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Phosphoenolpyruvate (PEP) carboxylase (PEPC) has an anaplerotic role in central plant metabolism but also initiates the carbon concentrating mechanism during C 4 photosynthesis. The C 4 PEPC has different binding affinities (K m ) for PEP (K 0.5PEP ) and HCO 3 - (K 0.5HCO3 ), and allosteric regulation by glucose-6-phosphate (G6-P) compared to non-photosynthetic isoforms. These differences are linked to specific changes in amino acids within PEPC. For example, region II (residues 302-433, Zea mays numbering) has been identified as important for G6-P regulation and within this region residue 353 may be conserved in C 4 PEPC enzymes. Additionally, residue 780 influences the C 4 PEPC kinetic properties and may interact with region II as well as residue 353 to influence G6-P regulation. We test the hypothesis that variation within region II, including residue 353, and their interactions with residue 780 influence the kinetic and allosteric regulation by G6-P of two C 4 PEPC isozymes from two C 4 grasses. The data does not support a kinetic tradeoff between K 0.5HCO3 and K 0.5PEP in these PEPC isozymes. Additionally, these enzymes had different response to G6-P that was only partially attributed to region II, residue 353 and residue 780. This data provides new insights into factors influencing the kinetic variation of C 4 PEPC isozymes., Competing Interests: Declarations. Competing interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

The ratio of net CO2 uptake (Anet) and stomatal conductance (gs) is an intrinsic measurement of leaf water use efficiency (WUEi); however, its measurement can be challenging for large phenotypic screens. Measurements of the leaf carbon isotope composition (δ13Cleaf) may be a scalable tool to approximate WUEi for screening because it in part reflects the competing influences of Anet and gs on the CO2 partial pressure (pCO2) inside the leaf over time. However, in C4 photosynthesis, the CO2-concentrating mechanism complicates the relationship between δ13Cleaf and WUEi. Despite this complicated relationship, several studies have shown genetic variation in δ13Cleaf across C4 plants. Yet there has not been a clear demonstration of if Anet or gs are the causal mechanisms controlling WUEi and δ13Cleaf. Our approach was to characterize leaf photosynthetic traits of two Zea mays recombinant inbred lines (Z007E0067 and Z007E0150) which consistently differ for δ13Cleaf even though they have minimal confounding genetic differences. We demonstrate that these two genotypes contrasted in WUEi driven by differences in the speed of stomatal responses to changes in pCO2 and light that lead to unproductive leaf water loss. These findings provide support that differences in δ13Cleaf in closely related genotypes do reflect greater WUEi and further suggest that differences in stomatal kinetic response to changing environmental conditions is a key target to improve WUEi., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 μm 3 . Wild-type guard cell plastids are approximately 18 μm 3 . Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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.)

Background and Aims: Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the carbon-concentrating mechanism (CCM) in plants that perform C4 photosynthesis. This CCM is generally driven by a single PEPC gene product that is highly expressed in the cytosol of mesophyll cells. We found two C4 grasses, Panicum miliaceum and Echinochloa colona, that each have two highly expressed PEPC genes. We characterized the kinetic properties of the two most abundant PEPCs in E. colona and P. miliaceum to better understand how the enzyme's amino acid structure influences its function., Methods: Coding sequences of the two most abundant PEPC proteins in E. colona and P. miliaceum were synthesized by GenScript and were inserted into bacteria expression plasmids. Point mutations resulting in substitutions at conserved amino acid residues (e.g. N-terminal serine and residue 890) were created via site-directed PCR mutagenesis. The kinetic properties of semi-purified plant PEPCs from Escherichia coli were analysed using membrane-inlet mass spectrometry and a spectrophotometric enzyme-coupled reaction., Key Results: The two most abundant P. miliaceum PEPCs (PmPPC1 and PmPPC2) have similar sequence identities (>95 %), and as a result had similar kinetic properties. The two most abundant E. colona PEPCs (EcPPC1 and EcPPC2) had identities of ~78 % and had significantly different kinetic properties. The PmPPCs and EcPPCs had different responses to allosteric inhibitors and activators, and substitutions at the conserved N-terminal serine and residue 890 resulted in significantly altered responses to allosteric regulators., Conclusions: The two, significantly expressed C4Ppc genes in P. miliaceum were probably the result of genomes combining from two closely related C4Panicum species. We found natural variation in PEPC's sensitivity to allosteric inhibition that seems to bypass the conserved 890 residue, suggesting alternative evolutionary pathways for increased malate tolerance and other kinetic properties., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Measurements of oxygen isotope enrichment of leaf water above source water (Δ 18 O LW ) can improve our understanding of the interaction between leaf anatomy and physiology on leaf water transport. Models have been developed to predict Δ 18 O LW such as the string-of-lakes model, which describes the mixing of leaf water pools, and the Péclet effect model, which incorporates transpiration rate and the mixing length between unenriched xylem and enriched mesophyll water in the mesophyll (L m ) or veins (L v ). Here we compare measurements and models of Δ 18 O LW on two cell wall composition mutants grown under two light intensities and relative humidities to evaluate cell wall properties on leaf water transport. In maize (Zea mays), the compromised ultrastructure of the suberin lamellae in the bundle sheath of the ALIPHATIC SUBERIN FERULOYL TRANSFERASE mutant (Zmasft) reduced barriers to apoplastic water movement, resulting in higher E and, potentially, L v and, consequently, lower Δ 18 O LW . The difference in Δ 18 O LW in cellulose synthase-like F6 (CslF6) mutants and wild-type of rice (Oryza sativa) grown under two light intensities co-varied with stomatal density. These results show that cell wall composition and stomatal density influence Δ 18 O LW and that stable isotopes can facilitate the development of a physiologically and anatomically explicit water transport model., (© 2023 John Wiley & Sons Ltd.)

Mesophyll CO 2 conductance (g m ) in C 3 species responds to short-term (minutes) changes in environment potentially due to changes in leaf anatomical and biochemical properties and measurement artefacts. Compared with C 3 species, there is less information on g m responses to short-term changes in environmental conditions such as partial pressure of CO 2 (pCO 2 ) across diverse C 4 species and the potential determinants of these responses. Using 16 C 4 grasses we investigated the response of g m to short-term changes in pCO 2 and its relationship with leaf anatomy and biochemistry. In general, g m increased as pCO 2 decreased (statistically significant increase in 12 species), with percentage increases in g m ranging from +13% to +250%. Greater increase in g m at low pCO 2 was observed in species exhibiting relatively thinner mesophyll cell walls along with greater mesophyll surface area exposed to intercellular air spaces, leaf N, photosynthetic capacity and activities of phosphoenolpyruvate carboxylase and Rubisco. Species with greater CO 2 responses of g m were also able to maintain their leaf water-use efficiencies (TE i ) under low CO 2 . Our study advances understanding of CO 2 response of g m in diverse C 4 species, identifies the key leaf traits related to this response and has implications for improving C 4 photosynthetic models and TE i through modification of g m ., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

In C4 plants, the pyruvate (Pyr), phosphate dikinase regulatory protein (PDRP) regulates the activity of the C4 pathway enzyme Pyr, phosphate dikinase (PPDK) in a light-/dark-dependent manner. The importance of this regulatory action to C4 pathway function and overall C4 photosynthesis is unknown. To resolve this question, we assessed in vivo PPDK phospho-regulation and whole leaf photophysiology in a CRISPR-Cas9 PDRP knockout (KO) mutant of the NADP-ME C4 grass green millet (Setaria viridis). PDRP enzyme activity was undetectable in leaf extracts from PDRP KO lines. Likewise, PPDK phosphorylated at the PDRP-regulatory Thr residue was immunologically undetectable in leaf extracts. PPDK enzyme activity in rapid leaf extracts was constitutively high in the PDRP KO lines, irrespective of light or dark pretreatment of leaves. Gas exchange analysis of net CO2 assimilation revealed PDRP KO leaves had markedly slower light induction kinetics when leaves transition from dark to high-light or low-light to high-light. In the initial 30 min of the light induction phase, KO leaves had an ∼15% lower net CO2 assimilation rate versus the wild-type (WT). Despite the impaired slower induction kinetics, we found growth and vigor of the KO lines to be visibly indistinguishable from the WT when grown in normal air and under standard growth chamber conditions. However, the PDRP KO plants grown under a fluctuating light regime exhibited a gradual multi-day decline in Fv/Fm, indicative of progressive photosystem II damage due to the absence of PDRP. Collectively, our results demonstrate that one of PDRP's functions in C4 photosynthesis is to ensure optimal photosynthetic light induction kinetics during dynamic changes in incident light., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C3 plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO3−) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO2 and HCO3−) or active HCO3− transport can be of great value in the process of introducing CCM components into terrestrial C3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO2-dependent phenotype (5% (v/v) CO2 in air). CAs produce HCO3− for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO3− for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO2 (0.04% (v/v) CO2 in air). ΔCA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur−Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.

Carbonic anhydrase (CA) performs the first enzymatic step of C 4 photosynthesis by catalysing the reversible hydration of dissolved CO 2 that diffuses into mesophyll cells from intercellular airspaces. This CA-catalysed reaction provides the bicarbonate used by phosphoenolpyruvate carboxylase to generate products that flow into the C 4 carbon-concentrating mechanism (CCM). It was previously demonstrated that the Zea mays ca1ca2 double mutant lost 97% of leaf CA activity, but there was little difference in the growth phenotype under ambient CO 2 partial pressures (pCO 2 ). We hypothesise that since CAs are among the fastest enzymes, minimal activity from a third CA, CA8, can provide the inorganic carbon needed to drive C 4 photosynthesis. We observed that removing CA8 from the maize ca1ca2 background resulted in plants that had 0.2% of wild-type leaf CA activity. These ca1ca2ca8 plants had reduced photosynthetic parameters and could only survive at elevated pCO 2 . Photosynthetic and carbon isotope analysis combined with modelling of photosynthesis and carbon isotope discrimination was used to determine if ca1ca2ca8 plants had a functional C 4 cycle or were relying on direct CO 2 diffusion to ribulose 1,5-bisphosphate carboxylase/oxygenase within bundle sheath cells. The results suggest that leaf CA activity in ca1ca2ca8 plants was not sufficient to sustain the C 4 CCM., (© 2022 John Wiley & Sons Ltd.)

The CO2-concentrating mechanism (CCM) in C4 plants is initiated by the uptake of bicarbonate (HCO3-) via phosphoenolpyruvate carboxylase (PEPC). Generation of HCO3- for PEPC is determined by the interaction between mesophyll CO2 conductance and the hydration of CO2 to HCO3- by carbonic anhydrase (CA). Genetic reduction of CA was previously shown not to limit C4 photosynthesis under ambient atmospheric partial pressures of CO2 (pCO2). However, CA activity varies widely across C4 species and it is unknown if there are specific environmental conditions (e.g. high temperature) where CA may limit HCO3- production for C4 photosynthesis. Additionally, CA activity has been suggested to influence mesophyll conductance, but this has not been experimentally tested. We hypothesize that CA activity can limit PEPC at high temperatures, particularly at low pCO2, but does not directly influence gm. Here we tested the influence of genetically reduced CA activity on photosynthesis and gm in the C4 plant Zea mays under a range of pCO2 and temperatures. Reduced CA activity limited HCO3- production for C4 photosynthesis at low pCO2 as temperatures increased, but did not influence mesophyll conductance. Therefore, high leaf CA activity may enhance C4 photosynthesis under high temperature when stomatal conductance restricts the availability of atmospheric CO2., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

With increased demand on freshwater resources for agriculture, it is imperative that more water-use efficient crops are developed. Leaf stable carbon isotope composition, δ13C, is a proxy for transpiration efficiency and a possible tool for breeders, but the underlying mechanisms effecting δ13C in C4 plants are not known. It has been suggested that differences in specific leaf area (SLA), which potentially reflects variation in internal CO2 diffusion, can impact leaf δ13C. Furthermore, although it is known that water movement is important for elemental uptake, it is not clear how manipulation of transpiration for increased water-use efficiency may impact nutrient accumulation. Here, we characterize the genetic architecture of leaf δ13C and test its relationship to SLA and the ionome in five populations of maize. Five significant QTL for leaf δ13C were identified, including novel QTL as well as some that were identified previously in maize kernels. One of the QTL regions contains an Erecta-like gene, the ortholog of which has been shown to regulate transpiration efficiency and leaf δ13C in Arabidopsis. QTL for δ13C were located in the same general chromosome region, but slightly shifted, when comparing data from two different years. Our data does not support a relationship between δ13C and SLA, and of the 19 elements analyzed, only a weak correlation between molybdenum and δ13C was detected. Together these data add to the genetic understanding of leaf δ13C in maize and suggest that improvements to plant water use may be possible without significantly influencing elemental homeostasis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

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.)

Plateauing yield and stressful environmental conditions necessitate selecting crops for superior physiological traits with untapped potential to enhance crop performance. Plant productivity is often limited by carbon fixation rates that could be improved by increasing maximum photosynthetic carboxylation capacity (Vcmax). However, Vcmax measurements using gas exchange and biochemical assays are slow and laborious, prohibiting selection in breeding programs. Rapid hyperspectral reflectance measurements show potential for predicting Vcmax using regression models. While several hyperspectral models have been developed, contributions from different spectral regions to predictions of Vcmax have not been clearly identified or linked to biochemical variation contributing to Vcmax. In this study, hyperspectral reflectance data from 350-2500 nm were used to build partial least squares regression models predicting in vivo and in vitro Vcmax. Wild-type and transgenic tobacco plants with antisense reductions in Rubisco content were used to alter Vcmax independent from chlorophyll, carbon, and nitrogen content. Different spectral regions were used to independently build partial least squares regression models and identify key regions linked to Vcmax and other leaf traits. The greatest Vcmax prediction accuracy used a portion of the shortwave infrared region from 2070 nm to 2470 nm, where the inclusion of fewer spectral regions resulted in more accurate models., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Canopy water use efficiency (above-ground biomass over lifetime water loss, WUE canopy ) can influence yield in wheat and other crops. Breeding for WUE canopy is difficult because it is influenced by many component traits. For example, intrinsic water use efficiency (WUE i ), the ratio of net carbon assimilation (A net ) over stomatal conductance, contributes to WUE canopy and can be estimated from carbon isotope discrimination (Δ). However, Δ is not sensitive to differences in the water vapor pressure deficit between the air and leaf (VPD leaf ). Alternatively, measurements of instantaneous leaf water use efficiency (WUE leaf ) are defined as A net over transpiration and can be determined with gas exchange, but the dynamic nature of field conditions are not represented. Specifically, fluctuations in canopy temperature lead to changes in VPD leaf that impact transpiration but not A net . This alters WUE leaf and in turn affects WUE canopy . To test this relationship, WUE canopy was measured in conjunction with WUE i , WUE canopy , and canopy temperature under well-watered and water-limited conditions in two drought-tolerant wheat cultivars that differ in canopy architecture. In this experiment, boundary layer conductance was low and significant changes in leaf temperature occurred between cultivars and treatments that correlated with WUE canopy likely because of the effect of canopy temperature on VPD leaf driving T. However, deviations between WUE i , WUE leaf , and WUE canopy were present because measurements made at the leaf level do not account for variations in leaf temperature. This uncoupled the relationship of measured WUE leaf and WUE i from WUE canopy and emphasizes the importance of canopy temperature on carbon uptake and transpired water loss., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Introduction of a C 4 photosynthetic mechanism into C 3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C 4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13 CO 2 labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C 4 proteins introduced thus far, a functional C 4 pathway is achievable in rice., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C 4 plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C 4 plants is driven by PEPC's affinity for bicarbonate (K HCO3 ), which can be rate limiting when atmospheric CO 2 availability is restricted due to low stomatal conductance. We hypothesize that natural variation in K HCO3 across C 4 plants is driven by specific amino acid substitutions to impact rates of C 4 photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured K HCO3 from 20 C 4 grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in K HCO3 across these C 4 grasses (24.3 ± 1.5 to 46.3 ± 2.4 μm), which significantly impacts modeled rates of C 4 photosynthesis. Additionally, molecular engineering of a low-HCO 3 - affinity PEPC identified key domains that confer variation in K HCO3 . This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO 3 - affinity and C 4 photosynthetic efficiency in important C 4 crops., (© 2020 Society for Experimental Biology and 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.)

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.)

Genetic selection for whole-plant water use efficiency (yield per transpiration; WUE plant ) in any crop-breeding programme requires high-throughput phenotyping of component traits of WUE plant such as intrinsic water use efficiency (WUE i ; CO 2 assimilation rate per stomatal conductance). Measuring WUE i by gas exchange measurements is laborious and time consuming and may not reflect an integrated WUE i over the life of the leaf. Alternatively, leaf carbon stable isotope composition (δ 13 C leaf ) has been suggested as a potential time-integrated proxy for WUE i that may provide a tool to screen for WUE plant . However, a genetic link between δ 13 C leaf and WUE plant in a C 4 species has not been well established. Therefore, to determine if there is a genetic relationship in a C 4 plant between δ 13 C leaf and WUE plant under well watered and water-limited growth conditions, a high-throughput phenotyping facility was used to measure WUE plant in a recombinant inbred line (RIL) population created between the C 4 grasses Setaria viridis and S. italica. Three quantitative trait loci (QTL) for δ 13 C leaf were found and co-localized with transpiration, biomass accumulation, and WUE plant . Additionally, WUE plant for each of the δ 13 C leaf QTL allele classes was negatively correlated with δ 13 C leaf , as would be predicted when WUE i influences WUE plant . These results demonstrate that δ 13 C leaf is genetically linked to WUE plant , likely to be through their relationship with WUE i , and can be used as a high-throughput proxy to screen for WUE plant in these C 4 species., (© 2020 The Authors The Plant Journal © 2020 John Wiley & Sons Ltd.)

The cold acclimations of mesophyll conductance (g m ), bundle-sheath conductance (g bs ) and the CO 2 concentrating mechanism (CCM) of C 4 plants have not been well studied. Here, we estimated the temperature response of g m , g bs and leakiness (ϕ), the amount of concentrated CO 2 that escapes the bundle-sheath cells, for the chilling-tolerant C 4 plant Miscanthus × giganteus grown at 14 and 25°C. To estimate these parameters, we combined the C 4 -enzyme-limited photosynthesis model and the Δ 13 C discrimination model. These combined models were parameterised using in vitro activities of carbonic anhydrase (CA), pyruvate, phosphate dikinase (PPDK), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and phosphoenolpyruvate carboxylase (PEPc). Cold-grown Miscanthus plants increased in vitro activities of RuBisCO and PPDK but decreased PEPc activity compared with warm-grown plants. Mesophyll conductance and g bs responded strongly to measurement temperatures but did not differ between plants from the two growth temperatures. Furthermore, modelling showed that ϕ increased with measurement temperatures for both cold-grown and warm-grown plants, but was only marginally larger in cold-grown compared with warm-grown plants. Our results in Miscanthus support that g m and g bs are unresponsive to growth temperature and that the CCM is able to acclimate to cold through increased activity of PPDK and RuBisCO., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

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.)

Mesophyll conductance (g m ) is the diffusion of CO 2 from intercellular air spaces (IAS) to the first site of carboxylation in the mesophyll cells. In C 3 species, g m is influenced by diverse leaf structural and anatomical traits; however, little is known about traits affecting g m in C 4 species. To address this knowledge gap, we used online oxygen isotope discrimination measurements to estimate g m and microscopy techniques to measure leaf structural and anatomical traits potentially related to g m in 18 C 4 grasses. In this study, g m scaled positively with photosynthesis and intrinsic water-use efficiency (TE i ), but not with stomatal conductance. Also, g m was not determined by a single trait but was positively correlated with adaxial stomatal densities (SD ada ), stomatal ratio (SR), mesophyll surface area exposed to IAS (S mes ) and leaf thickness. However, g m was not related to abaxial stomatal densities (SD aba ) and mesophyll cell wall thickness (T CW ). Our study suggests that greater SD ada and SR increased g m by increasing S mes and creating additional parallel pathways for CO 2 diffusion inside mesophyll cells. Thus, SD ada , SR and S mes are important determinants of C 4 -g m and could be the target traits selected or modified for achieving greater g m and TE i in C 4 species., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The engineering process of C 4 photosynthesis into C 3 plants requires an increased activity of phosphoenolpyruvate carboxylase (PEPC) in the cytosol of leaf mesophyll cells. The literature varies on the physiological effect of transgenic maize (Zea mays) PEPC (ZmPEPC) leaf expression in Oryza sativa (rice). Therefore, to address this issue, leaf-atmosphere CO 2 and 13 CO 2 exchanges were measured, both in the light (at atmospheric O 2 partial pressure of 1.84 kPa and at different CO 2 levels) and in the dark, in transgenic rice expressing ZmPEPC and wild-type (WT) plants. The in vitro PEPC activity was 25 times higher in the PEPC overexpressing (PEPC-OE) plants (~20% of maize) compared to the negligible activity in WT. In the PEPC-OE plants, the estimated fraction of carboxylation by PEPC (β) was ~6% and leaf net biochemical discrimination against 13 CO 2 [Formula: see text] was ~ 2‰ lower than in WT. However, there were no differences in leaf net CO 2 assimilation rates (A) between genotypes, while the leaf dark respiration rates (R d ) over three hours after light-dark transition were enhanced (~ 30%) and with a higher 13 C composition [Formula: see text] in the PEPC-OE plants compared to WT. These data indicate that ZmPEPC in the PEPC-OE rice plants contributes to leaf carbon metabolism in both the light and in the dark. However, there are some factors, potentially posttranslational regulation and PEP availability, which reduce ZmPEPC activity in vivo.

C 4 plants achieve higher photosynthesis (A n ) and intrinsic water use efficiency (iWUE) than C 3 plants, but processes underpinning the variability in A n and iWUE across the three C 4 subtypes remain unclear, partly because we lack an integrated framework for quantifying the contribution of diffusional and biochemical limitations to C 4 photosynthesis. We exploited the natural diversity among C 4 grasses to develop an original mathematical approach for estimating eight key processes of C 4 photosynthesis and their relative limitations to A n . We also developed a new formulation to estimate mesophyll conductance (g m ) based on actual hydration rates of CO 2 by carbonic anhydrases. We found a positive relationship between g m and iWUE and an inverse correlation with g sw among C 4 grasses. We also revealed subtype-specific regulatory processes of iWUE that may be related to known anatomical traits characterising each C 4 subtype. Leaf width was an important determinant of iWUE and showed significant correlations with key limitations of A n , especially among NADP-ME species. In conclusion, incorporating leaf width in breeding trials may unlock new opportunities for C 4 crops because the revealed negative relationship between leaf width and iWUE may translate into higher crop and canopy WUE., (© 2019 Western Sydney University New Phytologist © 2019 New Phytologist Trust.)

The arrangement of mitochondria and chloroplasts, together with the relative resistances of cell wall and chloroplast, determine the path of diffusion out of the leaf for (photo)respired CO 2 . Traditional photosynthesis models have assumed a tight arrangement of chloroplasts packed together against the cell wall with mitochondria located behind the chloroplasts, deep inside the cytosol. Accordingly, all (photo)respired CO 2 must cross the chloroplast before diffusing out of the leaf. Different arrangements have recently been considered, where all or part of the (photo)respired CO 2 diffuses through the cytosol without ever entering the chloroplast. Assumptions about the path for the (photo)respiratory flux are particularly relevant for the calculation of mesophyll conductance (g m ). If (photo)respired CO 2 can diffuse elsewhere besides the chloroplast, apparent g m is no longer a mere physical resistance but a flux-weighted variable sensitive to the ratio of (photo)respiration to net CO 2 assimilation. We discuss existing photosynthesis models in conjunction with their treatment of the (photo)respiratory flux and present new equations applicable to the generalized case where (photo)respired CO 2 can diffuse both into the chloroplast and through the cytosol. Additionally, we present a new generalized Δ 13 C model that incorporates this dual diffusion pathway. We assess how assumptions about the fate of (photo)respired CO 2 affect the interpretation of photosynthetic data and the challenges it poses for the application of different models.

The influence of reduced glycine decarboxylase complex (GDC) activity on leaf atmosphere CO2 and 13CO2 exchange was tested in transgenic Oryza sativa with the GDC H-subunit knocked down in leaf mesophyll cells. Leaf measurements on transgenic gdch knockdown and wild-type plants were carried out in the light under photorespiratory and low photorespiratory conditions (i.e. 18.4 kPa and 1.84 kPa atmospheric O2 partial pressure, respectively), and in the dark. Under approximately current ambient O2 partial pressure (18.4 kPa pO2), the gdch knockdown plants showed an expected photorespiratory-deficient phenotype, with lower leaf net CO2 assimilation rates (A) than the wild-type. Additionally, under these conditions, the gdch knockdown plants had greater leaf net discrimination against 13CO2 (Δo) than the wild-type. This difference in Δo was in part due to lower 13C photorespiratory fractionation (f) ascribed to alternative decarboxylation of photorespiratory intermediates. Furthermore, the leaf dark respiration rate (Rd) was enhanced and the 13CO2 composition of respired CO2 (δ13CRd) showed a tendency to be more depleted in the gdch knockdown plants. These changes in Rd and δ13CRd were due to the amount and carbon isotopic composition of substrates available for dark respiration. These results demonstrate that impairment of the photorespiratory pathway affects leaf 13CO2 exchange, particularly the 13C decarboxylation fractionation associated with photorespiration., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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.)

Background: Carbonic anhydrase (CA) catalyzes the hydration of CO 2 in the first biochemical step of C 4 photosynthesis, and has been considered a potentially rate-limiting step when CO 2 availability within a leaf is low. Previous work in Zea mays (maize) with a double knockout of the two highest-expressed β-CA genes, CA1 and CA2, reduced total leaf CA activity to less than 3% of wild-type. Surprisingly, this did not limit photosynthesis in maize at ambient or higher CO 2 concentrations. However, the ca1ca2 mutants exhibited reduced rates of photosynthesis at sub-ambient CO 2 , and accumulated less biomass when grown under sub-ambient CO 2 (9.2 Pa). To further clarify the importance of CA for C 4 photosynthesis, we assessed gene expression changes in wild-type, ca1 and ca1ca2 mutants in response to changes in pCO 2 from 920 to 9.2 Pa., Results: Leaf samples from each genotype were collected for RNA-seq analysis at high CO 2 and at two time points after the low CO 2 transition, in order to identify early and longer-term responses to CO 2 deprivation. Despite the existence of multiple isoforms of CA, no other CA genes were upregulated in CA mutants. Although photosynthetic genes were downregulated in response to low CO 2 , differential expression was not observed between genotypes. However, multiple indicators of carbon starvation were present in the mutants, including amino acid synthesis, carbohydrate metabolism, and sugar signaling. In particular, multiple genes previously implicated in low carbon stress such as asparagine synthetase, amino acid transporters, trehalose-6-phosphate synthase, as well as many transcription factors, were strongly upregulated. Furthermore, genes in the CO 2 stomatal signaling pathway were differentially expressed in the CA mutants under low CO 2 ., Conclusions: Using a transcriptomic approach, we showed that carbonic anhydrase mutants do not compensate for the lack of CA activity by upregulating other CA or photosynthetic genes, but rather experienced extreme carbon stress when grown under low CO 2 . Our results also support a role for CA in the CO 2 stomatal signaling pathway. This study provides insight into the importance of CA for C 4 photosynthesis and its role in stomatal signaling.

Phosphoenolpyruvate (PEP) carboxylase (PEPc) catalyzes the first committed step of C4 photosynthesis generating oxaloacetate from bicarbonate (HCO3-) and PEP. It is hypothesized that PEPc affinity for HCO3- has undergone selective pressure for a lower KHCO3 (Km for HCO3-) to increase the carbon flux entering the C4 cycle, particularly during conditions that limit CO2 availability. However, the decrease in KHCO3 has been hypothesized to cause an unavoidable increase in KPEP (Km for PEP). Therefore, the amino acid residue S774 in the C4 enzyme, which has been shown to increase KPEP, should lead to a decrease in KHCO3. Several studies reported the effect S774 has on KPEP; however, the influence of this amino acid substitution on KHCO3 has not been tested. To test these hypotheses, membrane-inlet mass spectrometry (MIMS) was used to measure the KHCO3 of the photosynthetic PEPc from the C4Flaveria trinervia and the non-photosynthetic PEPc from the C3F. pringlei. The cDNAs for these enzymes were overexpressed and purified from the PEPc-less PCR1 Escherichia coli strain. Our work in comparison with previous reports suggests that KHCO3 and KPEP are linked by specific amino acids, such as S774; however, these kinetic parameters respond differently to the tested allosteric regulators, malate and glucose-6-phosphate.

Enhancement of Rubisco kinetics could improve photosynthetic efficiency, ultimately resulting in increased crop yield. However, imprecise knowledge of the reaction mechanism and the individual rate constants limits our ability to optimize the enzyme. Membrane inlet mass spectrometry (MIMS) may offer benefits over traditional methods for determining individual rate constants of the Rubisco reaction mechanism, as it can directly monitor concentration changes in CO2, O2, and their isotopologs during assays. However, a direct comparison of MIMS with the traditional radiolabel method of determining Rubisco kinetic parameters has not been made. Here, the temperature responses of Rubisco kinetic parameters from Arabidopsis thaliana were measured using radiolabel and MIMS methods. The two methods provided comparable parameters above 25 °C, but temperature responses deviated at low temperature as MIMS-derived catalytic rates of carboxylation, oxygenation, and CO2/O2 specificity showed thermal breakpoints. Here, we discuss the variability and uncertainty surrounding breakpoints in the Rubisco temperature response and the relevance of individual rate constants of the reaction mechanisms to potential breakpoints.

Plant growth and water use are interrelated processes influenced by genetically controlled morphological and biochemical characteristics. Improving plant water use efficiency (WUE) to sustain growth in different environments is an important breeding objective that can improve crop yields and enhance agricultural sustainability. However, genetic improvement of WUE using traditional methods has proven difficult due to the low throughput and environmental heterogeneity of field settings. To overcome these limitations, this study utilizes a high-throughput phenotyping platform to quantify plant size and water use of an interspecific Setaria italica × Setaria viridis recombinant inbred line population at daily intervals in both well-watered and water-limited conditions. Our findings indicate that measurements of plant size and water use are correlated strongly in this system; therefore, a linear modeling approach was used to partition this relationship into predicted values of plant size given water use and deviations from this relationship at the genotype level. The resulting traits describing plant size, water use, and WUE all were heritable and responsive to soil water availability, allowing for a genetic dissection of the components of plant WUE under different watering treatments. Linkage mapping identified major loci underlying two different pleiotropic components of WUE. This study indicates that alleles controlling WUE derived from both wild and domesticated accessions can be utilized to predictably modulate trait values given a specified precipitation regime in the model C 4 genus Setaria ., (© 2018 American Society of Plant Biologists. All rights reserved.)

Phospho enol pyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C 4 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 CO 2 concentrations for growth, consistent with the essential role of PEPC in C 4 photosynthesis. The combination of estimating direct CO 2 fixation by the bundle sheath using gas-exchange measurements and modeling C 4 photosynthesis with low PEPC activity allowed the calculation of bundle sheath conductance to CO 2 diffusion ( g bs ) in the progeny of these plants. Measurements made at a range of temperatures suggested no or negligible effect of temperature on g bs depending on the technique used to calculate g bs 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 g bs 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 g bs It is intriguing to speculate that PD development is responsive to changes in C 4 photosynthetic flux., (© 2018 American Society of Plant Biologists. All rights reserved.)

Diffusion of CO 2 from the leaf intercellular air space to the site of carboxylation (g m ) is a potential trait for increasing net rates of CO 2 assimilation (A net ), photosynthetic efficiency, and crop productivity. Leaf anatomy plays a key role in this process; however, there are few investigations into how cell wall properties impact g m and A net . Online carbon isotope discrimination was used to determine g m and A net in Oryza sativa wild-type (WT) plants and mutants with disruptions in cell wall mixed-linkage glucan (MLG) production (CslF6 knockouts) under high- and low-light growth conditions. Cell wall thickness (T cw ), surface area of chloroplast exposed to intercellular air spaces (S c ), leaf dry mass per area (LMA), effective porosity, and other leaf anatomical traits were also analyzed. The g m of CslF6 mutants decreased by 83% relative to the WT, with c. 28% of the reduction in g m explained by S c . Although A net /LMA and A net /Chl partially explained differences in A net between genotypes, the change in cell wall properties influenced the diffusivity and availability of CO 2 . The data presented here indicate that the loss of MLG in CslF6 plants had an impact on g m and demonstrate the importance of cell wall effective porosity and liquid path length on g m ., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Stomata regulate transpirational water loss and CO 2 uptake for photosynthesis in response to changing environmental conditions. Research investigating stomatal movement has mostly been conducted in C 3 eudicot species, which have very different CO 2 requirements for photosynthesis relative to C 4 grasses. Carbonic anhydrase (CA) catalyzes the hydration of CO 2 , and its activity has been linked to stomatal aperture regulation in eudicots. The number of Ca genes and their evolutionary history differ between monocots and dicots, and many questions remain unanswered about potential neofunctionalization and subfunctionalization of grass Ca paralogs and their roles in photosynthesis and stomatal conductance. To investigate the roles of different Ca genes in maize ( Zea mays ), we examined stomatal responses in ca1 and ca2 single mutants as well as a ca1ca2 double mutant. The ca1 and ca2 single mutants had 10% and 87% of the CA activity exhibited by the wild type, respectively, while ca1ca2 had less than 5% of wild-type CA activity. The ca mutants had higher stomatal conductance than the wild type and slower stomatal closure in response to increases in CO 2 partial pressure. Contrary to previous reports in eudicots, ca mutants showed slowed stomatal closure in response to the light-dark transition and did not show differences in stomatal density compared with the wild type. These results implicate CA-mediated signaling in the control of stomatal movement but not stomatal development. Drought experiments with ca1ca2 mutant plants suggest a role for CA in water-use efficiency and reveal that Z. mays is not optimized for water-use efficiency under well-watered conditions., (© 2018 American Society of Plant Biologists. All rights reserved.)

Carbon isotope discrimination is used to study CO2 diffusion, substrate availability for photosynthesis, and leaf biochemistry, but the intraspecific drivers of leaf carbon isotope composition (δ13C) in C4 species are not well understood. In this study, the role of photosynthetic enzymes and post-photosynthetic fractionation on δ13C (‰) was explored across diverse maize inbred lines. A significant 1.3‰ difference in δ13C was observed between lines but δ13C did not correlate with in vitro leaf carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), or ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity. RNA-sequencing was used to identify potential differences in post-photosynthetic metabolism that would influence δ13C; however, no correlations were identified that would indicate significant differences in post-photosynthetic fractionation between lines. Variation in δ13C has been observed between C4 subtypes, but differential expression of NADP-ME and PEP-CK pathways within these lines did not correlate with δ13C. However, co-expression network analysis provided novel evidence for isoforms of C4 enzymes and putative transporters. Together, these data indicate that diversity in maize δ13C cannot be fully explained by variation in CA, PEPC, or Rubisco activity or gene expression. The findings further emphasise the need for future work exploring the influence of stomatal sensitivity and mesophyll conductance on δ13C in maize.

Mesophyll conductance (g m ) describes the movement of CO 2 from the intercellular air spaces below the stomata to the site of initial carboxylation in the mesophyll. In contrast with C 3 -g m , little is currently known about the intraspecific variation in C 4 -g m or its responsiveness to environmental stimuli. To address these questions, g m was measured on five maize (Zea mays) lines in response to CO 2 , employing three different estimates of g m . Each of the methods indicated a significant response of g m to CO 2 . Estimates of g m were similar between methods at ambient and higher CO 2 , but diverged significantly at low partial pressures of CO 2 . These differences are probably driven by incomplete chemical and isotopic equilibrium between CO 2 and bicarbonate under these conditions. Carbonic anhydrase and phosphoenolpyruvate carboxylase in vitro activity varied significantly despite similar values of g m and leaf anatomical traits. These results provide strong support for a CO 2 response of g m in Z. mays, and indicate that g m in maize is probably driven by anatomical constraints rather than by biochemical limitations. The CO 2 response of g m indicates a potential role for facilitated diffusion in C 4 -g m . These results also suggest that water-use efficiency could be enhanced in C 4 species by targeting g m ., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Mesophyll conductance (gm) limits rates of C3 photosynthesis but little is known about its role in C4 photosynthesis. If gm were to limit C4 photosynthesis, it would likely be at low CO2 concentrations (pCO2). However, data on C4-gm across ranges of pCO2 are scarce. We describe the response of C4-gm to short-term variation in pCO2, at three temperatures in Setaria viridis, and at 25 °C in Zea mays. Additionally, we quantified the effect of finite gm calculations of leakiness (ϕ) and the potential limitations to photosynthesis imposed by stomata, mesophyll, and carbonic anhydrase (CA) across pCO2. In both species, gm increased with decreasing pCO2. Including a finite gm resulted in either no change or increased ϕ compared with values calculated with infinite gm depending on whether the observed 13C discrimination was high (Setaria) or low (Zea). Post-transitional regulation of the maximal PEP carboxylation rate and PEP regeneration limitation could influence estimates of gm and ϕ. At pCO2 below ambient, the photosynthetic rate was limited by CO2 availability. In this case, the limitation imposed by the mesophyll was similar or slightly lower than stomata limitation. At very low pCO2, CA further constrained photosynthesis. High gm could increase CO2 assimilation at low pCO2 and improve photosynthetic efficiency under situations when CO2 is limited, such as drought.

Leaf carbon and oxygen isotope ratios can potentially provide a time-integrated proxy for stomatal conductance (gs) and transpiration rate (E), and can be used to estimate transpiration efficiency (TE). In this study, we found significant relationships of bulk leaf carbon isotopic signature (δ13CBL) and bulk leaf oxygen enrichment above source water (Δ18OBL) with gas exchange and TE in the model C4 grasses Setaria viridis and S. italica. Leaf δ13C had strong relationships with E, gs, water use, biomass, and TE. Additionally, the consistent difference in δ13CBL between well-watered and water-limited plants suggests that δ13CBL is effective in separating C4 plants with different availability of water. Alternatively, the use of Δ18OBL as a proxy for E and TE in S. viridis and S. italica was problematic. First, the oxygen isotopic composition of source water, used to calculate leaf water enrichment (Δ18OLW), was variable with time and differed across water treatments. Second, water limitations changed leaf size and masked the relationship of Δ18OLW and Δ18OBL with E. Therefore, the data collected here suggest that δ13CBL but not Δ18OBL may be an effective proxy for TE in C4 grasses., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Mesophyll conductance (g m ) is an important factor limiting rates of C 3 photosynthesis. However, its role in C 4 photosynthesis is poorly understood because it has been historically difficult to estimate. We use two methods to derive the temperature responses of g m in C 4 species. The first (Δ 18 O) combines measurements of gas exchange with models and measurements of 18 O discrimination. The second method (in vitro V pmax ) derives g m by retrofitting models of C 4 photosynthesis and 13 C discrimination with gas exchange, kinetic constants and in vitro V pmax measurements. The two methods produced similar g m for Setaria viridis and Zea mays. Additionally, we present the first temperature response (10-40°C) of C 4 g m in S. viridis, Z. mays and Miscanthus × giganteus. Values for g m at 25°C ranged from 2.90 to 7.85 μmol m -2  s -1  Pa -1 . Our study demonstrated that: the two described methods are suitable to calculate g m in C 4 species; g m values in C 4 are similar to high-end values reported for C 3 species; and g m increases with temperature analogous to reports for C 3 species and the response is species specific. These results improve our mechanistic understanding of C 4 photosynthesis., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

There are numerous studies describing how growth conditions influence the efficiency of C 4 photosynthesis. However, it remains unclear how changes in the biochemical capacity versus leaf anatomy drives this acclimation. Therefore, the aim of this study was to determine how growth light and nitrogen availability influence leaf anatomy, biochemistry and the efficiency of the CO 2 concentrating mechanism in Miscanthus × giganteus. There was an increase in the mesophyll cell wall surface area but not cell well thickness in the high-light (HL) compared to the low-light (LL) grown plants suggesting a higher mesophyll conductance in the HL plants, which also had greater photosynthetic capacity. Additionally, the HL plants had greater surface area and thickness of bundle-sheath cell walls compared to LL plants, suggesting limited differences in bundle-sheath CO 2 conductance because the increased area was offset by thicker cell walls. The gas exchange estimates of phosphoenolpyruvate carboxylase (PEPc) activity were significantly less than the in vitro PEPc activity, suggesting limited substrate availability in the leaf due to low mesophyll CO 2 conductance. Finally, leakiness was similar across all growth conditions and generally did not change under the different measurement light conditions. However, differences in the stable isotope composition of leaf material did not correlate with leakiness indicating that dry matter isotope measurements are not a good proxy for leakiness. Taken together, these data suggest that the CO 2 concentrating mechanism in Miscanthus is robust under low-light and limited nitrogen growth conditions, and that the observed changes in leaf anatomy and biochemistry likely help to maintain this efficiency.

Aquatic microalgae have evolved diverse CO 2 -concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO 2 -fixing enzyme rubisco. The limiting CO 2 -inducible B protein (LCIB) is known to be essential for CCM function in Chlamydomonas reinhardtii To assign a function to this previously uncharacterized protein family, we purified and characterized a phylogenetically diverse set of LCIB homologs. Three of the six homologs are functional carbonic anhydrases (CAs). We determined the crystal structures of LCIB and limiting CO 2 -inducible C protein (LCIC) from C. reinhardtii and a CA-functional homolog from Phaeodactylum tricornutum, all of which harbor motifs bearing close resemblance to the active site of canonical β-CAs. Our results identify the LCIB family as a previously unidentified group of β-CAs, and provide a biochemical foundation for their function in the microalgal CCMs., Competing Interests: The authors declare no conflict of interest.

Background: Comparisons between C 3 and C 4 grasses often utilize C 3 species from the subfamilies Ehrhartoideae or Pooideae and C 4 species from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the C 3 panicoid grass Dichanthelium oligosanthes from the independent C 4 lineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of C 3 and C 4 grasses., Results: We report the assembly of the nuclear and chloroplast genomes of D. oligosanthes, from high-throughput short read sequencing data and a comparative transcriptomics analysis of the developing leaf of D. oligosanthes, S. viridis, and S. bicolor. Physiological and anatomical characterizations verified that D. oligosanthes utilizes the C 3 pathway for carbon fixation and lacks Kranz anatomy. Expression profiles of transcription factors along developing leaves of D. oligosanthes and S. viridis were compared with previously published data from S. bicolor, Zea mays, and Oryza sativa to identify a small suite of transcription factors that likely acquired functions specifically related to C 4 photosynthesis., Conclusions: The phylogenetic location of D. oligosanthes makes it an ideal C 3 plant for comparative analysis of C 4 evolution in the panicoid grasses. This genome will not only provide a better C 3 species for comparisons with C 4 panicoid grasses, but also highlights the power of using high-throughput sequencing to address questions in evolutionary biology.

The group of homoiochlorophyllous resurrection plants evolved the unique capability to survive severe drought stress without dismantling the photosynthetic machinery. This implies that they developed efficient strategies to protect the leaves from reactive oxygen species (ROS) generated by photosynthetic side reactions. These strategies, however, are poorly understood. Here, we performed a detailed study of the photosynthetic machinery in the homoiochlorophyllous resurrection plant Craterostigma pumilum during dehydration and upon recovery from desiccation. During dehydration and rehydration, C. pumilum deactivates and activates partial components of the photosynthetic machinery in a specific order, allowing for coordinated shutdown and subsequent reinstatement of photosynthesis. Early responses to dehydration are the closure of stomata and activation of electron transfer to oxygen accompanied by inactivation of the cytochrome b6 f complex leading to attenuation of the photosynthetic linear electron flux (LEF). The decline in LEF is paralleled by a gradual increase in cyclic electron transport to maintain ATP production. At low water contents, inactivation and supramolecular reorganization of photosystem II becomes apparent, accompanied by functional detachment of light-harvesting complexes and interrupted access to plastoquinone. This well-ordered sequence of alterations in the photosynthetic thylakoid membranes helps prepare the plant for the desiccated state and minimize ROS production., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Drought is a major agricultural problem worldwide. Therefore, selection for increased water use efficiency (WUE) in food and biofuel crop species will be an important trait in plant breeding programs. The leaf carbon isotopic composition (δ(13)Cleaf) has been suggested to serve as a rapid and effective high throughput phenotyping method for WUE in both C3 and C4 species. This is because WUE, leaf carbon discrimination (Δ(13)Cleaf), and δ(13)Cleaf are correlated through their relationships with intercellular to ambient CO2 partial pressures (Ci/Ca). However, in C4 plants, changing environmental conditions may influence photosynthetic efficiency (bundle-sheath leakiness) and post-photosynthetic fractionation that will potentially alter the relationship between δ(13)Cleaf and Ci/Ca. Here we discuss how these factors influence the relationship between δ(13)Cleaf and WUE, and the potential of using δ(13)Cleaf as a meaningful proxy for WUE., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Water limitations decrease stomatal conductance (g(s)) and, in turn, photosynthetic rate (A(net)), resulting in decreased crop productivity. The current techniques for evaluating these physiological responses are limited to leaf-level measures acquired by measuring leaf-level gas exchange. In this regard, proximal sensing techniques can be a useful tool in studying plant biology as they can be used to acquire plant-level measures in a high-throughput manner. However, to confidently utilize the proximal sensing technique for high-throughput physiological monitoring, it is important to assess the relationship between plant physiological parameters and the sensor data. Therefore, in this study, the application of rapid sensing techniques based on thermal imaging and visual-near infrared spectroscopy for assessing water-use efficiency (WUE) in foxtail millet (Setaria italica (L.) P. Beauv) was evaluated. The visible-near infrared spectral reflectance (350-2500 nm) and thermal (7.5-14 µm) data were collected at regular intervals from well-watered and drought-stressed plants in combination with other leaf physiological parameters (transpiration rate-E, A(net), g(s), leaf carbon isotopic signature-δ(13)C(leaf), WUE). Partial least squares regression (PLSR) analysis was used to predict leaf physiological measures based on the spectral data. The PLSR modeling on the hyperspectral data yielded accurate and precise estimates of leaf E, gs, δ(13)C(leaf), and WUE with coefficient of determination in a range of 0.85-0.91. Additionally, significant differences in average leaf temperatures (~1°C) measured with a thermal camera were observed between well-watered plants and drought-stressed plants. In summary, the visible-near infrared reflectance data, and thermal images can be used as a potential rapid technique for evaluating plant physiological responses such as WUE., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The glycine decarboxylase complex (GDC) plays a critical role in the photorespiratory C2 cycle of C3 species by recovering carbon following the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase. Loss of GDC from mesophyll cells (MCs) is considered a key early step in the evolution of C4 photosynthesis. To assess the impact of preferentially reducing GDC in rice MCs, we decreased the abundance of OsGDCH (Os10g37180) using an artificial microRNA (amiRNA) driven by a promoter that preferentially drives expression in MCs. GDC H- and P-proteins were undetectable in leaves of gdch lines. Plants exhibited a photorespiratory-deficient phenotype with stunted growth, accelerated leaf senescence, reduced chlorophyll, soluble protein and sugars, and increased glycine accumulation in leaves. Gas exchange measurements indicated an impaired ability to regenerate ribulose 1,5-bisphosphate in photorespiratory conditions. In addition, MCs of gdch lines exhibited a significant reduction in chloroplast area and coverage of the cell wall when grown in air, traits that occur during the later stages of C4 evolution. The presence of these two traits important for C4 photosynthesis and the non-lethal, down-regulation of the photorespiratory C2 cycle positively contribute to efforts to produce a C4 rice prototype., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Temporal and spatial patterns of photosynthetic enzyme expression and structural maturation of chlorenchyma cells along longitudinal developmental gradients were characterized in young leaves of two single cell C4 species, Bienertia sinuspersici and Suaeda aralocaspica Both species partition photosynthetic functions between distinct intracellular domains. In the C4-C domain, C4 acids are formed in the C4 cycle during capture of atmospheric CO2 by phosphoenolpyruvate carboxylase. In the C4-D domain, CO2 released in the C4 cycle via mitochondrial NAD-malic enzyme is refixed by Rubisco. Despite striking differences in origin and intracellular positioning of domains, these species show strong convergence in C4 developmental patterns. Both progress through a gradual developmental transition towards full C4 photosynthesis, with an associated increase in levels of photosynthetic enzymes. Analysis of longitudinal sections showed undeveloped domains at the leaf base, with Rubisco rbcL mRNA and protein contained within all chloroplasts. The two domains were first distinguishable in chlorenchyma cells at the leaf mid-regions, but still contained structurally similar chloroplasts with equivalent amounts of rbcL mRNA and protein; while mitochondria had become confined to just one domain (proto-C4-D). The C4 state was fully formed towards the leaf tips, Rubisco transcripts and protein were compartmentalized specifically to structurally distinct chloroplasts in the C4-D domains indicating selective regulation of Rubisco expression may occur by control of transcription or stability of rbcL mRNA. Determination of CO2 compensation points showed young leaves were not functionally C4, consistent with cytological observations of the developmental progression from C3 default to intermediate to C4 photosynthesis., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The two carboxylation reactions performed by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are vital in the fixation of inorganic carbon for C4 plants. The abundance of PEPC is substantially elevated in C4 leaves, while the location of Rubisco is restricted to one of two chloroplast types. These differences compared with C3 leaves have been shown to result in convergent enzyme optimization in some C4 species. Investigation into the kinetic properties of PEPC and Rubisco from Kranz C4, single cell C4, and C3 species in Chenopodiaceae s. s. subfamily Suaedoideae showed that these major carboxylases in C4 Suaedoideae species lack the same mutations found in other C4 systems which have been examined; but still have similar convergent kinetic properties. Positive selection analysis on the N-terminus of PEPC identified residues 364 and 368 to be under positive selection with a posterior probability >0.99 using Bayes empirical Bayes. Compared with previous analyses on other C4 species, PEPC from C4 Suaedoideae species have different convergent amino acids that result in a higher K m for PEP and malate tolerance compared with C3 species. Kinetic analysis of Rubisco showed that C4 species have a higher catalytic efficiency of Rubisco (k catc in mol CO2 mol(-1) Rubisco active sites s(-1)), despite lacking convergent substitutions in the rbcL gene. The importance of kinetic changes to the two-carboxylation reactions in C4 leaves related to amino acid selection is discussed., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The photosynthetic assimilation of CO2 in C4 plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C4 biochemical models of leaf CO2 exchange in response to changes in CO2 availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C4 plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO3 (-), and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C4 plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO3 (-) limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C3 species and the C4 plant S. viridis., (© 2015 American Society of Plant Biologists. All Rights Reserved.)