonous species and 52 Arabidopsis experiments.Within and across species, mean primordium cell number and maximum relative cell proliferation rate were the strongest developmental determinants of LA. Trade‐offs between developmental traits, consistent with evolutionary and metabolic scaling theory, strongly constrain LA variation. These include trade‐offs between primordium cell number vs cell proliferation, primordium mean cell size vs cell expansion, and the durations vs maximum relative rates of cell proliferation and expansion. Mutant and wild‐type comparisons showed these trade‐offs have a genetic basis in Arabidopsis.Analyses of developmental traits underlying LA and its diversification highlight mechanisms for leaf evolution, and opportunities for breeding trait shifts. 
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  3. ABSTRACT Identifying the physiological mechanisms by which plants are adapted to drought is critical to predict species responses to climate change. We measured the responses of leaf hydraulic and stomatal conductances (Kleafandgs, respectively) to dehydration, and their association with anatomy, in seven species of CaliforniaCeanothusgrown in a common garden, including some of the most drought‐tolerant species in the semi‐arid flora. We tested for matching of maximum hydraulic supply and demand and quantified the role of decline ofKleafin driving stomatal closure. AcrossCeanothusspecies, maximumKleafandgswere negatively correlated, and bothKleafandgsshowed steep declines with decreasing leaf water potential (i.e., a high sensitivity to dehydration). The leaf water potential at 50% decline ingswas linked with a low ratio of maximum hydraulic supply to demand (i.e., maximumKleaf:gs). This sensitivity ofgs, combined with low minimum epidermal conductance and water storage, could contribute to prolonged leaf survival under drought. The specialized anatomy of subg.Cerastesincludes trichomous stomatal crypts and pronounced hypodermis, and was associated with higher water use efficiency and water storage. Combining our data with comparative literature of other California species, species of subg. Cerastesshow traits associated with greater drought tolerance and reliance on leaf water storage relative to other California species. In addition to drought resistance mechanisms such as mechanical protection and resistance to embolism, drought avoidance mechanisms such as sensitive stomatal closure could contribute importantly to drought tolerance in dry‐climate adapted species. 
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  4. Abstract Changes in leaf temperature are known to drive stomatal responses, because the leaf‐to‐air water vapour gradient (Δw) increases with temperature if ambient vapour pressure is held constant, and stomata respond to changes in Δw. However, the direct response of stomata to temperature (DRST; the response when Δwis held constant by adjusting ambient humidity) has been examined far less extensively. Though the meagre available data suggest the response is usually positive, results differ widely and defy broad generalisation. As a result, little is known about the DRST. This review discusses the current state of knowledge about the DRST, including numerous hypothesised biophysical mechanisms, potential implications of the response for plant adaptation, and possible impacts of the DRST on plant‐atmosphere carbon and water exchange in a changing climate. 
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  5. Abstract Plants differ widely in how soil drying affects stomatal conductance (gs) and leaf water potential (ψleaf), and in the underlying physiological controls. Efforts to breed crops for drought resilience would benefit from a better understanding of these mechanisms and their diversity. We grew 12 diverse genotypes of common bean (Phaseolus vulgarisL.) and four of tepary bean (P. acutifolius;a highly drought resilient species) in the field under irrigation and post‐flowering drought, and quantified responses ofgsandψleaf, and their controls (soil water potential [ψsoil], evaporative demand [Δw] and plant hydraulic conductance [K]). We hypothesised that (i) common beans would be more “isohydric” (i.e., exhibit strong stomatal closure in drought, minimisingψleafdecline) than tepary beans, and that genotypes with largerψleafdecline (more “anisohydric”) would exhibit (ii) smaller increases in Δw, due to less suppression of evaporative cooling by stomatal closure and hence less canopy warming, but (iii) largerKdeclines due toψleafdecline. Contrary to our hypotheses, we found that half of the common bean genotypes were similarly anisohydric to most tepary beans; canopy temperature was cooler in isohydric genotypes leading to smaller increases in Δwin drought; and that stomatal closure andKdecline were similar in isohydric and anisohydric genotypes.gsandψleafwere virtually insensitive to drought in one tepary genotype (G40068). Our results highlight the potential importance of non‐stomatal mechanisms for leaf cooling, and the variability in drought resilience traits among closely related crop legumes. 
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  6. Abstract PremiseThe adaptive significance of amphistomy (stomata on both upper and lower leaf surfaces) is unresolved. A widespread association between amphistomy and open, sunny habitats suggests the adaptive benefit of amphistomy may be greatest in these contexts, but this hypothesis has not been tested experimentally. Understanding amphistomy informs its potential as a target for crop improvement and paleoenvironment reconstruction. MethodsWe developed a method to quantify “amphistomy advantage” () as the log‐ratio of photosynthesis in an amphistomatous leaf to that of the same leaf but with gas exchange blocked through the upper surface (pseudohypostomy). Humidity modulated stomatal conductance and thus enabled comparing photosynthesis at the same total stomatal conductance. We estimated and leaf traits in six coastal (open, sunny) and six montane (closed, shaded) populations of the indigenous Hawaiian species ʻilima (Sida fallax). ResultsCoastal ʻilima leaves benefit 4.04 times more from amphistomy than montane leaves. Evidence was equivocal with respect to two hypotheses: (1) that coastal leaves benefit more because they are thicker and have lower CO2conductance through the internal airspace and (2) that they benefit more because they have similar conductance on each surface, as opposed to most conductance being through the lower surface. ConclusionsThis is the first direct experimental evidence that amphistomy increases photosynthesis, consistent with the hypothesis that parallel pathways through upper and lower mesophyll increase CO2supply to chloroplasts. The prevalence of amphistomatous leaves in open, sunny habitats can partially be explained by the increased benefit of amphistomy in “sun” leaves, but the mechanistic basis remains uncertain. 
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  7. Salter, William (Ed.)
    Abstract Photosynthesis is co-limited by multiple factors depending on the plant and its environment. These include biochemical rate limitations, internal and external water potentials, temperature, irradiance and carbon dioxide ( CO2). Amphistomatous leaves have stomata on both abaxial and adaxial leaf surfaces. This feature is considered an adaptation to alleviate CO2 diffusion limitations in productive environments as the diffusion path length from stomate to chloroplast is effectively halved in amphistomatous leaves. Plants may also reduce CO2 limitations through other aspects of optimal stomatal anatomy: stomatal density, distribution, patterning and size. Some studies have demonstrated that stomata are overdispersed compared to a random distribution on a single leaf surface; however, despite their prevalence in nature and near ubiquity among crop species, much less is known about stomatal anatomy in amphistomatous leaves, especially the coordination between leaf surfaces. Here, we use novel spatial statistics based on simulations and photosynthesis modelling to test hypotheses about how amphistomatous plants may optimize CO2 diffusion in the model angiosperm Arabidopsis thaliana grown in different light environments. We find that (i) stomata are overdispersed, but not ideally dispersed, on both leaf surfaces across all light treatments; (ii) the patterning of stomata on abaxial and adaxial leaf surfaces is independent and (iii) the theoretical improvements to photosynthesis from abaxial–adaxial stomatal coordination are miniscule (≪1%) across the range of feasible parameter space. However, we also find that (iv) stomatal size is correlated with the mesophyll volume that it supplies with CO2, suggesting that plants may optimize CO2 diffusion limitations through alternative pathways other than ideal, uniform stomatal spacing. We discuss the developmental, physical and evolutionary constraints that may prohibit plants from reaching this theoretical adaptive peak of uniform stomatal spacing and inter-surface stomatal coordination. These findings contribute to our understanding of variation in the anatomy of amphistomatous leaves. 
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  8. Abstract Stomata are the gatekeepers of plant water use and must quickly respond to changes in plant water status to ensure plant survival under fluctuating environmental conditions. The mechanism for their closure is highly sensitive to disturbances in leaf water status, which makes isolating their response to declining water content difficult to characterise and to compare responses among species. Using a small‐scale non‐destructive nuclear magnetic resonance spectrometer as a leaf water content sensor, we measure the stomatal response to rapid induction of water deficit in the leaves of nine species of eucalypt from contrasting climates. We found a strong linear correlation between relative water content at 50% stomatal conductance (RWCgs50) and mean annual temperature at the climate of origin of each species. We also show evidence for stomata to maintain control over water loss well below turgor loss point in species adapted to warmer climates and secondary increases in stomatal conductance despite declining water content. We propose that RWCgs50is a promising trait to guide future investigations comparing stomatal responses to water deficit. It may provide a useful phenotyping trait to delineate tolerance and adaption to hot temperatures and high leaf‐to‐air vapour pressure deficits. 
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  9. Abstract Leaf surface conductance to water vapor and CO2 across the epidermis (gleaf) strongly determines the rates of gas exchange. Thus, clarifying the drivers of gleaf has important implications for resolving the mechanisms of photosynthetic productivity and leaf and plant responses and tolerance to drought. It is well recognized that gleaf is a function of the conductances of the stomata (gs) and of the epidermis + cuticle (gec). Yet, controversies have arisen around the relative roles of stomatal density (d) and size (s), fractional stomatal opening (α; aperture relative to maximum), and gec in determining gleaf. Resolving the importance of these drivers is critical across the range of leaf surface conductances, from strong stomatal closure under drought (gleaf,min), to typical opening for photosynthesis (gleaf,op), to maximum achievable opening (gleaf,max). We derived equations and analyzed a compiled database of published and measured data for approximately 200 species and genotypes. On average, within and across species, higher gleaf,min was determined 10 times more strongly by α and gec than by d and negligibly by s; higher gleaf,op was determined approximately equally by α (47%) and by stomatal anatomy (45% by d and 8% by s), and negligibly by gec; and higher gleaf,max was determined entirely by d. These findings clarify how diversity in stomatal functioning arises from multiple structural and physiological causes with importance shifting with context. The rising importance of d relative to α, from gleaf,min to gleaf,op, enables even species with low gleaf,min, which can retain leaves through drought, to possess high d and thereby achieve rapid gas exchange in periods of high water availability. 
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