Mesophyll airspace unsaturation drives C4 plant success under vapor pressure deficit stress.

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 have. 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.

The role of thermodiffusion in transpiration.

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.

Fast dehydration reduces bundle sheath conductance in C4 maize and sorghum.

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.

Chloroplast NADH dehydrogenase-like complex-mediated cyclic electron flow is the main electron transport route in C4 bundle sheath cells.

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.

Unsaturation in the air spaces of leaves and its implications.

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.

C4 maize and sorghum are more sensitive to rapid dehydration than C3 wheat and sunflower.

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.

Assessing the CO2 concentration at the surface of photosynthetic mesophyll cells.

We present a robust estimation of the CO2 concentration at the surface of photosynthetic mesophyll cells (cw ), applicable under reasonable assumptions of assimilation distribution within the leaf. We used Capsicum annuum, Helianthus annuus and Gossypium hirsutumas model plants for our experiments. We introduce calculations to estimate cw using independent adaxial and abaxial gas exchange measurements, and accounting for the mesophyll airspace resistances. The cw was lower than adaxial and abaxial estimated intercellular CO2 concentrations (ci ). Differences between cw and the ci of each surface were usually larger than 10 µmol mol-1 . Differences between adaxial and abaxial ci 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 ci and the ci 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 CO2 concentrations. Accounting for cw improves the information that can be extracted from gas exchange experiments, allowing a more detailed description of the CO2 and water vapor gradients within the leaf.

Linking water use efficiency with water use strategy from leaves to communities.

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.

A cross-scale analysis to understand and quantify the effects of photosynthetic enhancement on crop growth and yield across environments.

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (Ac ) and electron transport-limited (Aj ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C3 wheat and C4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing Ac alone generate more consistent but smaller yield gains across all water and nitrogen environments, Aj enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both Ac and Aj generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.

Ribulose 1,5-bisphosphate carboxylase/oxygenase activates O2 by electron transfer.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of atmospheric CO2 fixation by the biosphere. It catalyzes the addition of CO2 onto enolized ribulose 1,5-bisphosphate (RuBP), producing 3-phosphoglycerate which is then converted to sugars. The major problem of this reaction is competitive O2 addition, which forms a phosphorylated product (2-phosphoglycolate) that must be recycled by a series of biochemical reactions (photorespiratory metabolism). However, the way the enzyme activates O2 is still unknown. Here, we used isotope effects (with 2H, 25Mg, and 18O) to monitor O2 activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O2/CO2 selectivity. Neither the Rubisco form nor the use of solvent D2O and deuterated RuBP changed the 16O/18O isotope effect of O2 addition, in clear contrast with the 12C/13C isotope effect of CO2 addition. Furthermore, substitution of light magnesium (24Mg) by heavy, nuclear magnetic 25Mg had no effect on O2 addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O2) does not seem to be involved in O2 activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O2•-) production, must be insensitive to deuteration, and yields a predicted 16O/18O isotope effect and energy barrier close to observed values. Overall, O2 undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate.

Cuticular conductance of adaxial and abaxial leaf surfaces and its relation to minimum leaf surface conductance.

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.

Multienvironment QTL analysis delineates a major locus associated with homoeologous exchanges for water-use efficiency and seed yield in canola.

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.

Integrating the evidence for a terrestrial carbon sink caused by increasing atmospheric CO2.

Atmospheric carbon dioxide concentration ([CO2 ]) is increasing, which increases leaf-scale photosynthesis and intrinsic water-use efficiency. These direct responses have the potential to increase plant growth, vegetation biomass, and soil organic matter; transferring carbon from the atmosphere into terrestrial ecosystems (a carbon sink). A substantial global terrestrial carbon sink would slow the rate of [CO2 ] increase and thus climate change. However, ecosystem CO2 responses are complex or confounded by concurrent changes in multiple agents of global change and evidence for a [CO2 ]-driven terrestrial carbon sink can appear contradictory. Here we synthesize theory and broad, multidisciplinary evidence for the effects of increasing [CO2 ] (iCO2 ) on the global terrestrial carbon sink. Evidence suggests a substantial increase in global photosynthesis since pre-industrial times. Established theory, supported by experiments, indicates that iCO2 is likely responsible for about half of the increase. Global carbon budgeting, atmospheric data, and forest inventories indicate a historical carbon sink, and these apparent iCO2 responses are high in comparison to experiments and predictions from theory. Plant mortality and soil carbon iCO2 responses are highly uncertain. In conclusion, a range of evidence supports a positive terrestrial carbon sink in response to iCO2 , albeit with uncertain magnitude and strong suggestion of a role for additional agents of global change.

The effects on isotopic composition of leaf water and transpiration of adding a gas-exchange cuvette.

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.

Can hydraulic design explain patterns of leaf water isotopic enrichment in C3 plants?

H2 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 H2 18 O develop within leaves, influencing bulk leaf stable oxygen isotope enrichment (ΔL ) and the degree to which the Péclet effect is relevant in leaves. Leaf hydraulic design predicted the relevance of a Péclet effect to ΔL 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 H2 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 H2 18 O are very small. Prior knowledge of leaf hydraulic design allows informed selection of the appropriate ΔL modelling framework.

Rainfall statistics, stationarity, and climate change.

There is a growing research interest in the detection of changes in hydrologic and climatic time series. Stationarity can be assessed using the autocorrelation function, but this is not yet common practice in hydrology and climate. Here, we use a global land-based gridded annual precipitation (hereafter P) database (1940-2009) and find that the lag 1 autocorrelation coefficient is statistically significant at around 14% of the global land surface, implying nonstationary behavior (90% confidence). In contrast, around 76% of the global land surface shows little or no change, implying stationary behavior. We use these results to assess change in the observed P over the most recent decade of the database. We find that the changes for most (84%) grid boxes are within the plausible bounds of no significant change at the 90% CI. The results emphasize the importance of adequately accounting for natural variability when assessing change.

Two-Source δ18O Method to Validate the CO18O-Photosynthetic Discrimination Model: Implications for Mesophyll Conductance.

Theoretical models of photosynthetic isotopic discrimination of CO2 (13C and 18O) are commonly used to estimate mesophyll conductance (g m). This requires making simplifying assumptions and assigning parameter values so that g m can be solved for as the residual term. Uncertainties in g m estimation occur due to measurement noise and assumptions not holding, including parameter uncertainty and model parametrization. Uncertainties in the 13C model have been explored previously, but there has been little testing undertaken to determine the reliability of g m estimates from the 18O model (g m18). In this study, we exploited the action of carbonic anhydrase in equilibrating CO2 with leaf water and manipulated the observed photosynthetic discrimination (Δ18O) by changing the oxygen isotopic composition of the source gas CO2 and water vapor. We developed a two-source δ18O method, whereby two measurements of Δ18O were obtained for a leaf with its gas-exchange characteristics otherwise unchanged. Measurements were performed in broad bean (Vicia faba) and Algerian oak (Quercus canariensis) in response to light and vapor pressure deficit. Despite manipulating the Δ18O by over 100‰, in most cases we observed consistency in the calculated g m18, providing confidence in the measurements and model theory. Where there were differences in g m18 estimates between source-gas measurements, we explored uncertainty associated with two model assumptions (the isotopic composition of water at the sites of CO2-water exchange, and the humidity of the leaf internal airspace) and found evidence for both. Finally, we provide experimental guidelines to minimize the sensitivity of g m18 estimates to measurement errors. The two-source δ18O method offers a flexible tool for model parameterization and provides an opportunity to refine our understanding of leaf water and CO2 fluxes.

Directional change in leaf dry matter δ 13C during leaf development is widespread in C3 plants.

BACKGROUND AND AIMS: The stable carbon isotope ratio of leaf dry matter (δ 13Cp) is generally a reliable recorder of intrinsic water-use efficiency in C3 plants. Here, we investigated a previously reported pattern of developmental change in leaf δ 13Cp during leaf expansion, whereby emerging leaves are initially 13C-enriched compared to mature leaves on the same plant, with their δ 13Cp decreasing during leaf expansion until they eventually take on the δ 13Cp of other mature leaves. METHODS: We compiled data to test whether the difference between mature and young leaf δ 13Cp differs between temperate and tropical species, or between deciduous and evergreen species. We also tested whether the developmental change in δ 13Cp is indicative of a concomitant change in intrinsic water-use efficiency. To gain further insight, we made online measurements of 13C discrimination (∆ 13C) in young and mature leaves. KEY RESULTS: We found that the δ 13Cp difference between mature and young leaves was significantly larger for deciduous than for evergreen species (-2.1 ‰ vs. -1.4 ‰, respectively). Counter to expectation based on the change in δ 13Cp, intrinsic water-use efficiency did not decrease between young and mature leaves; rather, it did the opposite. The ratio of intercellular to ambient CO2 concentrations (ci/ca) was significantly higher in young than in mature leaves (0.86 vs. 0.72, respectively), corresponding to lower intrinsic water-use efficiency. Accordingly, instantaneous ∆ 13C was also higher in young than in mature leaves. Elevated ci/ca and ∆ 13C in young leaves resulted from a combination of low photosynthetic capacity and high day respiration rates. CONCLUSION: The decline in leaf δ 13Cp during leaf expansion appears to reflect the addition of the expanding leaf's own 13C-depleted photosynthetic carbon to that imported from outside the leaf as the leaf develops. This mixing of carbon sources results in an unusual case of isotopic deception: less negative δ 13Cp in young leaves belies their low intrinsic water-use efficiency.

A leaf-level biochemical model simulating the introduction of C2 and C4 photosynthesis in C3 rice: gains, losses and metabolite fluxes.

This work aims at developing an adequate theoretical basis for comparing assimilation of the ancestral C3 pathway with CO2 concentrating mechanisms (CCM) that have evolved to reduce photorespiratory yield losses. We present a novel model for C3 , C2 , C2  + C4 and C4 photosynthesis simulating assimilatory metabolism, energetics and metabolite traffic at the leaf level. It integrates a mechanistic description of light reactions to simulate ATP and NADPH production, and a variable engagement of cyclic electron flow. The analytical solutions are compact and thus suitable for larger scale simulations. Inputs were derived with a comprehensive gas-exchange experiment. We show trade-offs in the operation of C4 that are in line with ecophysiological data. C4 has the potential to increase assimilation over C3 at high temperatures and light intensities, but this benefit is reversed under low temperatures and light. We apply the model to simulate the introduction of progressively complex levels of CCM into C3 rice, which feeds > 3.5 billion people. Increasing assimilation will require considerable modifications such as expressing the NAD(P)H Dehydrogenase-like complex and upregulating cyclic electron flow, enlarging the bundle sheath, and expressing suitable transporters to allow adequate metabolite traffic. The simpler C2 rice may be a desirable alternative.

Plant water-use strategy mediates stomatal effects on the light induction of photosynthesis.

More efficient gas exchange strategies under dynamic light environments have been hypothesised to contribute to the dominance of angiosperms in the vascular plant flora. However, we still lack a clear understanding of how stomatal dynamics affect photosynthetic dynamics and whether differences exist between lineages. Stomatal and photosynthetic dynamics following changes in irradiance were studied in 15 species, encompassing ferns, gymnosperms and angiosperms. We determined the effect of stomatal speed on dynamic photosynthesis and water loss. Moreover, we assessed whether dynamic behaviour followed evolutionary lineage divisions, or whether ecological adaptation to maximise light fleck use could describe dynamic behaviour. We found that species with fast stomatal opening, such as ferns, forgo less photosynthesis during photosynthetic induction. By contrast, there was no relationship between stomatal closure speed and the water wasted by transiently more-open stomata, because species with higher rates of gas exchange also showed faster stomatal closure. Shade-adapted species possessed fast-opening but slow-closing stomata, consistent with ecological adaptation to maximise light fleck use. Our results suggest dynamic behaviour follows adaptive ecological trends more strongly than evolutionary ones, but angiosperms may benefit from relatively faster photosynthetic induction by adopting a less conservative water-use strategy.