The model of C3 photosynthesis of Farquhar et al. (1980) integrated knowledge of the functioning of the biochemical components of photosynthetic carbon assimilation in C3 plants. The model linked equations describing activated Rubisco kinetics with those on the stoichiometry of the photosynthetic carbon reduction cycle and the photorespiratory carbon oxidation cycle, particularly on their energetic (electron transport, ATP synthesis and NADPH) requirements. It included temperature dependencies of these processes and combined them with a semi-empirical equation for the dependence of potential electron transport rate on absorbed irradiance. The model aimed to match generalized observations of photosynthetic gas exchange of leaves with predictions from this mathematical summary of photosynthesis. In this model, we introduced the hypothesis that the rate of Rubisco carboxylation could not exceed the capacity for RuP2 regeneration or the enzymatic capacity to consume RuP2, and that the system behaved as a teeter-totter (see-saw) with a sharp transition from one limiting state to the other. We suggested that to model genotypic and phenotypic variations amongst leaves most parameters could be assigned a priori and only the maximum Rubisco activity (Vcmax) and the maximal electron transport rate, Jmax, needed to be varied. This simplicity of use led to the wider spread application and success of the model.

Mesophyll cells exhibit a previously underappreciated capacity to regulate water loss via low plasma membrane conductance ( L p), offering a non-stomatal mechanism for transpiration control. However, the structural basis and regulation of L p remain poorly understood, limiting its integration into predictive models. In this study, we show that L p responds dynamically to changes in cytosolic water potential ( ψ cy), decreasing as ψ cy approaches the turgor loss point. This identifies ψ cy as the primary physiological signal regulating L p. We introduce a predictive, physiologically grounded model linking L p to ψ cy. Our model establishes a mechanistic connection between internal water status, leaf hydraulics, substomatal cavity unsaturation, and gas exchange. This framework opens new avenues for understanding and modelling plant water use under stress. ### Competing Interest Statement The authors have declared no competing interest.

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.

- We consider two assumptions of leaf isotope gas exchange measurements: that leaf air spaces are saturated with water vapour, and that this vapour is of a homogeneous isotopic composition. In particular, we consider whether these assumptions can concurrently hold and, if not, which assumption is preferable to retain. - We present two methods using independent measurements of both leaf surfaces to consider these assumptions. The first method determines the isotopic inhomogeneity between the abaxial and adaxial evaporative sites when saturation is assumed. The second method determines the unsaturation in the abaxial and adaxial substomatal cavities when isotopic homogeneity is assumed. The methods are applied on Gossypium hirsutum (cotton) under benign atmospheric demand conditions (1.0 kPa air saturation deficit). - We find evidence that assuming saturation contradicts isotopic homogeneity and vice-versa. We compare each assumption to pre-existing data and find that it is reasonable to assume isotopic homogeneity, but not leaf vapour saturation. Thus, we find that leaves experience unsaturation even under benign atmospheric demand conditions and have a spatial variation in their unsaturation, with lower humidities associated with the surface of least stomatal resistance. - We conclude that leaves cannot be considered to be both saturated and of a homogeneous vapour isotopic composition. They are best approximated as isotopically homogeneous. ### Competing Interest Statement The authors have declared no competing interest.

The superior productivity of C4 plants is achieved via a metabolic C4 cycle which acts as a CO2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. The importance of chloroplast NADH dehydrogenase-like complex (NDH) operating cyclic electron flow (CEF) around Photosystem I (PSI) for C4 photosynthesis has been shown in reverse genetics studies but the contribution of CEF and NDH to cell-level electron fluxes remained unknown. We have created gene-edited Setaria viridis with null ndhO alleles lacking functional NDH and developed methods for quantification of electron flow through NDH in BS and mesophyll cells. We show that CEF accounts for 84% of electrons reducing PSI in BS cells and most of those electrons are delivered through NDH while the contribution of the complex to electron transport in mesophyll cells is minimal. A decreased leaf CO2 assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO2. Our results indicate that NDH-mediated CEF is the primary electron transport route in BS chloroplasts highlighting the essential role of NDH in generating ATP required for CO2 fixation by the C3 cycle in BS cells.

In the face of anthropogenic warming, drought poses an escalating threat to food production. C4 plants offer promise in addressing this threat. C4 leaves operate a biochemical CO2 concentrating mechanism that exchanges metabolites between two partially isolated compartments (mesophyll and bundle sheath), which confers high-productivity potential in hot climates boosting water use efficiency. However, when C4 leaves experience dehydration, photosynthesis plummets. This paper explores the physiological mechanisms behind this decline. In a fast dehydration experiment, we measured the fluxes and isotopic composition of water and CO2 in the gas exchanged by leaves, and we interpreted results using a novel biochemical model and analysis of elasticity. Our findings show that, while CO2 supply to the mesophyll and to the bundle sheath persisted during dehydration, there was a decrease in CO2 conductance at the bundle sheath-mesophyll interface. We interpret this as causing a slowdown of intercellular metabolite exchange - an essential feature of C4 photosynthesis. This would impede the supply of reducing power to the bundle sheath, leading to phosphoglycerate accumulation and feedback inhibition of Rubisco carboxylation. The interplay between this rapid sensitivity and the effectiveness of coping strategies that C4 plants deploy may be an overlooked driver of their competitive performance.

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

A fundamental assumption in plant science posits that leaf air spaces remain vapor saturated, leading to the predominant view that stomata alone control leaf water loss. This concept has been pivotal in photosynthesis and water- use efficiency research. However, recent evidence has refuted this longstanding assumption by providing evidence of unsaturation in the leaf air space of C3 plants under relatively mild vapor pressure deficit (VPD) stress. This phenomenon represents a nonstomatal mechanism restricting water loss from the mesophyll. The potential ubiquity and physiological implications of this phenomenon, its driving mechanisms in different plant species and habitats, and its interaction with other ecological adaptations remain to be fully explored. In this context, C4 plants spark particular interest for their importance as crops, bundle sheath cells' unique anatomical characteristics and specialized functions, and notably higher water- use efficiency relative to C3 plants. Here, we confirm reduced relative humidities in the substomatal cavity of the C4 plants maize, sorghum, and proso millet down to 80% under mild VPD stress. We demonstrate the critical role of nonstomatal control in these plants, indicating that the role of the CO2 concentration mechanism in CO2 management at a high VPD may have been overestimated. Our findings offer a mechanistic reconciliation between discrepancies in CO2 and VPD responses reported in C4 species. They also reveal that nonstomatal control is integral to maintaining an advantageous microclimate of relatively higher CO2 concentrations in the mesophyll air space of C4 plants for carbon fixation, proving vital when these plants face VPD stress.

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

The superior productivity of C 4 plants is achieved via a metabolic C 4 cycle which acts as a CO 2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. Chloroplast NADH dehydrogenase-like complex (NDH) increases ATP production in C 3 plants by operating cyclic electron flow (CEF) around Photosystem I (PSI), and its importance for C 4 photosynthesis has been proposed from evolutionary and reverse genetics studies. We used the gene-edited C 4 species Setaria viridis with null ndhO alleles lacking NDH to study a contribution of the complex to the cell-level electron transport. Our results indicate that NDH is the primary PSI electron acceptor mediating the majority of CEF in BS cells whilst the contribution of the complex to CEF in mesophyll cells is minimal. Moreover, the reduced leaf CO 2 assimilation rate and growth of plants lacking the complex cannot be rescued by supplying additional CO 2 , indicating that NDH is essential for generating ATP required for CO 2 fixation by the C 3 cycle. Hereby we resolve a cell-level mechanism for the contribution of NDH to supporting high CO 2 assimilation rates in C 4 photosynthesis.

The high productive potential, heat resilience, and greater water use efficiency of C4 over C3 plants attract considerable interest in the face of global warming and increasing population, but C4 plants are often sensitive to dehydration, questioning the feasibility of their wider adoption. To resolve the primary effect of dehydration from slower from secondary leaf responses originating within leaves to combat stress, we conducted an innovative dehydration experiment. Four crops grown in hydroponics were forced to a rapid yet controlled decrease in leaf water potential by progressively raising roots of out of the solution while measuring leaf gas exchange. We show that, under rapid dehydration, assimilation decreased more steeply in C4 maize and sorghum than in C3 wheat and sunflower. This reduction was due to a rise of nonstomatal limitation at triple the rate in maize and sorghum than in wheat and sunflower. Rapid reductions in assimilation were previously measured in numerous C4 species across both laboratory and natural conditions. Hence, we deduce that high sensitivity to rapid dehydration might stem from the disturbance of an intrinsic aspect of C4 bicellular photosynthesis. We posit that an obstruction to metabolite transport between mesophyll and bundle sheath cells could be the cause.

Summary We present a robust estimation of the CO 2 concentration at the surface of photosynthetic mesophyll cells ( c w ), applicable under reasonable assumptions of assimilation distribution within the leaf. We used Capsicum annuum , Helianthus annuus and Gossypium hirsutum as model plants for our experiments. We introduce calculations to estimate c w using independent adaxial and abaxial gas exchange measurements, and accounting for the mesophyll airspace resistances. The c w was lower than adaxial and abaxial estimated intercellular CO 2 concentrations ( c i ). Differences between c w and the c i of each surface were usually larger than 10 μmol mol −1 . Differences between adaxial and abaxial c i ranged from a few μmol mol −1 to almost 50 μmol mol −1 , where the largest differences were found at high air saturation deficits (ASD). Differences between adaxial and abaxial c i and the c i estimated by mixing both fluxes ranged from −30 to +20 μmol mol −1 , where the largest differences were found under high ASD or high ambient CO 2 concentrations. Accounting for c w improves the information that can be extracted from gas exchange experiments, allowing a more detailed description of the CO 2 and water vapor gradients within the leaf.

Measurement of leaf carbon gain and water loss (gas exchange) in planta is a standard procedure in plant science research for attempting to understand physiological traits related to water use and photosynthesis. Leaves carry out gas exchange through the upper (adaxial) and lower (abaxial) surfaces at different magnitudes, depending on the stomatal density, stomatal aperture, cuticular permeability, etc., of each surface, which we account for in gas exchange parameters such as stomatal conductance. Most commercial devices measure leaf gas exchange by combining the adaxial and abaxial fluxes and calculating bulk gas exchange parameters, missing details of the plant's physiological response on each side. Additionally, the widely used equations to estimate gas exchange parameters neglect the contribution of small fluxes such as cuticular conductance, adding extra uncertainties to measurements performed in water-stress or low-light conditions. Accounting for the gas exchange fluxes from each side of the leaf allows us to better describe plants' physiological traits under different environmental conditions and account for genetic variability. Here, apparatus and materials are presented for adapting two LI-6800 Portable Photosynthesis Systems to work as one gas exchange system to measure adaxial and abaxial gas exchange simultaneously. The modification includes a template script with the equations to account for small fluxes. Instructions are provided for incorporating the add-on script into the device's computational sequence, display, variables, and spreadsheet results. We explain the method to obtain an equation to estimate boundary layer conductance to water for the new setup and how to embed this equation in the devices' calculations using the provided add-on script. The apparatus, methods, and protocols presented here provide a simple adaptation combining two LI-6800s to obtain an improved system to measure leaf gas exchange on adaxial and abaxial surfaces. Graphical overview Figure 1.Diagram of the connection of two LI-6800s.Figure adapted from Márquez et al. (2021).

Net photosynthetic CO 2 assimilation rate ( A n ) decreases at leaf temperatures above a relatively mild optimum ( T opt ) in most higher plants. This decline is often attributed to reduced CO 2 conductance, increased CO 2 loss from photorespiration and respiration, reduced chloroplast electron transport rate ( J ), or deactivation of Ribulose-1,5-bisphosphate Carboxylase Oxygenase (Rubisco). However, it is unclear which of these factors can best predict species independent declines in A n at high temperature. We show that independent of species, and on a global scale, the observed decline in A n with rising temperatures can be effectively accounted for by Rubisco deactivation and declines in J . Our finding that A n declines with Rubisco deactivation and J supports a coordinated down-regulation of Rubisco and chloroplast electron transport rates to heat stress. We provide a model that, in the absence of CO 2 supply limitations, can predict the response of photosynthesis to short-term increases in leaf temperature.

Limitations and utility of three measures of water use characteristics were evaluated: water use efficiency (WUE), intrinsic WUE and marginal water cost of carbon gain ( ∂E/∂A ) estimated, respectively, as ratios of assimilation (A) to transpiration (E), of A to stomatal conductance (gs ) and of sensitivities of E and A with variation in gs . Only the measure ∂E/∂A estimates water use strategy in a way that integrates carbon gain relative to water use under varying environmental conditions across scales from leaves to communities. This insight provides updated and simplified ways of estimating ∂E/∂A and adds depth to understanding ways that plants balance water expenditure against carbon gain, uniquely providing a mechanistic means of predicting water use characteristics under changing environmental scenarios.

Cuticular conductance to water (gcw ) is difficult to quantify for stomatous surfaces due to the complexity of separating cuticular and stomatal transpiration, and additional complications arise for determining adaxial and abaxial gcw . This has led to the neglect of gcw as a separate parameter in most common gas exchange measurements. Here, we describe a simple technique to simultaneously estimate adaxial and abaxial values of gcw , tested in two amphistomatous plant species. What we term the 'Red-Light method' is used to estimate gcw from gas exchange measurements and a known CO2 concentration inside the leaf during photosynthetic induction under red light. We provide an easy-to-use web application to assist with the calculation of gcw . While adaxial and abaxial gcw varies significantly between leaves of the same species we found that the ratio of adaxial/abaxial gcw (γn ) is stable within a plant species. This has implications for use of generic values of gcw when analysing gas exchange data. The Red-Light method can be used to estimate total cuticular conductance (gcw-T ) accurately with the most common setup of gas exchange instruments, i.e. a chamber mixing the adaxial and abaxial gases, allowing for a wide application of this technique.

Canola varieties exhibit variation in drought avoidance and drought escape traits, reflecting adaptation to water-deficit environments. Our understanding of underlying genes and their interaction across environments in improving crop productivity is limited. A doubled haploid population was analysed to identify quantitative trait loci (QTL) associated with water-use efficiency (WUE) related traits. High WUE in the vegetative phase was associated with low seed yield. Based on the resequenced parental genome data, we developed sequence-capture-based markers and validated their linkage with carbon isotope discrimination (Δ13 C) in an F2 population. RNA sequencing was performed to determine the expression of candidate genes underlying Δ13 C QTL. QTL contributing to main and QTL × environment interaction effects for Δ13 C and yield were identified. One multiple-trait QTL for Δ13 C, days to flower, plant height, and seed yield was identified on chromosome A09. Interestingly, this QTL region overlapped with a homoeologous exchange (HE) event, suggesting its association with the multiple traits. Transcriptome analysis revealed 121 significantly differentially expressed genes underlying Δ13 C QTL on A09 and C09, including in HE regions. Sorting out the negative relationship between vegetative WUE and seed yield is a priority. Genetic and genomic resources and knowledge so developed could improve canola WUE and yield.

Stomata are orifices that connect the drier atmosphere with the interconnected network of more humid air spaces that surround the cells within a leaf. Accurate values of the humidities inside the substomatal cavity, w i , and in the air, w a , are needed to estimate stomatal conductance and the CO 2 concentration in the internal air spaces of leaves. Both are vital factors in the understanding of plant physiology and climate, ecological and crop systems. However, there is no easy way to measure w i directly. Out of necessity, w i has been taken as the saturation water vapour concentration at leaf temperature, w sat , and applied to the whole leaf intercellular air spaces. We explored the occurrence of unsaturation by examining gas exchange of leaves exposed to various magnitudes of w sat − w a , or Δ w , using a double-sided, clamp-on chamber, and estimated degrees of unsaturation from the gradient of CO 2 across the leaf that was required to sustain the rate of CO 2 assimilation through the upper surface. The relative humidity in the substomatal cavities dropped to about 97% under mild Δ w and as dry as around 80% when Δ w was large. Measurements of the diffusion of noble gases across the leaf indicated that there were still regions of near 100% humidity distal from the stomatal pores. We suggest that as Δ w increases, the saturation edge retreats into the intercellular air spaces, accompanied by the progressive closure of mesophyll aquaporins to maintain the cytosolic water potential.

This article is a Commentary on Leppä et al. (2022), 236: 2044–2060.