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Aim: Within C3 plants, photosynthesis is a balance between CO2 supply from the atmosphere via stomata and demand by enzymes within chloroplasts. This process is dynamic and a complex but crucial aspect of photosynthesis. We sought to understand the spatial pattern in CO2 supply–demand balance on a global scale, via analysis of stable isotopes of carbon within leaves (Δ13C), which provide an integrative record of CO2 drawdown during photosynthesis. LocationGlobal. Time period1951–2011. Major taxa studiedVascular plants. Methods: We assembled a database of leaf carbon isotope ratios containing 3,979 species–site combinations from across the globe, including 3,645 for C3 species. We examined a wide array of potential climate and soil drivers of variation in Δ13C. Results: The strongest drivers of carbon isotope discrimination at the global scale included atmospheric pressure, potential evapotranspiration and soil pH, which explained 44% of the variation in Δ13C. Addition of eight more climate and soil variables (each explaining small but highly significant amounts of variation) increased the explained variation to 60%. On top of this, the largest plant trait effect was leaf nitrogen per area, which explained 11% of Δ13C variation. Main conclusions: By considering variation in Δ13C at a considerably larger scale than previously, we were able to identify and quantify key drivers in CO2 supply–demand balance previously unacknowledged. Of special note is the key role of soil properties, with greater discrimination on low-pH and high-silt soils. Unlike other plant traits, which show typically wide variation within sets of coexisting species, the global pattern in carbon stable isotope ratios is much more conservative; there is relatively narrow variation in time-integrated CO2 concentrations at the site of carboxylation among plants in a given soil and climate.

We tested an approach to estimate daily canopy net photosynthesis, A, based on estimates of transpiration, E, using measurements of sap flow and water-use efficiency, ω, by measuring δ13C in CO2 respired from shoots in the canopies of two conifers (Podocarpaceae) native to New Zealand. The trees were planted in adjacent 20-year-old stands with the same soil and environmental conditions. Leaf area index was lower for Dacrycarpus dacrydioides D.Don in Lamb (1.34 m2 m-2) than for Podocarpus totara G.Benn. ex D.Don var. totara (2.01 m2 m-2), but mean (± standard error) stem diameters were the same at 152 ± 21 mm for D. dacrydioides and 154 ± 25 mm for P. totara. Over a 28-day period, daily A (per unit ground area) ranged almost five-fold but there were no significant differences between species (mean 2.73 ± 1.02 gC m-2 day-1). This was attributable to higher daily values of E (2.63 ± 0.83 mm day-1) and lower ω (1.35 ± 0.53 gC kg H2O-1) for D. dacrydioides compared with lower E (1.82 ± 0.72 mm day-1) and higher ω (1.90 ± 0.77 gC kg H2O-1) for P. totara. We attributed this to higher nitrogen availability and nitrogen concentration per unit foliage area, Na, and greater exposure to irradiance in the D. dacrydioides canopy compared with P. totara. Our findings support earlier observations that D. dacrydioides is more adapted to sites with poor drainage. In contrast, the high retention of leaf area and maintaining low rates of transpiration by P. totara, resulting in higher water-use efficiency, is an adaptive response to survival in dry conditions. Our findings show that physiological adjustments for two species adapted to different environments led to similar canopy photosynthesis rates when the trees were grown in the same conditions. We demonstrated consistency between whole-tree and more intensive shoot-scale measurements, confirming that integrated approaches are appropriate for comparative estimates of carbon uptake in stands with different species., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Wicked problems such as climate change and the COVID-19 pandemic require authentically transdisciplinary approaches to achieving effective collaboration. There exist several research approaches for identifying the components and interactions of complex problems; however, collaborative autoethnography provides an empirical way to collect and analyze self-reflection that leads to transformative change. Here, we present a case study of collaborative autoethnography, applied as a tool to transform research practice among a group of natural and social scientists, by constructively revealing and resolving deep, often unseen, disciplinary divides. We ask, "How can natural and social scientists genuinely accept, respect, and share one another's approaches to work on the wicked problems that need to be solved?" This study demonstrates how disciplinary divisions can be successfully bridged by open-minded and committed collaborators who are prepared to recognize the academic bias they bring to their research and use this as a platform of strength., Competing Interests: The authors declare no competing interests., (© 2022 Elsevier Inc.)

Past studies have established mesophyll diffusion conductance to CO 2 (g m ) as a variable and significant limitation to plant photosynthesis under steady-state conditions. However, the role of g m in influencing photosynthesis (A) during the transient period of light induction is largely unknown. We combined gas exchange measurements with laser-enabled carbon isotope discrimination measurements to assess g m during photosynthetic induction, using Arabidopsis as the measurement species. Our measurements revealed three key findings: (1) we found that the rate at which g m approached steady state during induction was not necessarily faster than the induction rate of the carboxylation process, contradictory to what has been suggested in previous studies; (2) g m displayed a strong and consistent coordination with A under both induction and steady-state settings, hinting that the mechanism driving g m -A coupling does not require physiological stability as a prerequisite; and (3) photosynthetic limitation analysis of our data revealed that when integrated over the entire induction period, the relative limitation of A imposed by g m can be as high as > 35%. The present study provides the first demonstration of the important role of g m in limiting CO 2 assimilation during photosynthetic induction, thereby pointing to a need for more research attention to be devoted to g m in future induction studies., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Background: Being able to accurately assess the 3D architecture of plant canopies can allow us to better estimate plant productivity and improve our understanding of underlying plant processes. This is especially true if we can monitor these traits across plant development. Photogrammetry techniques, such as structure from motion, have been shown to provide accurate 3D reconstructions of monocot crop species such as wheat and rice, yet there has been little success reconstructing crop species with smaller leaves and more complex branching architectures, such as chickpea., Results: In this work, we developed a low-cost 3D scanner and used an open-source data processing pipeline to assess the 3D structure of individual chickpea plants. The imaging system we developed consists of a user programmable turntable and three cameras that automatically captures 120 images of each plant and offloads these to a computer for processing. The capture process takes 5-10 min for each plant and the majority of the reconstruction process on a Windows PC is automated. Plant height and total plant surface area were validated against "ground truth" measurements, producing R 2  > 0.99 and a mean absolute percentage error < 10%. We demonstrate the ability to assess several important architectural traits, including canopy volume and projected area, and estimate relative growth rate in commercial chickpea cultivars and lines from local and international breeding collections. Detailed analysis of individual reconstructions also allowed us to investigate partitioning of plant surface area, and by proxy plant biomass., Conclusions: Our results show that it is possible to use low-cost photogrammetry techniques to accurately reconstruct individual chickpea plants, a crop with a complex architecture consisting of many small leaves and a highly branching structure. We hope that our use of open-source software and low-cost hardware will encourage others to use this promising technique for more architecturally complex species., (© 2021. The Author(s).)

An expression was earlier derived for the non-steady state isotopic composition of a leaf when the composition of the water entering the leaf was not necessarily the same as that of the water being transpired (Farquhar and Cernusak 2005). This was relevant to natural conditions because the associated time constant is typically sufficiently long to ensure that the leaf water composition and fluxes of the isotopologues are rarely steady. With the advent of laser-based measurements of isotopologues, leaves have been enclosed in cuvettes and time courses of fluxes recorded. The enclosure modifies the time constant by effectively increasing the resistance to the one-way gross flux out of the stomata because transpiration increases the vapour concentration within the chamber. The resistance is increased from stomatal and boundary layer in series, to stomata, boundary layer and chamber resistance, where the latter is given by the ratio of leaf area to the flow rate out of the chamber. An apparent change in concept from one-way to net flux, introduced by Song, Simonin, Loucos and Barbour (2015) is resolved, and shown to be unnecessary, but the value of their data is reinforced., (© 2021 John Wiley & Sons Ltd.)

The leaf intercellular airspace is a tortuous environment consisting of cells of different shapes, packing densities, and orientation, all of which have an effect on the travelling distance of molecules from the stomata to the mesophyll cell surfaces. Tortuosity, the increase in displacement over the actual distance between two points, is typically defined as encompassing the whole leaf airspace, but heterogeneity in pore dimensions and orientation between the spongy and palisade mesophyll likely result in heterogeneity in tortuosity along different axes and would predict longer traveling distance along the path of least tortuosity, such as vertically within the columnar cell matrix of the palisade layer. Here, we compare a previously established geometric method to a random walk approach, novel for this analysis in plant leaves, in four different Eucalyptus species. The random walk method allowed us to quantify directional tortuosity across the whole leaf profile, and separately for the spongy and palisade mesophyll. For all species tortuosity was higher in the palisade mesophyll than the spongy mesophyll and horizontal (parallel to the epidermis) tortuosity was consistently higher than vertical (from epidermis to epidermis) tortuosity. We demonstrate that a random walk approach improves on previous geometric approaches and is valuable for investigating CO 2 and H 2 O transport within leaves., (© 2021 John Wiley & Sons Ltd.)

H 2 18 O enrichment develops when leaves transpire, but an accurate generalized mechanistic model has proven elusive. We hypothesized that leaf hydraulic architecture may affect the degree to which gradients in H 2 18 O develop within leaves, influencing bulk leaf stable oxygen isotope enrichment (Δ L ) and the degree to which the Péclet effect is relevant in leaves. Leaf hydraulic design predicted the relevance of a Péclet effect to Δ L in 19 of the 21 species tested. Leaves with well-developed hydraulic connections between the vascular tissue and the epidermal cells through bundle sheath extensions and clear distinctions between palisade and spongy mesophyll layers (while the mesophyll is hydraulically disconnected) may have velocities of the transpiration stream such that gradients in H 2 18 O develop and are expressed in the mesophyll. In contrast, in leaves where the vascular tissue is hydraulically disconnected from the epidermal layers, or where all mesophyll cells are well connected to the transpiration stream, velocities within the liquid transport pathways may be low enough that gradients in H 2 18 O are very small. Prior knowledge of leaf hydraulic design allows informed selection of the appropriate Δ L modelling framework., (© 2020 John Wiley & Sons Ltd.)

Thermoregulation of leaf temperature (T leaf ) may foster metabolic homeostasis in plants, but the degree to which T leaf is moderated, and under what environmental contexts, is a topic of debate. Isotopic studies inferred the temperature of photosynthetic carbon assimilation to be a constant value of c. 20°C; by contrast, leaf biophysical theory suggests a strong dependence of T leaf on environmental drivers. Can this apparent disparity be reconciled? We continuously measured T leaf and whole-crown net CO 2 uptake for Eucalyptus parramattensis trees growing in field conditions in whole-tree chambers under ambient and +3°C warming conditions, and calculated assimilation-weighted leaf temperature (T L-AW ) across 265 d, varying in air temperature (T air ) from -1 to 45°C. We compared these data to T L-AW derived from wood cellulose δ 18 O. T leaf exhibited substantial variation driven by T air , light intensity, and vapor pressure deficit, and T leaf was strongly linearly correlated with T air with a slope of c. 1.0. T L-AW values calculated from cellulose δ 18 O vs crown fluxes were remarkably consistent; both varied seasonally and in response to the warming treatment, tracking variation in T air . The leaves studied here were nearly poikilothermic, with no evidence of thermoregulation of T leaf towards a homeostatic value. Importantly, this work supports the use of cellulose δ 18 O to infer T L-AW , but does not support the concept of strong homeothermic regulation of T leaf ., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Foundation.)

Enhancing the photosynthetic induction response to fluctuating light has been suggested as a key target for improvement in crop breeding programmes, with the potential to substantially increase whole-canopy carbon assimilation and contribute to crop yield potential. Rubisco activation may be the main physiological process that will allow us to achieve such a goal. In this study, we assessed the phenotype of Rubisco activation rate in a doubled haploid (DH) barley mapping population [131 lines from a Yerong/Franklin (Y/F) cross] after a switch from moderate to saturating light. Rates of Rubisco activation were found to be highly variable across the mapping population, with a median activation rate of 0.1 min -1 in the slowest genotype and 0.74 min -1 in the fastest genotype. A unique quantitative trait locus (QTL) for Rubisco activation rate was identified on chromosome 7H. This is the first report on the identification of a QTL for Rubisco activation rate in planta and the discovery opens the door to marker-assisted breeding to improve whole-canopy photosynthesis of barley. This also suggests that genetic factors other than the previously characterized Rubisco activase (RCA) isoforms on chromosome 4H control Rubisco activity. Further strength is given to this finding as this QTL co-localized with QTLs identified for steady-state photosynthesis and stomatal conductance. Several other distinct QTLs were identified for these steady-state traits, with a common overlapping QTL on chromosome 2H, and distinct QTLs for photosynthesis and stomatal conductance identified on chromosomes 4H and 5H, respectively. Future work should aim to validate these QTLs under field conditions so that they can be used to aid plant breeding efforts., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under- and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF-SEM has strong potential to contribute to improved understanding of leaf form and function., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Variation in temperature (T) is usually accompanied by changes in leaf water potential (Ψ leaf ), which may influence mesophyll conductance (g m ). However, the effects of Ψ leaf on g m have not yet been considered in models of the g m response to temperature. Temperature responses of g m and Ψ leaf and the response of g m to Ψ leaf were studied in rice (Oryza sativa) and wheat (Triticum aestivum), and then an empirical model of Ψ leaf was incorporated into an existing g m -T model. In wheat, Ψ leaf was dramatically decreased with increasing T, whereas in rice Ψ leaf was less sensitive or insensitive to T. Without taking Ψ leaf into account, g m for wheat showed no response to T. However, at a given Ψ leaf , g m was significantly higher at high temperature compared with low. After incorporating the function of Ψ leaf into the g m -T model, we suggest that the g m -T relationship can be influenced by the activation and deactivation energy for membrane permeability, Ψ leaf gradient between temperatures, and the sensitivity of g m to Ψ leaf , below a threshold (Ψ leaf,0 ). The data presented here suggest that Ψ leaf plays an important role in the g m -T relationship and should be considered in future studies related to the temperature response of g m and photosynthesis., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The temperature response of mesophyll conductance to CO 2 diffusion (g m ) has been shown to vary considerably between species but remains poorly understood. Here, we tested the hypothesis that increases in chloroplast surface area with increasing temperature, due to the formation of chloroplast protrusions, caused observed positive responses of g m to temperature. We found no evidence of chloroplast protrusions. Using simultaneous measurements of carbon and oxygen isotope discrimination during photosynthesis to separate total g m (g m13 ) into cell wall and plasma membrane conductance (g m18 ) and chloroplast membrane conductance (g cm ) components, we explored the temperature response in genotypes of soybean and barley, and sunflower plants grown at differing CO 2 concentrations. Differences in the temperature sensitivity of g m18 were found between genotypes and between plants grown at differing CO 2 concentration but did not relate to measured anatomical features such as chloroplast surface area or cell wall thickness. The closest fit of modelled g m13 to estimated values was found when cell wall thickness was allowed to decline at higher temperatures and transpiration rates, but it remains to be tested if this decline is realistic. The temperature response of g cm (calculated from the difference between 1/g m13 and 1/g m18 ) varied between barley genotypes, and was best fitted by an optimal response in sunflower. Taken together, these results indicate that g m is a highly complex trait with unpredictable sensitivity to temperature that varies between species, between genotypes within a single species, with growth environment, between replicate leaves, and even with age for an individual leaf.

Leaves are a nexus for the exchange of water, carbon, and energy between terrestrial plants and the atmosphere. Research in recent decades has highlighted the critical importance of the underlying biophysical and anatomical determinants of CO 2 and H 2 O transport, but a quantitative understanding of how detailed 3D leaf anatomy mediates within-leaf transport has been hindered by the lack of a consensus framework for analyzing or simulating transport and its spatial and temporal dynamics realistically, and by the difficulty of measuring within-leaf transport at the appropriate scales. We discuss how recent technological advancements now make a spatially explicit 3D leaf analysis possible, through new imaging and modeling tools that will allow us to address long-standing questions related to plant carbon-water exchange., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

. Mesophyll conductance ( g m ) has been shown to vary between genotypes of a number of species and with growth environments, including nitrogen availability, but understanding of g m variability in legumes is limited. We might expect g m in legumes to respond differently to limited nitrogen availability, due to their ability to fix atmospheric N 2 . Using online stable carbon isotope discrimination method, we quantified genetic variability in g m under ideal conditions, investigated g m response to N source (N 2 -fixation or inorganic N) and determined the effects of N source and water availability on the rapid response of g m to photosynthetic photon flux density (PPFD) and radiation wavelength in three genotypes of chickpea ( Cicer arietinum ). Genotypes varied 2-fold in g m under non-limiting environments. N-fed plants had higher g m than N 2 -fixing plants in one genotype, while g m in the other two genotypes was unaffected. g m response to PPFD was altered by N source in one of three genotypes, in which the g m response to PPFD was statistically significant in N-fed plants but not in N 2 -fixing plants. There was no clear effect of moderate water stress on the g m response to PPFD and radiation wavelength. Genotypes of a single legume species differ in the sensitivity of g m to both long- and short-term environmental conditions, precluding utility in crop breeding programmes.

Contents 986 I. 987 II. 987 III. 988 IV. 991 V. 992 VI. 995 VII. 997 VIII. 998 References 998 SUMMARY: It has been 75 yr since leaf respiratory metabolism in the light (day respiration) was identified as a low-flux metabolic pathway that accompanies photosynthesis. In principle, it provides carbon backbones for nitrogen assimilation and evolves CO 2 and thus impacts on plant carbon and nitrogen balances. However, for a long time, uncertainties have remained as to whether techniques used to measure day respiratory efflux were valid and whether day respiration responded to environmental gaseous conditions. In the past few years, significant advances have been made using carbon isotopes, 'omics' analyses and surveys of respiration rates in mesocosms or ecosystems. There is substantial evidence that day respiration should be viewed as a highly dynamic metabolic pathway that interacts with photosynthesis and photorespiration and responds to atmospheric CO 2 mole fraction. The view of leaf day respiration as a constant and/or negligible parameter of net carbon exchange is now outdated and it should now be regarded as a central actor of plant carbon-use efficiency., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

How water moves through leaves, and where the phase change from liquid to vapour occurs within leaves, remain largely mysterious. Some time ago, we suggested that the stable isotope composition of leaf water may contain information on transport pathways beyond the xylem, through differences in the development of gradients in enrichment within the various pathways. Subsequent testing of this suggestion provided ambiguous results and even questioned the existence of gradients in enrichment within the mesophyll. In this review, we bring together recent theoretical developments in understanding leaf water transport pathways and stable isotope theory to map a path for future work into understanding pathways of water transport and leaf water stable isotope composition. We emphasize the need for a spatially, anatomically and isotopically explicit model of leaf water transport., (© 2016 John Wiley & Sons Ltd.)

Stomata represent one resistor in a series of resistances for carbon and water exchange between the leaf and the atmosphere; the remaining resistors occurring within the leaf, commonly represented as mesophyll conductance to CO 2 , g m , and leaf hydraulic conductance, k Leaf . Recent studies have proposed that g m and k Leaf may be coordinated across species because of shared pathways. We assessed the correlation between g m and k Leaf within cotton, under growth CO 2 partial pressure and irradiance treatments and also with short-term variation in irradiance and humidity. g m was estimated using two isotopic techniques that allowed partitioning of total g m (Δ 13 C-g m ) into cell wall plus plasma membrane conductance (Δ 18 O-g m ) and chloroplast membrane conductance (g cm ). A weak correlation was found between Δ 13 C-g m and k Leaf only when measured under growth conditions. However, Δ 18 O-g m was related to k Leaf under both short-term environmental variation and growth conditions. Partitioning g m showed that g cm was not affected by short-term changes in irradiance or correlated with k Leaf , but was strongly reduced at high growth CO 2 partial pressure. Thus, simultaneous measurements of g m , k Leaf and g cm suggest independent regulation of carbon and water transport across the chloroplast membrane with limited coordinated regulation across the cell wall and plasma membrane., (© 2016 John Wiley & Sons Ltd.)

83 I. 83 II. 84 III. 84 IV. 85 V. 86 VI. 86 VII. 86 87 References 87 SUMMARY: The balance of carbon dioxide (CO 2 ) and water vapour exchange between leaves and the atmosphere is strongly controlled by stomatal conductance. However, the influence of transport processes within leaves has recently been gaining prominence. Stable isotope techniques are at the forefront of understanding transport within leaves and the recent development of online, real-time optical isotope analysers has paved the way for new questions to be asked. In this insight, I outline these new techniques and the questions they can potentially address, including assessing possible coordination between mesophyll conductance to CO 2 and leaf hydraulic conductance., (© 2016 The Author. New Phytologist © 2016 New Phytologist Trust.)

Increased mesophyll conductance (gm) has been suggested as a target for selection for high productivity and high water-use efficiency in crop plants, and genotypic variability in gm has been reported in several important crop species. However, effective selection requires an understanding of how gm varies with growth conditions, to ensure that the ranking of genotypes is consistent across environments. We assessed the genotypic variability in gm and other leaf gas exchange traits, as well as growth and biomass allocation for six wheat genotypes under different water and nitrogen availabilities. The wheat genotypes differed in their response of gm to growth conditions, resulting in genotypic differences in the mesophyll limitation to photosynthesis and a significant increase in the mesophyll limitation to photosynthesis under drought. In this experiment, leaf intrinsic water-use efficiency was more closely related to stomatal conductance than to mesophyll conductance, and stomatal limitation to photosynthesis increased more in some genotypes than in others in response to drought. Screening for gm should be carried out under a range of growth conditions., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The oxygen isotope composition of leaf water imparts a signal to a range of molecules in the atmosphere and biosphere, but has been notoriously difficult to measure in studies requiring a large number of samples as a consequence of the labour-intensive extraction step. We tested a method of direct equilibration of water in fresh leaf samples with CO2 , and subsequent oxygen isotope analysis on an optical spectrometer. The oxygen isotope composition of leaf water measured by the direct equilibration technique was strongly linearly related to that of cryogenically extracted leaf water in paired samples for a wide range of species with differing anatomy, with an R(2) of 0.95. The somewhat more enriched values produced by the direct equilibration method may reflect lack of full equilibration with unenriched water in the vascular bundles, but the strong relationship across a wide range of species suggests that this difference can be adequately corrected for using a simple linear relationship., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Mesophyll conductance significantly, and variably, limits photosynthesis but we currently have no reliable method of measurement for C4 plants. An online oxygen isotope technique was developed to allow quantification of mesophyll conductance in C4 plants and to provide an alternative estimate in C3 plants. The technique is compared to an established carbon isotope method in three C3 species. Mesophyll conductance of C4 species was similar to that in the C3 species measured, and declined in both C4 and C3 species as leaves aged from fully expanded to senescing. In cotton leaves, simultaneous measurement of carbon and oxygen isotope discrimination allowed the partitioning of total conductance to the chloroplasts into cell wall and plasma membrane versus chloroplast membrane components, if CO2 was assumed to be isotopically equilibrated with cytosolic water, and the partitioning remained stable with leaf age. The oxygen isotope technique allowed estimation of mesophyll conductance in C4 plants and, when combined with well-established carbon isotope techniques, may provide additional information on mesophyll conductance in C3 plants., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Leaf water contains naturally occurring stable isotopes of oxygen and hydrogen in abundances that vary spatially and temporally. When sufficiently understood, these can be harnessed for a wide range of applications. Here, we review the current state of knowledge of stable isotope enrichment of leaf water, and its relevance for isotopic signals incorporated into plant organic matter and atmospheric gases. Models describing evaporative enrichment of leaf water have become increasingly complex over time, reflecting enhanced spatial and temporal resolution. We recommend that practitioners choose a model with a level of complexity suited to their application, and provide guidance. At the same time, there exists some lingering uncertainty about the biophysical processes relevant to patterns of isotopic enrichment in leaf water. An important goal for future research is to link observed variations in isotopic composition to specific anatomical and physiological features of leaves that reflect differences in hydraulic design. New measurement techniques are developing rapidly, enabling determinations of both transpired and leaf water δ(18) O and δ(2) H to be made more easily and at higher temporal resolution than previously possible. We expect these technological advances to spur new developments in our understanding of patterns of stable isotope fractionation in leaf water., (© 2016 JohnWiley & Sons Ltd.)

The combined use of a gas-exchange system and laser-based isotope measurement is a tool of growing interest in plant ecophysiological studies, owing to its relevance for assessing isotopic variability in leaf water and/or transpiration under non-steady-state (NSS) conditions. However, the current Farquhar & Cernusak (F&C) NSS leaf water model, originally developed for open-field scenarios, is unsuited for use in a gas-exchange cuvette environment where isotope composition of water vapour (δv ) is intrinsically linked to that of transpiration (δE ). Here, we modified the F&C model to make it directly compatible with the δv -δE dynamic characteristic of a typical cuvette setting. The resultant new model suggests a role of 'net-flux' (rather than 'gross-flux' as suggested by the original F&C model)-based leaf water turnover rate in controlling the time constant (τ) for the approach to steady sate. The validity of the new model was subsequently confirmed in a cuvette experiment involving cotton leaves, for which we demonstrated close agreement between τ values predicted from the model and those measured from NSS variations in isotope enrichment of transpiration. Hence, we recommend that our new model be incorporated into future isotope studies involving a cuvette condition where the transpiration flux directly influences δv . There is an increasing popularity among plant ecophysiologists to use a gas-exchange system coupled to laser-based isotope measurement for investigating non-steady state (NSS) isotopic variability in leaf water (and/or transpiration); however, the current Farquhar & Cernusak (F&C) NSS leaf water model is unsuited for use in a gas-exchange cuvette environment due to its implicit assumption of isotope composition of water vapor (δv ) being constant and independent of that of transpiration (δE ). In the present study, we modified the F&C model to make it compatible with the dynamic relationship between δv and δE as is typically associated with a cuvette setting. Using an experiment conducted on cotton leaves, we show that the modified NSS model performed well in predicting the time constant for the exponential approach of leaf water toward steady state under cuvette conditions. Such a result demonstrates the applicability of this new model to gas-exchange cuvette conditions where the transpiration flux directly influences δv , and therefore suggests the need to incorporate this model into future isotope studies that employ a laser-cuvette coupled system., (© 2015 John Wiley & Sons Ltd.)

The two-pool and Péclet effect models represent two theories describing mechanistic controls underlying leaf water oxygen isotope composition at the whole-leaf level (δ(18) OL ). To test these models, we used a laser spectrometer coupled to a gas-exchange cuvette to make online measurements of δ(18) O of transpiration (δ(18) Otrans ) and transpiration rate (E) in 61 cotton (Gossypium hirsutum) leaves. δ(18) Otrans measurements permitted direct calculation of δ(18) O at the sites of evaporation (δ(18) Oe ) which, combined with values of δ(18) OL from the same leaves, allowed unbiased estimation of the proportional deviation of enrichment of δ(18) OL from that of δ(18) Oe (f) under both steady-state (SS) and non-steady-state (NSS) conditions. Among all leaves measured, f expressed relative to both δ(18) O of transpired water (ftrans ) and source water (fsw ) remained relatively constant with a mean ± SD of 0.11 ± 0.05 and 0.13 ± 0.05, respectively, regardless of variation in E spanning 0.8-9.1 mmol m(-2)  s(-1) . Neither ftrans nor fsw exhibited a significant difference between the SS and NSS leaves at the P < 0.05 level. Our results suggest that the simpler two-pool model is adequate for predicting cotton leaf water enrichment at the whole-leaf level. We discuss the implications of adopting a two-pool concept for isotopic applications in ecological studies., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Leaf hydraulic conductance (k leaf) is a central element in the regulation of leaf water balance but the properties of k leaf remain uncertain. Here, the evidence for the following two models for k leaf in well-hydrated plants is evaluated: (i) k leaf is constant or (ii) k leaf increases as transpiration rate (E) increases. The difference between stem and leaf water potential (ΔΨstem-leaf), stomatal conductance (g s), k leaf, and E over a diurnal cycle for three angiosperm and gymnosperm tree species growing in a common garden, and for Helianthus annuus plants grown under sub-ambient, ambient, and elevated atmospheric CO₂ concentration were evaluated. Results show that for well-watered plants k leaf is positively dependent on E. Here, this property is termed the dynamic conductance, k leaf(E), which incorporates the inherent k leaf at zero E, which is distinguished as the static conductance, k leaf(0). Growth under different CO₂ concentrations maintained the same relationship between k leaf and E, resulting in similar k leaf(0), while operating along different regions of the curve owing to the influence of CO₂ on g s. The positive relationship between k leaf and E minimized variation in ΔΨstem-leaf. This enables leaves to minimize variation in Ψleaf and maximize g s and CO₂ assimilation rate over the diurnal course of evaporative demand., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Stable oxygen isotope techniques may be a useful tool to investigate the pathways of water movement within leaves. However, implementation of such methods is limited due to uncertainty in the effective path length (L) for the Péclet effect in leaf water enrichment models. Previous studies have found relationships between L and physiological parameters such as transpiration rate (E) and leaf hydraulic conductance (k(leaf)) both within and between species. However, these studies relied on assumptions in their calculation of L, which were not directly tested. Eucalyptus paniculata Smith was used to evaluate the relationships between L, k(leaf) and E under differing water availability and a range of leaf temperatures. Coupled gas exchange and transpiration isotope measurements allowed previous assumptions to be directly tested. L was significantly and negatively related to both k(leaf) and E when the isotopic signature of water vapour was assumed to be in equilibrium with source water, was equivalent to the room vapour or equal to source water. However, the relationship between L and k(leaf) was non-significant when measured δ( 18)O of transpired vapour was used and disappeared entirely when non-steady-state leaves were excluded, and when evaporation site water was calculated from coupled gas exchange and transpiration isotope values. These results suggest that great care must be taken when calculating L, particularly regarding assumptions of isotopic steady state and δ( 18)O of vapour. Previous suggestions of changes in pathways for water movement as transpiration rate varied need to be reassessed in light of these observations., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Certainty regarding the degree to which organic molecules exchange oxygen with local water during plant cellulose synthesis (p(ex)) is necessary for cellulose oxygen isotope (δ(18)O(cell))-based applications in environmental and ecological studies. However, the currently accepted notion that p(ex) is a constant of ca. 0.42 appears inconsistent with biochemical theory, which predicts that marked variation may be present in p(ex), in relation to variation in the turnover time (τ) of the carbohydrate pool available for cellulose synthesis. The above prediction was tested in the present study with the analysis of data collected from leaves of Ricinus communis grown in controlled environmental conditions that varied in light intensity and vapour pressure deficit. The results revealed the existence of considerable variation in both p(ex) and τ across plants in the various growth environments. Moreover, despite uncertainties in estimates of the proportion of source water in the synthesis water (p(x)) and of the biochemical fractionation factor (ε(o)), our experiment yielded strong evidence that p(ex) exhibits a significant, positive relationship with τ, consistent with biochemical theory. The observed variation in p(ex) in association with τ has important implications for the interpretation of δ(18)O(cell) data in environmental/ecological studies., (© 2014 John Wiley & Sons Ltd.)

CO2 diffusion from substomatal intercellular cavities to sites of carboxylation in chloroplasts (mesophyll conductance; gm) limits photosynthetic rate and influences leaf intrinsic water-use efficiency (A/gsw). We investigated genotypic variability of gm and effects of gm on A/gsw among eleven wheat (Triticum aestivum L.) genotypes under light-saturated conditions and at either 2 or 21% O2. Significant variation in gm and A/gsw was found between genotypes at both O2 concentrations, but there was no significant effect of O2 concentration on gm. Further, gm was correlated with photosynthetic rate among the 11 genotypes, but was unrelated to stomatal conductance. The effect of leaf age differed between genotypes, with gm being lower in older leaves for one genotype but not another. This study demonstrates a high level of variation in gm between wheat genotypes; 0.5 to 1.0μmolm-2s-1 bar-1. Further, leaf age effects indicate that great care must be taken to choose suitable leaves in studies of genotypic variation in gm and water-use efficiency.

During daylight hours, the isotope composition of leaf water generally approximates steady-state leaf water isotope enrichment model predictions. However, until very recently there was little direct confirmation that isotopic steady-state (ISS) transpiration in fact exists. Using isotope ratio infrared spectroscopy (IRIS) and leaf gas exchange systems we evaluated the isotope composition of transpiration and the rate of change in leaf water isotopologue storage (isostorage) when leaves were exposed to variable environments. In doing so, we developed a method for controlling the absolute humidity entering the gas exchange cuvette for a wide range of concentrations without changing the isotope composition of water vapour. The measurement system allowed estimation of (18)O enrichment both at the evaporation site and for bulk leaf water, in the steady state and the non-steady state. We show that non-steady-state effects dominate the transpiration isoflux even when leaves are at physiological steady state. Our results suggest that a variable environment likely prevents ISS transpiration from being achieved and that this effect may be exacerbated by lengthy leaf water turnover times due to high leaf water contents., (© 2013 John Wiley & Sons Ltd.)

High nocturnal transpiration rates (5-15% of total water loss in terrestrial plants) may be adaptive under limited fertility, by increasing nutrient uptake or transport via transpiration-induced mass flow, but the response of stomata in the dark to environmental variables is poorly understood. Here we tested the impact of soil phosphorous (P) concentration, atmospheric CO2 concentration and air temperature on stomatal conductance (gs) during early and late periods in the night, as well as at midday in naturally, sun-lit glasshouse-grown Eucalyptus tereticornis Sm. seedlings. Soil P was the main driver of nocturnal gs, which was consistently higher in low soil P (37.3-79.9 mmol m(-2) s(-1)) than in high soil P (17.7-49.3 mmol m(-2)(-1)). Elevated temperature had only a marginal (P = 0.07) effect on gs early in the night (gs decreased from 34.7 to 25.8 mmol m(-2) s(-1) with an increase in temperature of 4 °C). The effect of CO2 depended on its interaction with temperature. Stomatal conductance responses to soil P were apparently driven by indirect effects of soil P on plant anatomy, since gs was significantly and negatively correlated with wood density. However, the relationship of gs with environmental factors became weaker late in the night, relative to early in the night, likely due to apparent endogenous processes; gs late in the night was two times larger than gs observed early in the night. Time-dependent controls over nocturnal gs suggest that daytime stomatal models may not apply during the night, and that different types of regulation may occur even within a single night. We conclude that the enhancement of nocturnal gs under low soil P availability is unlikely to be adaptive in our species because of the relatively small amount of transpiration-induced mass flow that can be achieved through rates of nocturnal water loss (3-6% of daytime mass flow).

Stable oxygen isotope ratio of leaf water (δ(18)O(L)) yields valuable information on many aspects of plant-environment interactions. However, current understanding of the mechanistic controls on δ(18)O(L) does not provide complete characterization of effective path length (L) of the Péclet effect,--a key component of the leaf water model. In this study, we collected diurnal and seasonal series of leaf water enrichment and estimated L in six field-grown angiosperm and gymnosperm tree species. Our results suggest a pivotal role of leaf transpiration rate (E) in driving both within- and across-species variations in L. Our observation of the common presence of an inverse scaling of L with E in the different species therefore cautions against (1) the conventional treatment of L as a species-specific constant in leaf water or cellulose isotope (δ(18)O(p)) modelling; and (2) the use of δ(18)O(p) as a proxy for gs or E under low E conditions. Further, we show that incorporation of a multi-species L-E scaling into the leaf water model has the potential to both improve the prediction accuracy and simplify parameterization of the model when compared with the conventional approach. This has important implications for future modelling of oxygen isotope ratios., (© 2013 John Wiley & Sons Ltd.)

The rate of CO(2) assimilation by plants is directly influenced by the concentration of CO(2) in the atmosphere, c(a). As an environmental variable, c(a) also has a unique global and historic significance. Although relatively stable and uniform in the short term, global c(a) has varied substantially on the timescale of thousands to millions of years, and currently is increasing at seemingly an unprecedented rate. This may exert profound impacts on both climate and plant function. Here we utilise extensive datasets and models to develop an integrated, multi-scale assessment of the impact of changing c(a) on plant carbon dioxide uptake and water use. We find that, overall, the sensitivity of plants to rising or falling c(a) is qualitatively similar across all scales considered. It is characterised by an adaptive feedback response that tends to maintain 1 - c(i)/c(a), the relative gradient for CO(2) diffusion into the leaf, relatively constant. This is achieved through predictable adjustments to stomatal anatomy and chloroplast biochemistry. Importantly, the long-term response to changing c(a) can be described by simple equations rooted in the formulation of more commonly studied short-term responses., (© 2013 The Author. New Phytologist © 2012 New Phytologist Trust.)

Although there is now a considerable literature on the inhibition of leaf respiration (CO(2) evolution) by light, little is known about the effect of other environmental conditions on day respiratory metabolism. In particular, CO(2) and O(2) mole fractions are assumed to cause changes in the tricarboxylic acid pathway (TCAP) but the amplitude and even the direction of such changes are still a matter of debate. Here, we took advantage of isotopic techniques, new simple equations and instant freeze sampling to follow respiratory metabolism in illuminated cocklebur leaves (Xanthium strumarium L.) under different CO(2) /O(2) conditions. Gas exchange coupled to online isotopic analysis showed that CO(2) evolved by leaves in the light came from 'old' carbon skeletons and there was a slight decrease in (13) C natural abundance when [CO(2) ] increased. This suggested the involvement of enzymatic steps fractionating more strongly against (13) C and thus increasingly limiting for the metabolic respiratory flux as [CO(2) ] increased. Isotopic labelling with (13) C(2) -2,4-citrate lead to (13) C-enriched Glu and 2-oxoglutarate (2OG), clearly demonstrating poor metabolism of citrate by the TCAP. There was a clear relationship between the ribulose-1,5-bisphosphate oxygenation-to-carboxylation ratio (v(o) /v(c) ) and the (13) C commitment to 2OG, demonstrating that 2OG and Glu synthesis via the TCAP is positively influenced by photorespiration., (© 2012 Blackwell Publishing Ltd.)

Mesophyll diffusion conductance to CO(2) is a key photosynthetic trait that has been studied intensively in the past years. The intention of the present review is to update knowledge of g(m), and highlight the important unknown and controversial aspects that require future work. The photosynthetic limitation imposed by mesophyll conductance is large, and under certain conditions can be the most significant photosynthetic limitation. New evidence shows that anatomical traits, such as cell wall thickness and chloroplast distribution are amongst the stronger determinants of mesophyll conductance, although rapid variations in response to environmental changes might be regulated by other factors such as aquaporin conductance. Gaps in knowledge that should be research priorities for the near future include: how different is mesophyll conductance among phylogenetically distant groups and how has it evolved? Can mesophyll conductance be uncoupled from regulation of the water path? What are the main drivers of mesophyll conductance? The need for mechanistic and phenomenological models of mesophyll conductance and its incorporation in process-based photosynthesis models is also highlighted., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The idea that photosynthesis-weighted tree canopy leaf temperature (T(canδ)) can be resolved through analysis of oxygen isotope composition in tree wood cellulose (δ(18) O(wc)) has led to the observation of boreal-to-subtropical convergence of T(canδ) to c. 20°C. To further assess the validity of the large-scale convergence of T(canδ), we used the isotope approach to perform calculation of T(canδ) for independent δ(18) O(wc) data sets that have broad coverage of climates. For the boreal-to-subtropical data sets, we found that the deviation of T(canδ) from the growing season temperature systemically increases with the decreasing mean annual temperature. Across the whole data sets we calculated a mean T(canδ) of 19.48°C and an SD of 2.05°C, while for the tropical data set, the mean T(canδ) was 26.40 ± 1.03°C, significantly higher than the boreal-to-subtropical mean. Our study thus offers independent isotopic support for the concept that boreal-to-subtropical trees display conserved T(canδ) near 20°C. The isotopic analysis cannot distinguish between the possibility that leaf temperatures are generally elevated above ambient air temperatures in cooler environments and the possibility that leaf temperature equals air temperature, whereas the leaf/air temperature at which photosynthesis occurs has a weighted average of near 20°C in cooler environments. Future work will separate these potential explanations., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Mesophyll conductance, g(m), was estimated from measurements of stomatal conductance to carbon dioxide transfer, g(s), photosynthesis, A, and chlorophyll fluorescence for Year 0 (current-year) and Year 1 (1-year-old) fully sunlit leaves from short (2 m tall, 10-year-old) and tall (15 m tall, 120-year-old) Nothofagus solandrii var. cliffortiodes trees growing in adjacent stands. Rates of photosynthesis at saturating irradiance and ambient CO(2) partial pressure, A(satQ), were 25% lower and maximum rates of carboxylation, V(cmax), were 44% lower in Year 1 leaves compared with Year 0 leaves across both tree sizes. Although g(s) and g(m) were not significantly different between Year 0 and Year 1 leaves and g(s) was not significantly different between tree heights, g(m) was significantly (19%) lower for leaves on tall trees compared with leaves on short trees. Overall, V(cmax) was 60% higher when expressed on the basis of CO(2) partial pressure at the chloroplasts, C(c), compared with V(cmax) on the basis of intercellular CO(2) partial pressure, C(i), but this varied with leaf age and tree size. To interpret the relative stomatal and mesophyll limitations to photosynthesis, we used a model of carbon isotopic composition for whole leaves incorporating g(m) effects to generate a surface of 'operating values' of A over the growing season for all leaf classes. Our analysis showed that A was slightly higher for leaves on short compared with tall trees, but lower g(m) apparently reduced actual A substantially compared with A(satQ). Our findings showed that lower rates of photosynthesis in Year 1 leaves compared with Year 0 leaves were attributable more to increased biochemical limitation to photosynthesis in Year 1 leaves than differences in g(m). However, lower A in leaves on tall trees compared with those on short trees could be attributed in part to lower g(m) and higher stomatal, L(s), and mesophyll, L(m), limitations to photosynthesis, consistent with steeper hydraulic gradients in tall trees.

Carbon and oxygen isotope discrimination of CO(2) during photosynthesis (Δ(13)C(obs) and Δ(18)O(obs)) were measured along a monocot leaf, triticale (Triticum × Secale). Both Δ(13)C(obs) and Δ(18)O(obs) increased towards the leaf tip. While this was expected for Δ(18)O(obs) , because of progressive enrichment of leaf water associated with the Péclet effect, the result was surprising for Δ(13) C(obs). To explore parameters determining this pattern, we measured activities of key photosynthetic enzymes [ribulose bis-phosphate carboxylase-oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase) as well as maximum carboxylation and electron transport rates (V(cmax) and J(max)) along the leaf. Patterns in leaf internal anatomy along the leaf were also quantified. Mesophyll conductance (g(m)) is known to have a strong influence on Δ(13)C(obs) , so we used three commonly used estimation methods to quantify variation in g(m) along the leaf. Variation in Δ(13)C(obs) was correlated with g(m) and chloroplast surface area facing the intercellular air space, but unrelated to photosynthetic enzyme activity. The observed variation could cause errors at higher scales if the appropriate portion of a leaf is not chosen for leaf-level measurements and model parameterization. Our study shows that one-third of the way from the base of the leaf represents the most appropriate portion to enclose in the leaf chamber., (© 2011 Blackwell Publishing Ltd.)

Patterns in the natural abundance of nitrogen (N) isotopes (¹⁵N and ¹⁴N) can help in the understanding of ecosystem processes along environmental gradients, because some processes fractionate against the heavier isotope. We measured δ¹⁵N in many components of the Franz Josef soil chronosequence in New Zealand to see how each component varied along the sequence and within sites, and to see what this variation can tell us about how ecosystem processes such as N losses change with soil age. We analyzed δ¹⁵N in foliage from 18 woody species, abscised leaves from seven woody species, three soil horizons, bryophytes, lichens, bulk deposition, and nodules from the N-fixing tree Coriaria arborea (Coriariaceae). Foliar δ¹⁵N varied significantly across plant species. Foliage and bulk litter became ¹⁵N-depleted as soil age increased. Soil N from organic and mineral horizons was significantly more ¹⁵N-enriched than bulk litter N at each site. Increasing precipitation also decreased foliar and soil δ¹⁵N. Comparing input and whole ecosystem δ¹⁵N revealed limited evidence for net fractionation during N losses. These trends are consistent with some combination of increasing fractionation during plant N uptake, mycorrhizal transfer, within-plant processing, and soil decomposition as soils age., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

The CO₂ respired by darkened, light-adapted, leaves is enriched in ¹³C during the first minutes, and this effect may be related to rapid changes in leaf respiratory biochemistry upon darkening. We hypothesized that this effect would be evident at the ecosystem scale. High temporal resolution measurements of the carbon isotope composition of ecosystem respiration were made over 28 diel periods in an abandoned temperate pasture, and were compared with leaf-level measurements at differing levels of pre-illumination. At the leaf level, CO₂ respired by darkened leaves that had been preadapted to high light was strongly enriched in ¹³C, but such a ¹³C-enrichment rapidly declined over 60-100 min. The ¹³C-enrichment was less pronounced when leaves were preadapted to low light. These leaf-level responses were mirrored at the ecosystem scale; after sunset following clear, sunny days respired CO₂ was first ¹³C enriched, but the ¹³C-enrichment rapidly declined over 60-100 min. Further, this response was less pronounced following cloudy days. We conclude that the dynamics of leaf respiratory isotopic signal caused variations in ecosystem-scale ¹²CO₂/¹³) CO₂ exchange. Such rapid isotope kinetics should be considered when applying ¹³C-based techniques to elucidate ecosystem carbon cycling., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Leaf intrinsic water-use efficiency (WUE), the ratio of photosynthetic rate to stomatal conductance (A/g(s) ), is a key plant trait linking terrestrial carbon and water cycles. A rapid, integrative proxy for A/g(s) is of benefit to crop breeding programmes aiming to improve WUE, but also for ecologists interested in plant carbon-water balance in natural systems. We hypothesize that the carbon isotope composition of leaf-respired CO(2) (δ(13) C(Rl) ), two hours after leaves are transferred to the dark, records photosynthetic carbon isotope discrimination and so provides a proxy for A/g(s) . To test this hypothesis, δ(13) C(Rl) was measured in four barley cultivars grown in the field at two levels of water availability and compared to leaf-level gas exchange (the ratio of leaf intercellular to ambient CO(2) partial pressure, C(i) /C(a) , and A/g(s) ). Leaf-respired CO(2) was more (13) C-depleted in plants grown at higher water availability, varied between days as environmental conditions changed, and was significantly different between cultivars. A strong relationship between δ(13) C(Rl) and δ(13) C of sucrose was observed. δ(13) C(Rl) was converted into apparent photosynthetic discrimination (Δ(13) C(Rl) ) revealing strong relationships between Δ(13) C(Rl) and C(i) /C(a) and A/g(s) during the vegetative stage of growth. We therefore conclude that δ(13) C(Rl) may provide a rapid, integrative proxy for A/g(s) in barley., (© 2011 Blackwell Publishing Ltd.)