The importance of species-specific and temperature-sensitive parameterisation of A/Ci models: A case study using cotton (Gossypium hirsutum L.) and the automated 'OptiFitACi' R-package.

Leaf gas exchange measurements are an important tool for inferring a plant's photosynthetic biochemistry. In most cases, the responses of photosynthetic CO2 assimilation to variable intercellular CO2 concentrations (A/Ci response curves) are used to model the maximum (potential) rate of carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, Vcmax) and the rate of photosynthetic electron transport at a given incident photosynthetically active radiation flux density (PAR; JPAR). The standard Farquhar-von Caemmerer-Berry model is often used with default parameters of Rubisco kinetic values and mesophyll conductance to CO2 (gm) derived from tobacco that may be inapplicable across species. To study the significance of using such parameters for other species, here we measured the temperature responses of key in vitro Rubisco catalytic properties and gm in cotton (Gossypium hirsutum cv. Sicot 71) and derived Vcmax and J2000 (JPAR at 2000 µmol m-2 s-1 PAR) from cotton A/Ci curves incrementally measured at 15°C-40°C using cotton and other species-specific sets of input parameters with our new automated fitting R package 'OptiFitACi'. Notably, parameterisation by a set of tobacco parameters produced unrealistic J2000:Vcmax ratio of <1 at 25°C, two- to three-fold higher estimates of Vcmax above 15°C, up to 2.3-fold higher estimates of J2000 and more variable estimates of Vcmax and J2000, for our cotton data compared to model parameterisation with cotton-derived values. We determined that errors arise when using a gm,25 of 2.3 mol m-2 s-1 MPa-1 or less and Rubisco CO2-affinities in 21% O2 (KC 21%O2) at 25°C outside the range of 46-63 Pa to model A/Ci responses in cotton. We show how the A/Ci modelling capabilities of 'OptiFitACi' serves as a robust, user-friendly, and flexible extension of 'plantecophys' by providing simplified temperature-sensitivity and species-specificity parameterisation capabilities to reduce variability when modelling Vcmax and J2000.

Does the response of Rubisco and photosynthesis to elevated [CO2] change with unfavourable environmental conditions?

Climate change due to anthropogenic CO2 emissions affects plant performance globally. To improve crop resilience, we need to understand the effects of elevated CO2 concentration (e[CO2]) on CO2 assimilation and Rubisco biochemistry. However, the interactive effects of e[CO2] and abiotic stress are especially unclear. This study analyses the CO2 effect on photosynthetic capacity under different water availability and temperature conditions in 42 different crop species, varying in functional group, photosynthetic pathway and phenological stage. We analysed close to 3000 data points extracted from 120 published manuscripts. For C3 species, e[CO2] increases net photosynthesis and intercellular [CO2], while reducing stomatal conductance and transpiration. Vmaxc, Rubisco in vitro extractable maximal activity and content also decrease with e[CO2] in C3 species, while C4 crops are less responsive to e[CO2]. The interaction with drought and/or heat stress does not significantly alter these photosynthetic responses, indicating that the photosynthetic capacity of stressed plants responds to e[CO2]. Moreover, e[CO2] has strong effect on the photosynthetic capacity of grasses mainly in the final stages of development. This study provides insight into the intricate interactions within the plant photosynthetic apparatus under the influence of climate change, enhancing the understanding of mechanisms governing plant responses to environmental parameters.

Elucidating the role of SWEET13 in phloem loading of the C4 grass Setaria viridis.

Photosynthetic efficiency and sink demand are tightly correlated with rates of phloem loading, where maintaining low cytosolic sugar concentrations is paramount to prevent the downregulation of photosynthesis. Sugars Will Eventually be Exported Transporters (SWEETs) are thought to have a pivotal role in the apoplastic phloem loading of C4 grasses. SWEETs have not been well studied in C4 species, and their investigation is complicated by photosynthesis taking place across two cell types and, therefore, photoassimilate export can occur from either one. SWEET13 homologues in C4 grasses have been proposed to facilitate apoplastic phloem loading. Here, we provide evidence for this hypothesis using the C4 grass Setaria viridis. Expression analyses on the leaf gradient of C4 species Setaria and Sorghum bicolor show abundant transcript levels for SWEET13 homologues. Carbohydrate profiling along the Setaria leaf shows total sugar content to be significantly higher in the mature leaf tip compared with the younger tissue at the base. We present the first known immunolocalization results for SvSWEET13a and SvSWEET13b using novel isoform-specific antisera. These results show localization to the bundle sheath and phloem parenchyma cells of both minor and major veins. We further present the first transport kinetics study of C4 monocot SWEETs by using a Xenopus laevis oocyte heterologous expression system. We demonstrate that SvSWEET13a and SvSWEET13b are high-capacity transporters of glucose and sucrose, with a higher apparent Vmax for sucrose, compared with glucose, typical of clade III SWEETs. Collectively, these results provide evidence for an apoplastic phloem loading pathway in Setaria and possibly other C4 species.

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.

Rubisco, the imperfect winner: it's all about the base.

Rubisco catalysis is complex and includes an activation step through the formation of a carbamate at the conserved active site lysine residue and the formation of a highly reactive enediol that is the key to its catalytic reaction. The formation of this enediol is both the basis of its success and its Achilles' heel, creating imperfections to its catalytic efficiency. While Rubisco originally evolved in an atmosphere of high CO2, the earth's multiple oxidation events provided challenges to Rubisco through the fixation of O2 that competes with CO2 at the active site. Numerous catalytic screens across the Rubisco superfamily have identified significant variation in catalytic properties that have been linked to large and small subunit sequences. Despite this, we still have a rudimentary understanding of Rubisco's catalytic mechanism and how the evolution of kinetic properties has occurred. This review identifies the lysine base that functions both as an activator and a proton abstractor to create the enediol as a key to understanding how Rubisco may optimize its kinetic properties. The ways in which Rubisco and its partners have overcome catalytic and activation imperfections and thrived in a world of high O2, low CO2, and variable climatic regimes is remarkable.

The role of SWEET4 proteins in the post-phloem sugar transport pathway of Setaria viridis sink tissues.

In the developing seeds of all higher plants, filial cells are symplastically isolated from the maternal tissue supplying photosynthate to the reproductive structure. Photoassimilates must be transported apoplastically, crossing several membrane barriers, a process facilitated by sugar transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been proposed to play a crucial role in apoplastic sugar transport during phloem unloading and the post-phloem pathway in sink tissues. Evidence for this is presented here for developing seeds of the C4 model grass Setaria viridis. Using immunolocalization, SvSWEET4 was detected in various maternal and filial tissues within the seed along the sugar transport pathway, in the vascular parenchyma of the pedicel, and in the xylem parenchyma of the stem. Expression of SvSWEET4a in Xenopus laevis oocytes indicated that it functions as a high-capacity glucose and sucrose transporter. Carbohydrate and transcriptional profiling of Setaria seed heads showed that there were some developmental shifts in hexose and sucrose content and consistent expression of SvSWEET4 homologues. Collectively, these results provide evidence for the involvement of SWEETs in the apoplastic transport pathway of sink tissues and allow a pathway for post-phloem sugar transport into the seed to be proposed.

Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop an RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a rbcS gene whose codon use and 5' untranslated-region matched rbcL Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pST-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pST However, the ßA-ßB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15%, respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.

Mining for allelic gold: finding genetic variation in photosynthetic traits in crops and wild relatives.

Improvement of photosynthetic traits in crops to increase yield potential and crop resilience has recently become a major breeding target. Synthetic biology and genetic technologies offer unparalleled opportunities to create new genetics for photosynthetic traits driven by existing fundamental knowledge. However, large 'gene bank' collections of germplasm comprising historical collections of crop species and their relatives offer a wealth of opportunities to find novel allelic variation in the key steps of photosynthesis, to identify new mechanisms and to accelerate genetic progress in crop breeding programmes. Here we explore the available genetic resources in food and fibre crops, strategies to selectively target allelic variation in genes underpinning key photosynthetic processes, and deployment of this variation via gene editing in modern elite material.

Increased Rubisco content in maize mitigates chilling stress and speeds recovery.

Many C4 plants, including maize, perform poorly under chilling conditions. This phenomenon has been linked in part to decreased Rubisco abundance at lower temperatures. An exception to this is chilling-tolerant Miscanthus, which is able to maintain Rubisco protein content under such conditions. The goal of this study was to investigate whether increasing Rubisco content in maize could improve performance during or following chilling stress. Here, we demonstrate that transgenic lines overexpressing Rubisco large and small subunits and the Rubisco assembly factor RAF1 (RAF1-LSSS), which have increased Rubisco content and growth under control conditions, maintain increased Rubisco content and growth during chilling stress. RAF1-LSSS plants exhibited 12% higher CO2 assimilation relative to nontransgenic controls under control growth conditions, and a 17% differential after 2 weeks of chilling stress, although assimilation rates of all genotypes were ~50% lower in chilling conditions. Chlorophyll fluorescence measurements showed RAF1-LSSS and WT plants had similar rates of photochemical quenching during chilling, suggesting Rubisco may not be the primary limiting factor that leads to poor performance in maize under chilling conditions. In contrast, RAF1-LSSS had improved photochemical quenching before and after chilling stress, suggesting that increased Rubisco may help plants recover faster from chilling conditions. Relatively increased leaf area, dry weight and plant height observed before chilling in RAF1-LSSS were also maintained during chilling. Together, these results demonstrate that an increase in Rubisco content allows maize plants to better cope with chilling stress and also improves their subsequent recovery, yet additional modifications are required to engineer chilling tolerance in maize.

The role of leaf width and conductances to CO2 in determining water use efficiency in C4 grasses.

C4 plants achieve higher photosynthesis (An ) and intrinsic water use efficiency (iWUE) than C3 plants, but processes underpinning the variability in An and iWUE across the three C4 subtypes remain unclear, partly because we lack an integrated framework for quantifying the contribution of diffusional and biochemical limitations to C4 photosynthesis. We exploited the natural diversity among C4 grasses to develop an original mathematical approach for estimating eight key processes of C4 photosynthesis and their relative limitations to An . We also developed a new formulation to estimate mesophyll conductance (gm ) based on actual hydration rates of CO2 by carbonic anhydrases. We found a positive relationship between gm and iWUE and an inverse correlation with gsw among C4 grasses. We also revealed subtype-specific regulatory processes of iWUE that may be related to known anatomical traits characterising each C4 subtype. Leaf width was an important determinant of iWUE and showed significant correlations with key limitations of An , especially among NADP-ME species. In conclusion, incorporating leaf width in breeding trials may unlock new opportunities for C4 crops because the revealed negative relationship between leaf width and iWUE may translate into higher crop and canopy WUE.

Diffusion of CO2 across the Mesophyll-Bundle Sheath Cell Interface in a C4 Plant with Genetically Reduced PEP Carboxylase Activity.

Phosphoenolpyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C4 photosynthetic pathway. Here, we used RNA interference to target the cytosolic photosynthetic PEPC isoform in Setaria viridis and isolated independent transformants with very low PEPC activities. These plants required high ambient CO2 concentrations for growth, consistent with the essential role of PEPC in C4 photosynthesis. The combination of estimating direct CO2 fixation by the bundle sheath using gas-exchange measurements and modeling C4 photosynthesis with low PEPC activity allowed the calculation of bundle sheath conductance to CO2 diffusion (gbs ) in the progeny of these plants. Measurements made at a range of temperatures suggested no or negligible effect of temperature on gbs depending on the technique used to calculate gbs Anatomical measurements revealed that plants with reduced PEPC activity had reduced cell wall thickness and increased plasmodesmata (PD) density at the mesophyll-bundle sheath (M-BS) cell interface, whereas we observed little difference in these parameters at the mesophyll-mesophyll cell interface. The increased PD density at the M-BS interface was largely driven by an increase in the number of PD pit fields (cluster of PDs) rather than an increase in PD per pit field or the size of pit fields. The correlation of gbs with bundle sheath surface area per leaf area and PD area per M-BS area showed that these parameters and cell wall thickness are important determinants of gbs It is intriguing to speculate that PD development is responsive to changes in C4 photosynthetic flux.

Shade compromises the photosynthetic efficiency of NADP-ME less than that of PEP-CK and NAD-ME C4 grasses.

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.

Photosynthetic capacity and leaf nitrogen decline along a controlled climate gradient in provenances of two widely distributed Eucalyptus species.

Climate is an important factor limiting tree distributions and adaptation to different thermal environments may influence how tree populations respond to climate warming. Given the current rate of warming, it has been hypothesized that tree populations in warmer, more thermally stable climates may have limited capacity to respond physiologically to warming compared to populations from cooler, more seasonal climates. We determined in a controlled environment how several provenances of widely distributed Eucalyptus tereticornis and E. grandis adjusted their photosynthetic capacity to +3.5°C warming along their native distribution range (~16-38°S) and whether climate of seed origin of the provenances influenced their response to different growth temperatures. We also tested how temperature optima (Topt ) of photosynthesis and Jmax responded to higher growth temperatures. Our results showed increased photosynthesis rates at a standardized temperature with warming in temperate provenances, while rates in tropical provenances were reduced by about 40% compared to their temperate counterparts. Temperature optima of photosynthesis increased as provenances were exposed to warmer growth temperatures. Both species had ~30% reduced photosynthetic capacity in tropical and subtropical provenances related to reduced leaf nitrogen and leaf Rubisco content compared to temperate provenances. Tropical provenances operated closer to their thermal optimum and came within 3% of the Topt of Jmax during the daily temperature maxima. Hence, further warming may negatively affect C uptake and tree growth in warmer climates, whereas eucalypts in cooler climates may benefit from moderate warming.

Engineering chloroplasts to improve Rubisco catalysis: prospects for translating improvements into food and fiber crops.

494 I. 495 II. 496 III. 496 IV. 499 V. 499 VI. 501 VII. 501 VIII. 502 IX. 505 X. 506 507 References 507 SUMMARY: The uncertainty of future climate change is placing pressure on cropping systems to continue to provide stable increases in productive yields. To mitigate future climates and the increasing threats against global food security, new solutions to manipulate photosynthesis are required. This review explores the current efforts available to improve carbon assimilation within plant chloroplasts by engineering Rubisco, which catalyzes the rate-limiting step of CO2 fixation. Fixation of CO2 and subsequent cycling of 3-phosphoglycerate through the Calvin cycle provides the necessary carbohydrate building blocks for maintaining plant growth and yield, but has to compete with Rubisco oxygenation, which results in photorespiration that is energetically wasteful for plants. Engineering improvements in Rubisco is a complex challenge and requires an understanding of chloroplast gene regulatory pathways, and the intricate nature of Rubisco catalysis and biogenesis, to transplant more efficient forms of Rubisco into crops. In recent times, major advances in Rubisco engineering have been achieved through improvement of our knowledge of Rubisco synthesis and assembly, and identifying amino acid catalytic switches in the L-subunit responsible for improvements in catalysis. Improving the capacity of CO2 fixation in crops such as rice will require further advances in chloroplast bioengineering and Rubisco biogenesis.

Linking photosynthesis and leaf N allocation under future elevated CO2 and climate warming in Eucalyptus globulus.

Leaf-level photosynthetic processes and their environmental dependencies are critical for estimating CO2 uptake from the atmosphere. These estimates use biochemical-based models of photosynthesis that require accurate Rubisco kinetics. We investigated the effects of canopy position, elevated atmospheric CO2 [eC; ambient CO2 (aC)+240 ppm] and elevated air temperature (eT; ambient temperature (aT)+3 °C) on Rubisco content and activity together with the relationship between leaf N and Vcmax (maximal Rubisco carboxylation rate) of 7 m tall, soil-grown Eucalyptus globulus trees. The kinetics of E. globulus and tobacco Rubisco at 25 °C were similar. In vitro estimates of Vcmax derived from measures of E. globulus Rubisco content and kinetics were consistent, although slightly lower, than the in vivo rates extrapolated from gas exchange. In E. globulus, the fraction of N invested in Rubisco was substantially lower than for crop species and varied with treatments. Photosynthetic acclimation of E. globulus leaves to eC was underpinned by reduced leaf N and Rubisco contents; the opposite occurred in response to eT coinciding with growth resumption in spring. Our findings highlight the adaptive capacity of this key forest species to allocate leaf N flexibly to Rubisco and other photosynthetic proteins across differing canopy positions in response to future, warmer and elevated [CO2] climates.

Short-term thermal photosynthetic responses of C4 grasses are independent of the biochemical subtype.

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.

Effects of reduced carbonic anhydrase activity on CO2 assimilation rates in Setaria viridis: a transgenic analysis.

In C4 species, the major ß-carbonic anhydrase (ß-CA) localized in the mesophyll cytosol catalyses the hydration of CO2 to HCO3-, which phosphoenolpyruvate carboxylase uses in the first step of C4 photosynthesis. To address the role of CA in C4 photosynthesis, we generated transgenic Setaria viridis depleted in ß-CA. Independent lines were identified with as little as 13% of wild-type CA. No photosynthetic defect was observed in the transformed lines at ambient CO2 partial pressure (pCO2). At low pCO2, a strong correlation between CO2 assimilation rates and CA hydration rates was observed. C18O16O isotope discrimination was used to estimate the mesophyll conductance to CO2 diffusion from the intercellular air space to the mesophyll cytosol (gm) in control plants, which allowed us to calculate CA activities in the mesophyll cytosol (Cm). This revealed a strong relationship between the initial slope of the response of the CO2 assimilation rate to cytosolic pCO2 (ACm) and cytosolic CA activity. However, the relationship between the initial slope of the response of CO2 assimilation to intercellular pCO2 (ACi) and cytosolic CA activity was curvilinear. This indicated that in S. viridis, mesophyll conductance may be a contributing limiting factor alongside CA activity to CO2 assimilation rates at low pCO2.

The role of Rubisco kinetics and pyrenoid morphology in shaping the CCM of haptophyte microalgae.

The haptophyte algae are a cosmopolitan group of primary producers that contribute significantly to the marine carbon cycle and play a major role in paleo-climate studies. Despite their global importance, little is known about carbon assimilation in haptophytes, in particular the kinetics of their Form 1D CO2-fixing enzyme, Rubisco. Here we examine Rubisco properties of three haptophytes with a range of pyrenoid morphologies (Pleurochrysis carterae, Tisochrysis lutea, and Pavlova lutheri) and the diatom Phaeodactylum tricornutum that exhibit contrasting sensitivities to the trade-offs between substrate affinity (Km) and turnover rate (kcat) for both CO2 and O2. The pyrenoid-containing T. lutea and P. carterae showed lower Rubisco content and carboxylation properties (KC and kCcat) comparable with those of Form 1D-containing non-green algae. In contrast, the pyrenoid-lacking P. lutheri produced Rubisco in 3-fold higher amounts, and displayed a Form 1B Rubisco kCcat-KC relationship and increased CO2/O2 specificity that, when modeled in the context of a C3 leaf, supported equivalent rates of photosynthesis to higher plant Rubisco. Correlation between the differing Rubisco properties and the occurrence and localization of pyrenoids with differing intracellular CO2:O2 microenvironments has probably influenced the divergent evolution of Form 1B and 1D Rubisco kinetics.

Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C4 grasses at inter-glacial CO2.

C4 photosynthesis evolved multiple times in diverse lineages. Most physiological studies comparing C4 plants were not conducted at the low atmospheric CO2 prevailing during their evolution. Here, 24 C4 grasses belonging to three biochemical subtypes [nicotinamide adenine dinucleotide malic enzyme (NAD-ME), phosphoenolpyruvate carboxykinase (PCK) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP-ME)] and six major evolutionary lineages were grown under ambient (400 µL L(-1) ) and inter-glacial (280 µL L(-1) ) CO2 . We hypothesized that nitrogen-related and water-related physiological traits are associated with subtypes and lineages, respectively. Photosynthetic rate and stomatal conductance were constrained by the shared lineage, while variation in leaf mass per area (LMA), leaf N per area, plant dry mass and plant water use efficiency were influenced by the subtype. Subtype and lineage were equally important for explaining variations in photosynthetic nitrogen use efficiency (PNUE) and photosynthetic water use efficiency (PWUE). CO2 treatment impacted most parameters. Overall, higher LMA and leaf N distinguished the Chloridoideae/NAD-ME group, while NADP-ME and PCK grasses were distinguished by higher PNUE regardless of lineage. Plants were characterized by high photosynthesis and PWUE when grown at ambient CO2 and by high conductance at inter-glacial CO2 . In conclusion, the evolutionary and biochemical diversity among C4 grasses was aligned with discernible leaf physiology, but it remains unknown whether these traits represent ecophysiological adaptation.