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Leaf-level gas exchange data support the mechanistic understanding of plant fluxes of carbon and water. These fluxes inform our understanding of ecosystem function, are an important constraint on parameterization of terrestrial biosphere models, are necessary to understand the response of plants to global environmental change, and are integral to efforts to improve crop production. Collection of these data using gas analyzers can be both technically challenging and time consuming, and individual studies generally focus on a small range of species, restricted time periods, or limited geographic regions. The high value of these data is exemplified by the many publications that reuse and synthesize gas exchange data, however the lack of metadata and data reporting conventions make full and efficient use of these data difficult. Here we propose a reporting format for leaf-level gas exchange data and metadata to provide guidance to data contributors on how to store data in repositories to maximize their discoverability, facilitate their efficient reuse, and add value to individual datasets. For data users, the reporting format will better allow data repositories to optimize data search and extraction, and more readily integrate similar data into harmonized synthesis products. The reporting format specifies data table variable naming and unit conventions, as well as metadata characterizing experimental conditions and protocols. For common data types that were the focus of this initial version of the reporting format, i.e., survey measurements, dark respiration, carbon dioxide and light response curves, and parameters derived from those measurements, we took a further step of defining required additional data and metadata that would maximize the potential reuse of those data types. To aid data contributors and the development of data ingest tools by data repositories we provided a translation table comparing the outputs of common gas exchange instruments. Extensive consultation with data collectors, data users, instrument manufacturers, and data scientists was undertaken in order to ensure that the reporting format met community needs. The reporting format presented here is intended to form a foundation for future development that will incorporate additional data types and variables as gas exchange systems and measurement approaches advance in the future. The reporting format is published in the U.S. Department of Energy's ESS-DIVE data repository, with documentation and future development efforts being maintained in a version control system.

Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall (CW) properties might help improve photosynthesis and crop water use efficiency (WUE). We tested this using 2 independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a "BAHD" CoA acyltransferase), which alters CW hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, CW composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll CW thickness (-14%) and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic WUE (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in aboveground biomass (AGB) (+12.5%) and canopy-level WUE (+8.8%; ratio of AGB to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in CW composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic WUE will need to enhance mesophyll conductance and photosynthesis while maintaining or decreasing stomatal conductance., Competing Interests: Conflict of interest statement. The authors declare no conflict of interests., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Environmental variability poses a major challenge to any field study. Researchers attempt to mitigate this challenge through replication. Thus, the ability to detect experimental signals is determined by the degree of replication and the amount of environmental variation, noise, within the experimental system. A major source of noise in field studies comes from the natural heterogeneity of soil properties which create microtreatments throughout the field. In addition, the variation within different soil properties is often nonrandomly distributed across a field. We explore this challenge through a sorghum field trial dataset with accompanying plant, microbiome, and soil property data. Diverse sorghum genotypes and two watering regimes were applied in a split-plot design. We describe a process of identifying, estimating, and controlling for the effects of spatially distributed soil properties on plant traits and microbial communities using minimal degrees of freedom. Importantly, this process provides a method with which sources of environmental variation in field data can be identified and adjusted, improving our ability to resolve effects of interest and to quantify subtle phenotypes., Competing Interests: JB, MQ, BS, AS, AC, JP, DS, PL No competing interests declared, RB Reviewing editor, eLife, (© 2022, Berry et al.)

Leaf hydraulic and mesophyll CO 2 conductance are both influenced by leaf anatomical traits, however it is poorly understood how the temperature response of these conductances differs between C 4 and C 3 species with distinct leaf anatomy. This study investigated the temperature response of leaf hydraulic conductance (K leaf ), stomatal (g s ) and mesophyll (g m ) conductance to CO 2 , and leaf anatomical traits in phylogenetically related Panicum antidotale (C 4 ) and P. bisulcatum (C 3 ) grasses. The C 4 species had lower hydraulic conductance outside xylem (K ox ) and K leaf compared with the C 3 species. However, the C 4 species had higher g m compared with the C 3 species. Traits associated with leaf water movement, K leaf and K ox , increased with temperature more in the C 3 than in the C 4 species, whereas traits related to carbon uptake, A net and g m , increased more with temperature in the C 4 than the C 3 species. Our findings demonstrate that, in addition to a CO 2 concentrating mechanism, outside-xylem leaf anatomy in the C 4 species P. antidotale favours lower water movement through the leaf and stomata that provides an additional advantage for greater leaf carbon uptake relative to water loss with increasing leaf temperature than in the C 3 species P. bisulcatum., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

In habitats with low water availability, a fundamental challenge for plants will be to maximize photosynthetic C-gain while minimizing transpirational water-loss. This trade-off between C-gain and water-loss can in part be achieved through the coordination of leaf-level photosynthetic and hydraulic traits. To test the relationship of photosynthetic C-gain and transpirational water-loss, we grew, under common growth conditions, 18 C 4 grasses adapted to habitats with different mean annual precipitation (MAP) and measured leaf-level structural and anatomical traits associated with mesophyll conductance (g m ) and leaf hydraulic conductance (K leaf ). The C 4 grasses adapted to lower MAP showed greater mesophyll surface area exposed to intercellular air spaces (S mes ) and adaxial stomatal density (SD ada ) which supported greater g m . These grasses also showed greater leaf thickness and vein-to-epidermis distance, which may lead to lower K leaf . Additionally, grasses with greater g m and lower K leaf also showed greater photosynthetic rates (A net ) and leaf-level water-use efficiency (WUE). In summary, we identify a suite of leaf-level traits that appear important for adaptation of C 4 grasses to habitats with low MAP and may be useful to identify C 4 species showing greater A net and WUE in drier conditions., (© 2020 John Wiley & Sons Ltd.)

Breeding economically important C 4 crops for enhanced whole-plant water-use efficiency (WUE plant ) is needed for sustainable agriculture. WUE plant is a complex trait and an efficient phenotyping method that reports on components of WUE plant , such as intrinsic water-use efficiency (WUE i , the rate of leaf CO 2 assimilation relative to water loss via stomatal conductance), is needed. In C 4 plants, theoretical models suggest that leaf carbon isotope composition (δ 13 C), when the efficiency of the CO 2 -concentrating mechanism (leakiness, ϕ) remains constant, can be used to screen for WUE i . The limited information about how ϕ responds to water limitations confines the application of δ 13 C for WUE i screening of C 4 crops. The current research aimed to test the response of ϕ to short- or long-term moderate water limitations, and the relationship of δ 13 C with WUE i and WUE plant , by addressing potential mesophyll CO 2 conductance (g m ) and biochemical limitations in the C 4 plant Sorghum bicolor. We demonstrate that g m and ϕ are not responsive to short- or long-term water limitations. Additionally, δ 13 C was not correlated with gas-exchange estimates of WUE i under short- and long-term water limitations, but showed a significant negative relationship with WUE plant . The observed association between the δ 13 C and WUE plant suggests an intrinsic link of δ 13 C with WUE i in this C 4 plant, and can potentially be used as a screening tool for WUE plant in sorghum., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

The conductance of carbon dioxide (CO 2 ) from the substomatal cavities to the initial sites of CO 2 fixation (g m ) can significantly reduce the availability of CO 2 for photosynthesis. There have been many recent reviews on: (i) the importance of g m for accurately modelling net rates of CO 2 assimilation, (ii) on how leaf biochemical and anatomical factors influence g m , (iii) the technical limitation of estimating g m , which cannot be directly measured, and (iv) how g m responds to long- and short-term changes in growth and measurement environmental conditions. Therefore, this review will highlight these previous publications but will attempt not to repeat what has already been published. We will instead initially focus on the recent developments on the two-resistance model of g m that describe the potential of photorespiratory and respiratory CO 2 released within the mitochondria to diffuse directly into both the chloroplast and the cytosol. Subsequently, we summarize recent developments in the three-dimensional (3-D) reaction-diffusion models and 3-D image analysis that are providing new insights into how the complex structure and organization of the leaf influences g m . Finally, because most of the reviews and literature on g m have traditionally focused on C 3 plants we review in the final sections some of the recent developments, current understanding and measurement techniques of g m in C 4 and crassulacean acid metabolism (CAM) plants. These plants have both specialized leaf anatomy and either a spatially or temporally separated CO 2 concentrating mechanisms (C 4 and CAM, respectively) that influence how we interpret and estimate g m compared with a C 3 plants., (© 2020 The Authors The Plant Journal © 2020 John Wiley & Sons Ltd.)

The internal CO 2 gradient imposed by mesophyll conductance (g m ) reduces substrate availability for C 3 photosynthesis. With several assumptions, estimates of g m can be made from coupled leaf gas exchange with isoflux analysis of carbon ∆ 13 C-g m and oxygen in CO 2 , coupled with transpired water (H 2 O) ∆ 18 O-g m to partition g m into its biochemical and anatomical components. However, these assumptions require validation under changing leaf temperatures. To test these assumptions, we measured and modeled the temperature response (15-40°C) of ∆ 13 C-g m and ∆ 18 O-g m along with leaf biochemistry in the C 3 grass Panicum bisulcatum, which has naturally low carbonic anhydrase activity. Our study suggests that assumptions regarding the extent of isotopic equilibrium (θ) between CO 2 and H 2 O at the site of exchange, and that the isotopic composition of the H 2 O at the sites of evaporation ( δ w - e 18 ) and at the site of exchange ( δ w - ce 18 ) are similar, may lead to errors in estimating the ∆ 18 O-g m temperature response. The input parameters for ∆ 13 C-g m appear to be less sensitive to temperature. However, this needs to be tested in species with diverse carbonic anhydrase activity. Additional information on the temperature dependency of cytosolic and chloroplastic pH may clarify uncertainties used for ∆ 18 O-g m under changing leaf temperatures., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

The high energy cost and apparently low plasticity of C4 photosynthesis compared with C3 photosynthesis may limit the productivity and distribution of C4 plants in low light (LL) environments. C4 photosynthesis evolved numerous times, but it remains unclear how different biochemical subtypes perform under LL. We grew eight C4 grasses belonging to three biochemical subtypes [NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK)] under shade (16% sunlight) or control (full sunlight) conditions and measured their photosynthetic characteristics at both low and high light. We show for the first time that LL (during measurement or growth) compromised the CO2-concentrating mechanism (CCM) to a greater extent in NAD-ME than in PEP-CK or NADP-ME C4 grasses by virtue of a greater increase in carbon isotope discrimination (∆P) and bundle sheath CO2 leakiness (ϕ), and a greater reduction in photosynthetic quantum yield (Φmax). These responses were partly explained by changes in the ratios of phosphoenolpyruvate carboxylase (PEPC)/initial Rubisco activity and dark respiration/photosynthesis (Rd/A). Shade induced a greater photosynthetic acclimation in NAD-ME than in NADP-ME and PEP-CK species due to a greater Rubisco deactivation. Shade also reduced plant dry mass to a greater extent in NAD-ME and PEP-CK relative to NADP-ME grasses. In conclusion, LL compromised the co-ordination of the C4 and C3 cycles and, hence, the efficiency of the CCM to a greater extent in NAD-ME than in PEP-CK species, while CCM efficiency was less impacted by LL in NADP-ME species. Consequently, NADP-ME species are more efficient at LL, which could explain their agronomic and ecological dominance relative to other C4 grasses.

C4 photosynthesis evolved independently many times, resulting in multiple biochemical pathways; however, little is known about how these different pathways respond to temperature. We investigated the photosynthetic responses of eight C4 grasses belonging to three biochemical subtypes (NADP-ME, PEP-CK, and NAD-ME) to four leaf temperatures (18, 25, 32, and 40 °C). We also explored whether the biochemical subtype influences the thermal responses of (i) in vitro PEPC (Vpmax) and Rubisco (Vcmax) maximal activities, (ii) initial slope (IS) and CO2-saturated rate (CSR) derived from the A-Ci curves, and (iii) CO2 leakage out of the bundle sheath estimated from carbon isotope discrimination. We focussed on leakiness and the two carboxylases because they determine the coordination of the CO2-concentrating mechanism and are important for parameterizing the semi-mechanistic C4 photosynthesis model. We found that the thermal responses of Vpmax and Vcmax, IS, CSR, and leakiness varied among the C4 species independently of the biochemical subtype. No correlation was observed between Vpmax and IS or between Vcmax and CSR; while the ratios Vpmax/Vcmax and IS/CSR did not correlate with leakiness among the C4 grasses. Determining mesophyll and bundle sheath conductances in diverse C4 grasses is required to further elucidate how C4 photosynthesis responds to temperature., (© Society for Experimental Biology 2017.)

The intimate association between the arbuscular mycorrhizal fungi and host plants helps the latter in phosphate acquisition in exchange of carbohydrates and in enhanced stress tolerance. Similarly, the ubiquitous 14-3-3 protein family is known to be a major regulator of plant metabolism and stress responses. However, the involvement of mycorrhiza and plant 14-3-3 proteins interaction in plant response to environmental stimuli, such as arsenic (As) stress, is yet unknown. In this study, we analysed the impact of the As stress on the expression profile of 14-3-3 genes in the shoot of mycorrhiza colonized rice ( Oryza sativa ) plants. Ten day old rice seedlings were kept for 45 days for mycorrhizal colonisation (10 g inoculum per 120 g soilrite) and were then subjected to 12.5 µM arsenate [As(V)] exposure for 1 and 3 days, in hydroponics. Arsenate stress resulted in significant change in expression of 14-3-3 protein genes in non-colonized and mycorrhiza colonized rice plants which indicated As mediated effects on 14-3-3 proteins as well as interactive impact of mycorrhiza colonization. Indeed, mycorrhiza colonization itself induced up-regulation of all 14-3-3 genes in the absence of As stress. The results thus indicate that 14-3-3 proteins might be involved in As stress signalling and the mycorrhiza induced As stress response of the rice plants., Competing Interests: Authors declare that they have no conflict of interest.

Plants operating C3 and C4 photosynthetic pathways exhibit differences in leaf anatomy and photosynthetic carbon fixation biochemistry. Fully understanding this underpinning biochemical variation is requisite to identifying solutions for improving photosynthetic efficiency and growth. Here we refine assay methods for accurately measuring the carboxylase and decarboxylase activities in C3 and C4 plant soluble protein. We show that differences in plant extract preparation and assay conditions are required to measure NADP-malic enzyme and phosphoenolpyruvate carboxylase (pH 8, Mg(2+), 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2mM Mn(2+), no Mg(2+)) maximal activities accurately. We validate how the omission of MgCl2 during leaf protein extraction, lengthy (>1min) centrifugation times, and the use of non-pure ribulose-1,5-bisphosphate (RuBP) significantly underestimate Rubisco activation status. We show how Rubisco activation status varies with leaf ontogeny and is generally lower in mature C4 monocot leaves (45-60% activation) relative to C3 monocots (55-90% activation). Consistent with their >3-fold lower Rubisco contents, full Rubisco activation in soluble protein from C4 leaves (<5min) was faster than in C3 plant samples (<10min), with addition of Rubisco activase not required for full activation. We conclude that Rubisco inactivation in illuminated leaves primarily stems from RuBP binding to non-carbamylated enzyme, a state readily reversible by dilution during cellular protein extraction., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

C4 photosynthesis involves a close collaboration of the C3 and C4 metabolic cycles across the mesophyll and bundle-sheath cells. This study investigated the coordination of C4 photosynthesis in maize plants subjected to two salinity (50 and 100mM NaCl) treatments and one shade (20% of full sunlight) treatment. Photosynthetic efficiency was probed by combining leaf gas-exchange measurements with carbon isotope discrimination and assaying the key carboxylases [ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [nicotinamide adenine dinucleotide phosphate malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] operating in maize leaves. Generally, salinity inhibited plant growth and photosynthesis to a lesser extent than shade. Salinity reduced photosynthesis primarily by reducing stomatal conductance and secondarily by equally reducing Rubisco and PEPC activities; the decarboxylases were inhibited more than the carboxylases. Salinity increased photosynthetic carbon isotope discrimination (Δp) and reduced leaf dry-matter carbon isotope composition ((13)δ) due to changes in p i/p a (intercellular to ambient CO2 partial pressure), while CO2 leakiness out of the bundle sheath (ϕ) was similar to that in control plants. Acclimation to shade was underpinned by a greater downregulation of PEPC relative to Rubisco activity, and a lesser inhibition of NADP-ME (primary decarboxylase) relative to PEP-CK (secondary decarboxylase). Shade reduced Δp and ɸ without significantly affecting leaf (13)δ or p i/p a relative to control plants. Accordingly, shade perturbed the balance between the C3 and C4 cycles during photosynthesis in maize, and demonstrated the flexible partitioning of C4 acid decarboxylation activity between NADP-ME and PEP-CK in response to the environment. This study highlights the need to improve our understanding of the links between leaf (13)δ and photosynthetic Δp, and the role of the secondary decarboxylase PEP-CK in NADP-ME plants such as maize., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)