Decametric-scale buffering of climate extremes in forest understory within a riparian microrefugia: the key role of microtopography.

Riparian corridors often act as low-land climate refugia for temperate tree species in their southern distribution range. A plausible mechanism is the buffering of regional climate extremes by local physiographic and biotic factors. We tested this idea using a 3-year-long microclimate dataset collected along the Ciron river, a refugia for European beech (Fagus sylvatica) in southwestern France. Across the whole network, canopy gap fraction was the main predictor for spatial microclimatic variations, together with two other landscape features (elevation above the river and woodland fraction within a 300m radius). However, within the riparian forest only (canopy gap fraction < 25%, distance to the river < 150m), variations of up to -4°C and + 15% in summertime daily maximum air temperature and minimum relative humidity, respectively, were still found from the plateau to the cooler, moister river banks, only ~ 5-10m below. Elevation above the river was then identified as the main predictor, and explained the marked variations from the plateau to the banks much better than canopy gap fraction. The microclimate measured near the river is as cool but moister than the macroclimate encountered at 700-1000m asl further east in F. sylvatica's main distribution range. Indeed, at all locations, we found that air relative humidity was higher than expected from a temperature-only effect, suggesting that extra moisture is brought by the river. Our results explain well why beech trees in this climate refugium are restricted to the river gorges where microtopographic variations are the strongest and canopy gaps are rare.

Using Carbon Stable Isotopes to Study C3 and C4 Photosynthesis: Models and Calculations.

Stable carbon isotopes are a powerful tool to study photosynthesis. Initial applications consisted of determining isotope ratios of plant biomass using mass spectrometry. Subsequently, theoretical models relating C isotope values to gas exchange characteristics were introduced and tested against instantaneous online measurements of 13C photosynthetic discrimination. Beginning in the twenty-first century, laser absorption spectroscopes with sufficient precision for determining isotope mixing ratios became commercially available. This has allowed collection of large data sets at lower cost and with unprecedented temporal resolution. More data and accompanying knowledge have permitted refinement of 13C discrimination model equations, but often at the expense of increased model complexity and difficult parametrization. This chapter describes instantaneous online measurements of 13C photosynthetic discrimination, provides recommendations for experimental setup, and presents a thorough compilation of equations available to researchers. We update our previous 2018 version of this chapter by including recently improved descriptions of (photo)respiratory processes and associated fractionations. We discuss the capabilities and limitations of the diverse 13C discrimination model equations and provide guidance for selecting the model complexity needed for different applications.

Half of the 18O enrichment of leaf sucrose is conserved in leaf cellulose of a C3 grass across atmospheric humidity and CO2 levels.

The 18O enrichment (Δ18O) of cellulose (Δ18OCel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (pex) that exchanges post-photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C3 grasses demonstrated that the Δ18O of leaf sucrose (Δ18OSuc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ18O of leaf water (Δ18OLW), which could alter conclusions on photosynthetic versus post-photosynthetic effects on Δ18OCel via pex. Here, we assessed pex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO2 levels, by determinations of Δ18OCel in leaves, Δ18OLGDZW (the Δ18O of water in the leaf growth-and-differentiation zone) and both Δ18OSuc and Δ18OLW (adjusted for εbio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ18OLGDZW was always close to irrigation water, and pex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ18OSuc. Conversely, pex was erroneously and variably underestimated (range 0.02-0.44) when based on Δ18OLW. The photosynthetic signal fraction in Δ18OCel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

18 O enrichment of sucrose and photosynthetic and nonphotosynthetic leaf water in a C3 grass-atmospheric drivers and physiological relations.

The 18 O enrichment (Δ18 O) of leaf water affects the Δ18 O of photosynthetic products such as sucrose, generating an isotopic archive of plant function and past climate. However, uncertainty remains as to whether leaf water compartmentation between photosynthetic and nonphotosynthetic tissue affects the relationship between Δ18 O of bulk leaf water (Δ18 OLW ) and leaf sucrose (Δ18 OSucrose ). We grew Lolium perenne (a C3 grass) in mesocosm-scale, replicated experiments with daytime relative humidity (50% or 75%) and CO2 level (200, 400 or 800 µmol mol-1 ) as factors, and determined Δ18 OLW , Δ18 OSucrose and morphophysiological leaf parameters, including transpiration (Eleaf ), stomatal conductance (gs ) and mesophyll conductance to CO2 (gm ). The Δ18 O of photosynthetic medium water (Δ18 OSSW ) was estimated from Δ18 OSucrose and the equilibrium fractionation between water and carbonyl groups (εbio ). Δ18 OSSW was well predicted by theoretical estimates of leaf water at the evaporative site (Δ18 Oe ) with adjustments that correlated with gas exchange parameters (gs or total conductance to CO2 ). Isotopic mass balance and published work indicated that nonphotosynthetic tissue water was a large fraction (~0.53) of bulk leaf water. Δ18 OLW was a poor proxy for Δ18 OSucrose , mainly due to opposite Δ18 O responses of nonphotosynthetic tissue water (Δ18 Onon-SSW ) relative to Δ18 OSSW , driven by atmospheric conditions.

Contrasting stem water uptake and storage dynamics of water-saver and water-spender species during drought and recovery.

Drought is projected to occur more frequently and intensely in the coming decades, and the extent to which it will affect forest functioning will depend on species-specific responses to water stress. Aiming to understand the hydraulic traits and water dynamics behind water-saver and water-spender strategies in response to drought and recovery, we conducted a pot experiment with two species with contrasting physiological strategies, Scots pine (Pinus sylvestris L.) and Portuguese oak (Quercus faginea L.). We applied two cycles of soil drying and recovery and irrigated with isotopically different water to track fast changes in soil and stem water pools, while continuously measuring physiological status and xylem water content from twigs. Our results provide evidence for a tight link between the leaf-level response and the water uptake and storage patterns in the stem. The water-saver strategy of pines prevented stem dehydration by rapidly closing stomata which limited their water uptake during the early stages of drought and recovery. Conversely, oaks showed a less conservative strategy, maintaining transpiration and physiological activity under dry soil conditions, and consequently becoming more dehydrated at the stem level. We interpreted this dehydration as the release of water from elastic storage tissues as no major loss of hydraulic conductance occurred for this species. After soil rewetting, pines recovered pre-drought leaf water potential rapidly, but it took longer to replace the water from conductive tissues (slower labeling speed). In contrast, water-spender oaks were able to quickly replace xylem water during recovery (fast labeling speed), but it took longer to refill stem storage tissues, and hence to recover pre-drought leaf water potential. These different patterns in sap flow rates, speed and duration of the labeling reflected a combination of water-use and storage traits, linked to the leaf-level strategies in response to drought and recovery.

Explicitly accounting for needle sugar pool size crucial for predicting intra-seasonal dynamics of needle carbohydrates δ18 O and δ13 C.

We explore needle sugar isotopic compositions (δ18 O and δ13 C) in boreal Scots pine (Pinus sylvestris) over two growing seasons. A leaf-level dynamic model driven by environmental conditions and based on current understanding of isotope fractionation processes was built to predict δ18 O and δ13 C of two hierarchical needle carbohydrate pools, accounting for the needle sugar pool size and the presence of an invariant pinitol pool. Model results agreed well with observed needle water δ18 O, δ18 O and δ13 C of needle water-soluble carbohydrates (sugars + pinitol), and needle sugar δ13 C (R2 = 0.95, 0.84, 0.60, 0.73, respectively). Relative humidity (RH) and intercellular to ambient CO2 concentration ratio (Ci /Ca ) were the dominant drivers of δ18 O and δ13 C variability, respectively. However, the variability of needle sugar δ18 O and δ13 C was reduced on diel and intra-seasonal timescales, compared to predictions based on instantaneous RH and Ci /Ca , due to the large needle sugar pool, which caused the signal formation period to vary seasonally from 2 d to more than 5 d. Furthermore, accounting for a temperature-sensitive biochemical 18 O-fractionation factor and mesophyll resistance in 13 C-discrimination were critical. Interpreting leaf-level isotopic signals requires understanding on time integration caused by mixing in the needle sugar pool.

Do 2 H and 18 O in leaf water reflect environmental drivers differently?

We compiled hydrogen and oxygen stable isotope compositions (δ2 H and δ18 O) of leaf water from multiple biomes to examine variations with environmental drivers. Leaf water δ2 H was more closely correlated with δ2 H of xylem water or atmospheric vapour, whereas leaf water δ18 O was more closely correlated with air relative humidity. This resulted from the larger proportional range for δ2 H of meteoric waters relative to the extent of leaf water evaporative enrichment compared with δ18 O. We next expressed leaf water as isotopic enrichment above xylem water (Δ2 H and Δ18 O) to remove the impact of xylem water isotopic variation. For Δ2 H, leaf water still correlated with atmospheric vapour, whereas Δ18 O showed no such correlation. This was explained by covariance between air relative humidity and the Δ18 O of atmospheric vapour. This is consistent with a previously observed diurnal correlation between air relative humidity and the deuterium excess of atmospheric vapour across a range of ecosystems. We conclude that 2 H and 18 O in leaf water do indeed reflect the balance of environmental drivers differently; our results have implications for understanding isotopic effects associated with water cycling in terrestrial ecosystems and for inferring environmental change from isotopic biomarkers that act as proxies for leaf water.

Maintaining forest cover to enhance temperature buffering under future climate change.

Forest canopies buffer macroclimatic temperature fluctuations. However, we do not know if and how the capacity of canopies to buffer understorey temperature will change with accelerating climate change. Here we map the difference (offset) between temperatures inside and outside forests in the recent past and project these into the future in boreal, temperate and tropical forests. Using linear mixed-effect models, we combined a global database of 714 paired time series of temperatures (mean, minimum and maximum) measured inside forests vs. in nearby open habitats with maps of macroclimate, topography and forest cover to hindcast past (1970-2000) and to project future (2060-2080) temperature differences between free-air temperatures and sub-canopy microclimates. For all tested future climate scenarios, we project that the difference between maximum temperatures inside and outside forests across the globe will increase (i.e. result in stronger cooling in forests), on average during 2060-2080, by 0.27 ± 0.16 °C (RCP2.6) and 0.60 ± 0.14 °C (RCP8.5) due to macroclimate changes. This suggests that extremely hot temperatures under forest canopies will, on average, warm less than outside forests as macroclimate warms. This knowledge is of utmost importance as it suggests that forest microclimates will warm at a slower rate than non-forested areas, assuming that forest cover is maintained. Species adapted to colder growing conditions may thus find shelter and survive longer than anticipated at a given forest site. This highlights the potential role of forests as a whole as microrefugia for biodiversity under future climate change.

Evidence for distinct isotopic compositions of sap and tissue water in tree stems: consequences for plant water source identification.

The long-standing hypothesis that the isotopic composition of plant stem water reflects that of source water is being challenged by studies reporting bulk water from woody stems with an isotopic composition that cannot be attributed to any potential water source. The mechanism behind such source-stem water isotopic offsets is still poorly understood. Using a novel technique to extract selectively sap water from xylem conduits, we show that, in cut stems and potted plants, the isotopic composition of sap water reflects that of irrigation water, demonstrating unambiguously that no isotopic fractionation occurs during root water uptake or sap water extraction. By contrast, water in nonconductive xylem tissues is always depleted in deuterium compared with sap water, irrespective of wood anatomy. Previous studies have shown that isotopic heterogeneity also exists in soils at the pore scale in which water adsorbed onto soil particles is more depleted in deuterium than unbound water. Data collected at a riparian forest indicated that sap water matches best unbound soil water from depth below -70 cm, while bulk stem and soil water differ markedly. We conclude that source-stem isotopic offsets can be explained by micrometre-scale heterogeneity in the isotope ratios of water within woody stems and soil micro-pores.

Forest microclimates and climate change: Importance, drivers and future research agenda.

Forest microclimates contrast strongly with the climate outside forests. To fully understand and better predict how forests' biodiversity and functions relate to climate and climate change, microclimates need to be integrated into ecological research. Despite the potentially broad impact of microclimates on the response of forest ecosystems to global change, our understanding of how microclimates within and below tree canopies modulate biotic responses to global change at the species, community and ecosystem level is still limited. Here, we review how spatial and temporal variation in forest microclimates result from an interplay of forest features, local water balance, topography and landscape composition. We first stress and exemplify the importance of considering forest microclimates to understand variation in biodiversity and ecosystem functions across forest landscapes. Next, we explain how macroclimate warming (of the free atmosphere) can affect microclimates, and vice versa, via interactions with land-use changes across different biomes. Finally, we perform a priority ranking of future research avenues at the interface of microclimate ecology and global change biology, with a specific focus on three key themes: (1) disentangling the abiotic and biotic drivers and feedbacks of forest microclimates; (2) global and regional mapping and predictions of forest microclimates; and (3) the impacts of microclimate on forest biodiversity and ecosystem functioning in the face of climate change. The availability of microclimatic data will significantly increase in the coming decades, characterizing climate variability at unprecedented spatial and temporal scales relevant to biological processes in forests. This will revolutionize our understanding of the dynamics, drivers and implications of forest microclimates on biodiversity and ecological functions, and the impacts of global changes. In order to support the sustainable use of forests and to secure their biodiversity and ecosystem services for future generations, microclimates cannot be ignored.

Temperature-sensitive biochemical 18 O-fractionation and humidity-dependent attenuation factor are needed to predict δ18 O of cellulose from leaf water in a grassland ecosystem.

We explore here our mechanistic understanding of the environmental and physiological processes that determine the oxygen isotope composition of leaf cellulose (δ18 Ocellulose ) in a drought-prone, temperate grassland ecosystem. A new allocation-and-growth model was designed and added to an 18 O-enabled soil-vegetation-atmosphere transfer model (MuSICA) to predict seasonal (April-October) and multi-annual (2007-2012) variation of δ18 Ocellulose and 18 O-enrichment of leaf cellulose (Δ18 Ocellulose ) based on the Barbour-Farquhar model. Modelled δ18 Ocellulose agreed best with observations when integrated over c. 400 growing-degree-days, similar to the average leaf lifespan observed at the site. Over the integration time, air temperature ranged from 7 to 22°C and midday relative humidity from 47 to 73%. Model agreement with observations of δ18 Ocellulose (R2  = 0.57) and Δ18 Ocellulose (R2  = 0.74), and their negative relationship with canopy conductance, was improved significantly when both the biochemical 18 O-fractionation between water and substrate for cellulose synthesis (εbio , range 26-30‰) was temperature-sensitive, as previously reported for aquatic plants and heterotrophically grown wheat seedlings, and the proportion of oxygen in cellulose reflecting leaf water 18 O-enrichment (1 - pex px , range 0.23-0.63) was dependent on air relative humidity, as observed in independent controlled experiments with grasses. Understanding physiological information in δ18 Ocellulose requires quantitative knowledge of climatic effects on pex px and εbio .

Asymmetric responses of ecosystem productivity to rainfall anomalies vary inversely with mean annual rainfall over the conterminous United States.

The CONterminous United States (CONUS) presents a large range of climate conditions and biomes where terrestrial primary productivity and its inter-annual variability are controlled regionally by rainfall and/or temperature. Here, the response of ecosystem productivity to those climate variables was investigated across different biomes from 2010 to 2018 using three climate datasets of precipitation, air temperature or drought severity, combined with several proxies of ecosystem productivity: a remote sensing product of aboveground biomass, an net primary productivity (NPP) remote sensing product, an NPP model-based product and four gross primary productivity products. We used an asymmetry index (AI) where positive AI indicates a greater increase of ecosystem productivity in wet years compared to the decline in dry years, and negative AI indicates a greater decline of ecosystem productivity in dry years compared to the increase in wet years. We found consistent spatial patterns of AI across the CONUS for the different products, with negative asymmetries over the Great Plains and positive asymmetries over the southwestern CONUS. Shrubs and, to a lesser extent, evergreen forests show a persistent positive asymmetry, whilst (natural) grasslands appear to have transitioned from positive to negative anomalies during the last decade. The general tendency of dominant negative asymmetry response for ecosystem productivity across the CONUS appears to be influenced by the negative asymmetry of precipitation anomalies. AI was found to be a function of mean rainfall: more positive AIs were found in dry areas where plants are adapted to drought and take advantage of rainfall pulses, and more negative AIs were found in wet areas, with a threshold delineating the two regimes corresponding to a mean annual rainfall of 200-400 mm/year.

Partitioning net carbon dioxide fluxes into photosynthesis and respiration using neural networks.

The eddy covariance (EC) technique is used to measure the net ecosystem exchange (NEE) of CO2 between ecosystems and the atmosphere, offering a unique opportunity to study ecosystem responses to climate change. NEE is the difference between the total CO2 release due to all respiration processes (RECO), and the gross carbon uptake by photosynthesis (GPP). These two gross CO2 fluxes are derived from EC measurements by applying partitioning methods that rely on physiologically based functional relationships with a limited number of environmental drivers. However, the partitioning methods applied in the global FLUXNET network of EC observations do not account for the multiple co-acting factors that modulate GPP and RECO flux dynamics. To overcome this limitation, we developed a hybrid data-driven approach based on combined neural networks (NNC-part ). NNC-part incorporates process knowledge by introducing a photosynthetic response based on the light-use efficiency (LUE) concept, and uses a comprehensive dataset of soil and micrometeorological variables as fluxes drivers. We applied the method to 36 sites from the FLUXNET2015 dataset and found a high consistency in the results with those derived from other standard partitioning methods for both GPP (R2  > .94) and RECO (R2  > .8). High consistency was also found for (a) the diurnal and seasonal patterns of fluxes and (b) the ecosystem functional responses. NNC-part performed more realistic than the traditional methods for predicting additional patterns of gross CO2 fluxes, such as: (a) the GPP response to VPD, (b) direct effects of air temperature on GPP dynamics, (c) hysteresis in the diel cycle of gross CO2 fluxes, (d) the sensitivity of LUE to the diffuse to direct radiation ratio, and (e) the post rain respiration pulse after a long dry period. In conclusion, NNC-part is a valid data-driven approach to provide GPP and RECO estimates and complementary to the existing partitioning methods.

µgreen-db: a reference database for the 23S rRNA gene of eukaryotic plastids and cyanobacteria.

Studying the ecology of photosynthetic microeukaryotes and prokaryotic cyanobacterial communities requires molecular tools to complement morphological observations. These tools rely on specific genetic markers and require the development of specialised databases to achieve taxonomic assignment. We set up a reference database, called µgreen-db, for the 23S rRNA gene. The sequences were retrieved from generalist (NCBI, SILVA) or Comparative RNA Web (CRW) databases, in addition to a more original approach involving recursive BLAST searches to obtain the best possible sequence recovery. At present, µgreen-db includes 2,326 23S rRNA sequences belonging to both eukaryotes and prokaryotes encompassing 442 unique genera and 736 species of photosynthetic microeukaryotes, cyanobacteria and non-vascular land plants based on the NCBI and AlgaeBase taxonomy. When PR2/SILVA taxonomy is used instead, µgreen-db contains 2,217 sequences (399 unique genera and 696 unique species). Using µgreen-db, we were able to assign 96% of the sequences of the V domain of the 23S rRNA gene obtained by metabarcoding after amplification from soil DNA at the genus level, highlighting good coverage of the database. µgreen-db is accessible at http://microgreen-23sdatabase.ea.inra.fr.

An explanation for the isotopic offset between soil and stem water in a temperate tree species.

A growing number of field studies report isotopic offsets between stem water and its potential sources that prevent the unambiguous identification of plant water origin using water isotopes. We explored the causes of this isotopic offset by conducting a controlled experiment on the temperate tree species Fagus sylvatica. We measured δ2 H and δ18 O of soil and stem water from potted saplings growing on three soil substrates and subjected to two watering regimes. Regardless of substrate, soil and stem water δ2 H were similar only near permanent wilting point. Under moister conditions, stem water δ2 H was 11 ± 3‰ more negative than soil water δ2 H, coherent with field studies. Under drier conditions, stem water δ2 H became progressively more enriched than soil water δ2 H. Although stem water δ18 O broadly reflected that of soil water, soil-stem δ2 H and δ18 O differences were correlated (r = 0.76) and increased with transpiration rates indicated by proxies. Soil-stem isotopic offsets are more likely to be caused by water isotope heterogeneities within the soil pore and stem tissues, which would be masked under drier conditions as a result of evaporative enrichment, than by fractionation under root water uptake. Our results challenge our current understanding of isotopic signals in the soil-plant continuum.

A novel optimization approach incorporating non-stomatal limitations predicts stomatal behaviour in species from six plant functional types.

The primary function of stomata is to minimize plant water loss while maintaining CO2 assimilation. Stomatal water loss incurs an indirect cost to photosynthesis in the form of non-stomatal limitations (NSL) via reduced carboxylation capacity (CAP) and/or mesophyll conductance (MES). Two optimal formulations for stomatal conductance (gs) arise from the assumption of each type of NSL. In reality, both NSL could coexist, but one may prevail for a given leaf ontogenetic stage or plant functional type, depending on leaf morphology. We tested the suitability of two gs formulations (CAP versus MES) on species from six plant functional types (C4 crop, C3 grass, fern, conifer, evergreen, and deciduous angiosperm trees). MES and CAP parameters (the latter proportional to the marginal water cost to carbon gain) decreased with water availability only in deciduous angiosperm trees, while there were no clear differences between leaf ontogenetic stages. Both CAP and MES formulations fit our data in most cases, particularly under low water availability. For ferns, stomata appeared to operate optimally only when subjected to water stress. Overall, the CAP formulation provided a better fit across all species, suggesting that sub-daily stomatal responses minimize NSL by reducing carboxylation capacity predominantly, regardless of leaf morphology and ontogenetic stage.

Soil exchange rates of COS and CO18O differ with the diversity of microbial communities and their carbonic anhydrase enzymes.

Differentiating the contributions of photosynthesis and respiration to the global carbon cycle is critical for improving predictive climate models. Carbonic anhydrase (CA) activity in leaves is responsible for the largest biosphere-atmosphere trace gas fluxes of carbonyl sulfide (COS) and the oxygen-18 isotopologue of carbon dioxide (CO18O) that both reflect gross photosynthetic rates. However, CA activity also occurs in soils and will be a source of uncertainty in the use of COS and CO18O as carbon cycle tracers until process-based constraints are improved. In this study, we measured COS and CO18O exchange rates and estimated the corresponding CA activity in soils from a range of biomes and land use types. Soil CA activity was not uniform for COS and CO2, and patterns of divergence were related to microbial community composition and CA gene expression patterns. In some cases, the same microbial taxa and CA classes catalyzed both COS and CO2 reactions in soil, but in other cases the specificity towards the two substrates differed markedly. CA activity for COS was related to fungal taxa and ß-D-CA expression, whereas CA activity for CO2 was related to algal and bacterial taxa and α-CA expression. This study integrates gas exchange measurements, enzyme activity models, and characterization of soil taxonomic and genetic diversity to build connections between CA activity and the soil microbiome. Importantly, our results identify kinetic parameters to represent soil CA activity during application of COS and CO18O as carbon cycle tracers.

Coupling of ecosystem-scale plant water storage and leaf phenology observed by satellite.

Plant water storage is fundamental to the functioning of terrestrial ecosystems by participating in plant metabolism, nutrient and sugar transport, and maintenance of the integrity of the hydraulic system of the plant. However, a global view of the size and dynamics of the water pools stored in plant tissues is still lacking. Here, we report global patterns of seasonal variations in ecosystem-scale plant water storage and their relationship with leaf phenology, based on space-borne measurements of L-band vegetation optical depth. We find that seasonal variations in plant water storage are highly synchronous with leaf phenology for the boreal and temperate forests, but asynchronous for the tropical woodlands, where the seasonal development of plant water storage lags behind leaf area by up to 180 days. Contrasting patterns of the time lag between plant water storage and terrestrial groundwater storage are also evident in these ecosystems. A comparison of the water cycle components in seasonally dry tropical woodlands highlights the buffering effect of plant water storage on the seasonal dynamics of water supply and demand. Our results offer insights into ecosystem-scale plant water relations globally and provide a basis for an improved parameterization of eco-hydrological and Earth system models.

Estimating Mesophyll Conductance from Measurements of C18OO Photosynthetic Discrimination and Carbonic Anhydrase Activity.

Carbonic anhydrase (CA) activity in leaves catalyzes the 18O exchange between CO2 and water during photosynthesis. This feature has been used to estimate the mesophyll conductance to CO2 (g m) from measurements of online C18OO photosynthetic discrimination (∆18O). Based on CA assays on leaf extracts, it has been argued that CO2 in mesophyll cells should be in isotopic equilibrium with water in most C3 species as well as many C4 dicot species. However, this seems incompatible with ∆18O data that would indicate a much lower degree of equilibration, especially in C4 plants under high light intensity. This apparent contradiction is resolved here using a new model of C3 and C4 photosynthetic discrimination that includes competition between CO2 hydration and carboxylation and the contribution of respiratory fluxes. This new modeling framework is used to revisit previously published data sets on C3 and C4 species, including CA-deficient plants. We conclude that (1) newly ∆18O-derived g m values are usually close but significantly higher (typically 20% and up to 50%) than those derived assuming full equilibration and (2) despite the uncertainty associated with the respiration rate in light, or the water isotope gradient between mesophyll and bundle sheath cells, robust estimates of ∆18O-derived g m can be achieved in both C3 and C4 plants.