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Oxygen isotopic composition (Δ18OLW) of leaf water can help improve our understanding of how anatomy interacts with physiology to influence leaf water transport. Leaf water isotope models of Δ18OLW such as the Péclet effect model have been developed to predict Δ18OLW, and it incorporates transpiration rate (E) and the mixing length between unenriched xylem water and enriched mesophyll water, which can occur in the mesophyll (Lm) or veins (Lv). Here we used two cell wall composition mutants grown under two light intensities and RH to evaluate the effect of cell wall composition on Δ18OLW. 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 Lv and, consequently, lower Δ18OLW. In cellulose synthase-like F6 (Cslf6) mutants and wildtype of rice (Oryza sativa), the difference in Δ18OLW in plants grown under high and low growth light intensity co-varied with their differences in stomatal density. These results show that cell wall composition and stomatal density influence Δ18OLW by altering the Péclet effect and that stable isotopes can facilitate the development of a physiologically and anatomically explicit water transport model.

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.

Leaf hydraulic and mesophyll CO2 conductance are both influenced by leaf anatomical traits, however it is poorly understood how the temperature response of these conductances differs between C4 and C3 species with distinct leaf anatomy. This study investigated the temperature response of leaf hydraulic conductance (Kleaf ), stomatal (gs ) and mesophyll (gm ) conductance to CO2 , and leaf anatomical traits in phylogenetically related Panicum antidotale (C4 ) and P. bisulcatum (C3 ) grasses. The C4 species had lower hydraulic conductance outside xylem (Kox ) and Kleaf compared with the C3 species. However, the C4 species had higher gm compared with the C3 species. Traits associated with leaf water movement, Kleaf and Kox , increased with temperature more in the C3 than in the C4 species, whereas traits related to carbon uptake, Anet and gm , increased more with temperature in the C4 than the C3 species. Our findings demonstrate that, in addition to a CO2 concentrating mechanism, outside-xylem leaf anatomy in the C4 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 C3 species P. bisulcatum.

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.

SummaryMesophyll CO2 conductance (gm) in C3 species responds to short-term (minutes) changes in environment potentially due to changes in some leaf anatomical and biochemical properties and due to measurement artifacts. Compared to C3 species, there is less information about gm responses to short-term changes in environment conditions like pCO2 across diverse C4 species and the potential determinants of these responses.Using 16 diverse C4 grasses we investigated the response of gm to short-term changes in CO2 and how this response related to the leaf anatomical and biochemical traits.For all the measured C4-grasses gm increased as CO2 decreased; however, the percent change in gm varied (+13% to +250%) and significantly related to percent changes in leaf transpiration efficiency (TEi). The percent increase in gm was highest in grasses with thinner mesophyll cell walls and greater leaf nitrogen, activities of phosphoenolpyruvate carboxylase (PEPC), Rubisco and carbonic anhydrase, and a higher affinity of PEPC for bicarbonate.Our study demonstrates that CO2 response of gm varies greatly across diverse C4 grasses and identifies the key leaf anatomical and biochemical traits related to this variation. These findings have implications for improving C4 photosynthetic models, and in attempts to improve TEi through manipulation of gm.

Mesophyll conductance (gm ) is the diffusion of CO2 from intercellular air spaces (IAS) to the first site of carboxylation in the mesophyll cells. In C3 species, gm is influenced by diverse leaf structural and anatomical traits; however, little is known about traits affecting gm in C4 species. To address this knowledge gap, we used online oxygen isotope discrimination measurements to estimate gm and microscopy techniques to measure leaf structural and anatomical traits potentially related to gm in 18 C4 grasses. In this study, gm scaled positively with photosynthesis and intrinsic water-use efficiency (TEi ), but not with stomatal conductance. Also, gm was not determined by a single trait but was positively correlated with adaxial stomatal densities (SDada ), stomatal ratio (SR), mesophyll surface area exposed to IAS (Smes ) and leaf thickness. However, gm was not related to abaxial stomatal densities (SDaba ) and mesophyll cell wall thickness (TCW ). Our study suggests that greater SDada and SR increased gm by increasing Smes and creating additional parallel pathways for CO2 diffusion inside mesophyll cells. Thus, SDada , SR and Smes are important determinants of C4 -gm and could be the target traits selected or modified for achieving greater gm and TEi in C4 species.

The engineering process of C4 photosynthesis into C3 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 CO2 and 13CO2 exchanges were measured, both in the light (at atmospheric O2 partial pressure of 1.84 kPa and at different CO2 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 13CO2$$\left( {\Delta_{\text{bio}} } \right)$$ was ~ 2‰ lower than in WT. However, there were no differences in leaf net CO2 assimilation rates (A) between genotypes, while the leaf dark respiration rates (Rd) over three hours after light–dark transition were enhanced (~ 30%) and with a higher 13C composition $$\left( {\delta^{ 1 3} {\text{C}}_{\text{Rd}} } \right)$$ 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.

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 CO2. 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 CO2 must cross the chloroplast before diffusing out of the leaf. Different arrangements have recently been considered, where all or part of the (photo)respired CO2 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 (gm). If (photo)respired CO2 can diffuse elsewhere besides the chloroplast, apparent gm is no longer a mere physical resistance but a flux-weighted variable sensitive to the ratio of (photo)respiration to net CO2 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 CO2 can diffuse both into the chloroplast and through the cytosol. Additionally, we present a new generalized Δ13C model that incorporates this dual diffusion pathway. We assess how assumptions about the fate of (photo)respired CO2 affect the interpretation of photosynthetic data and the challenges it poses for the application of different models.

The disruption of photorespiration in GDC knockdown rice plants alters leaf photorespiratory 13CO2 fractionation and carbon isotope exchange., 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 internal CO2 gradient imposed by mesophyll conductance (gm ) reduces substrate availability for C3 photosynthesis. With several assumptions, estimates of gm can be made from coupled leaf gas exchange with isoflux analysis of carbon ∆13 C-gm and oxygen in CO2 , coupled with transpired water (H2 O) ∆18 O-gm to partition gm 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-gm and ∆18 O-gm along with leaf biochemistry in the C3 grass Panicum bisulcatum, which has naturally low carbonic anhydrase activity. Our study suggests that assumptions regarding the extent of isotopic equilibrium (θ) between CO2 and H2 O at the site of exchange, and that the isotopic composition of the H2 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-gm temperature response. The input parameters for ∆13 C-gm 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-gm under changing leaf temperatures.

Flaveria trinervia C4 phosphoenolpyruvate carboxylase (PEPc) has higher affinity for HCO3− than its closely related Flaveria pringlei C3 PEPc, which drives higher modeled rates of C4 photosynthesis under low CO2 partial pressures., 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.

C4 grasses often outperform C3 species under hot, arid conditions due to superior water and nitrogen use efficiencies and lower rates of photorespiration. A method of concentrating CO2 around the site of carbon fixation in the bundle sheath (BS) is required to realize these gains. In NADP-malic enzyme (NADP-ME)-type C4 grasses such as maize, suberin deposition in the BS cell wall is hypothesized to act as a diffusion barrier to CO2 escape and O2 entry from surrounding mesophyll cells. Suberin is a heteropolyester comprised of acyl-lipid-derived aliphatic and phenylpropanoid-derived aromatic components. To disrupt BS suberization, we mutated two paralogously duplicated, unlinked maize orthologues of Arabidopsis thaliana ALIPHATIC SUBERIN FERULOYL TRANSFERASE, ZmAsft1 and ZmAsft2, using closely linked Dissociation transposons. Loss-of-function double mutants revealed a 97% reduction in suberin-specific omega-hydroxy fatty acids without a stoichiometric decrease in ferulic acid. However, BS suberin lamellae were deficient in electron opaque material, and cohesion between the suberin lamellae and polysaccharide cell walls was attenuated in double mutants. There were no other morphological phenotypes under ambient conditions. Furthermore, there was no significant effect on net CO2 assimilation at any intercellular CO2 concentration, and no effect on 13C isotope discrimination relative to wild type. Thus, ZmAsft expression is not required to establish a functional CO2 concentrating mechanism in in maize. Double mutant leaves exhibit elevated cell wall elasticity, transpirational, and stomatal conductance relative to WT. Thus, the ZmAsft genes are dispensable for gas exchange barrier function but may be involved in regulation of leaf water movement.One-sentence SummaryDouble mutants of two paralogously duplicated maize Aliphatic Suberin Feruloyl Transferase (ZmAsft) genes exhibit reduced aliphatic suberin content, cell wall cohesion defects, and elevated leaf transpiration, but no changes in CO2 assimilation relative to wild type.

A distinctive structural feature of the higher plant plastidial starch phosphorylase (Pho1) is a 50 to 82 amino acid long peptide (L50 - L82), which is absent in phosphorylases from non-plant organisms. To study the function of the rice Pho1 L80 peptide, we complemented apho1−rice mutant (BMF136) with the wildtype Pho1 gene or with a Pho1 gene lacking the L80 region (Pho1ΔL80). While expression of Pho1 in BMF136 restored normal wildtype phenotype, the introduction of Pho1ΔL80 enhanced growth rate and plant productivity above wildtype levels. Mass spectrometry analysis of proteins captured by anti-Pho1 showed the surprising presence of PsaC, the terminal electron acceptor/donor subunit of photosystem I (PSI). This unexpected interaction was substantiated by reciprocal immobilized protein pulldown assays of seedling extracts and supported by the presence of Pho1 on isolated PSI complexes resolved by blue native gels. Spectrophotometric studies showed that Pho1ΔL80 plants exhibited modified PSI and enhanced CO2assimilation properties. Collectively, these findings indicate that the higher plant Pho1 has dual roles as a potential modulator of source and sink processes.

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., Rubisco kinetic parameters from Arabidopsis thaliana were measured using radiolabel and membrane inlet mass spectrometry to determine how the reaction mechanisms and the individual rate constants change with temperature.

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.

In C4 photosynthesis, pyruvate orthophosphate dikinase (PPDK) catalyzes the regeneration of phosphoenolpyruvate in the carbon shuttle pathway. Although the biochemical function of PPDK in maize is well characterized, a genetic analysis of PPDK has not been reported. In this study, we use the maize transposable elements Mutator and Ds to generate multiple mutant alleles of PPDK. Loss-of-function mutants are seedling lethal, even when plants were grown under 2% CO2 , and they show very low capacity for CO2 assimilation, indicating C4 photosynthesis is essential in maize. Using RNA-seq and GC-MS technologies, we examined the transcriptional and metabolic responses to a deficiency in PPDK activity. These results indicate loss of PPDK results in downregulation of gene expression of enzymes of the C4 cycle, the Calvin cycle, and components of photochemistry. Furthermore, the loss of PPDK did not change Kranz anatomy, indicating that this metabolic defect in the C4 cycle did not impinge on the morphological differentiation of C4 characters. However, sugar metabolism and nitrogen utilization were altered in the mutants. An interaction between light intensity and genotype was also detected from transcriptome profiling, suggesting altered transcriptional and metabolic responses to environmental and endogenous signals in the PPDK mutants.

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.

Diffusion of CO2 from the leaf intercellular air space to the site of carboxylation (gm ) is a potential trait for increasing net rates of CO2 assimilation (Anet ), 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 gm and Anet . Online carbon isotope discrimination was used to determine gm and Anet 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 (Tcw ), surface area of chloroplast exposed to intercellular air spaces (Sc ), leaf dry mass per area (LMA), effective porosity, and other leaf anatomical traits were also analyzed. The gm of CslF6 mutants decreased by 83% relative to the WT, with c. 28% of the reduction in gm explained by Sc . Although Anet /LMA and Anet /Chl partially explained differences in Anet between genotypes, the change in cell wall properties influenced the diffusivity and availability of CO2 . The data presented here indicate that the loss of MLG in CslF6 plants had an impact on gm and demonstrate the importance of cell wall effective porosity and liquid path length on gm .

In two C4 species, mesophyll conductance increases with short-term exposure to decreasing pCO2 and limits photosynthetic capacity below ambient levels, whilst carbonic anhydrase imposes a further limitation only at very low pCO2., 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.

Mesophyll conductance (gm ) describes the movement of CO2 from the intercellular air spaces below the stomata to the site of initial carboxylation in the mesophyll. In contrast with C3 -gm , little is currently known about the intraspecific variation in C4 -gm or its responsiveness to environmental stimuli. To address these questions, gm was measured on five maize (Zea mays) lines in response to CO2 , employing three different estimates of gm . Each of the methods indicated a significant response of gm to CO2 . Estimates of gm were similar between methods at ambient and higher CO2 , but diverged significantly at low partial pressures of CO2 . These differences are probably driven by incomplete chemical and isotopic equilibrium between CO2 and bicarbonate under these conditions. Carbonic anhydrase and phosphoenolpyruvate carboxylase in vitro activity varied significantly despite similar values of gm and leaf anatomical traits. These results provide strong support for a CO2 response of gm in Z. mays, and indicate that gm in maize is probably driven by anatomical constraints rather than by biochemical limitations. The CO2 response of gm indicates a potential role for facilitated diffusion in C4 -gm . These results also suggest that water-use efficiency could be enhanced in C4 species by targeting gm .

Leaf carbon isotopic composition correlated well with transpiration efficiency, while leaf oxygen isotopic composition was confounded by leaf length in the C4 grasses Setaria viridis and Setaria italica., 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 cold acclimations of mesophyll conductance (gm ), bundle-sheath conductance (gbs ) and the CO2 concentrating mechanism (CCM) of C4 plants have not been well studied. Here, we estimated the temperature response of gm , gbs and leakiness (ϕ), the amount of concentrated CO2 that escapes the bundle-sheath cells, for the chilling-tolerant C4 plant Miscanthus × giganteus grown at 14 and 25°C. To estimate these parameters, we combined the C4 -enzyme-limited photosynthesis model and the Δ13 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 gbs 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 gm and gbs are unresponsive to growth temperature and that the CCM is able to acclimate to cold through increased activity of PPDK and RuBisCO.

Background Carbonic anhydrase (CA) catalyzes the hydration of CO2 in the first biochemical step of C4 photosynthesis, and has been considered a potentially rate-limiting step when CO2 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 CO2concentrations. However, the ca1ca2 mutants exhibited reduced rates of photosynthesis at sub-ambient CO2, and accumulated less biomass when grown under sub-ambient CO2 (9.2 Pa). To further clarify the importance of CA for C4 photosynthesis, we assessed gene expression changes in wild-type, ca1 and ca1ca2 mutants in response to changes in pCO2 from 920 to 9.2 Pa. Results Leaf samples from each genotype were collected for RNA-seq analysis at high CO2 and at two time points after the low CO2 transition, in order to identify early and longer-term responses to CO2 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 CO2, 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 CO2 stomatal signaling pathway were differentially expressed in the CA mutants under low CO2. 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 CO2. Our results also support a role for CA in the CO2 stomatal signaling pathway. This study provides insight into the importance of CA for C4 photosynthesis and its role in stomatal signaling.

Mixotrophic growth of microalgae offers great potential as an efficient strategy for biofuel production. In this study, photosynthetic regulation of mixotrophically cultured Chlorella sorokiniana cells was systematically evaluated. Mixotrophic cells in the exponential growth phase showed the highest photosynthetic activity, where maximum photosynthetic O 2 evolution was approximately 3- and 4-fold higher than cells in the same phase grown photoautotrophically in 1% CO 2 (in air) and air, respectively. Additionally, characteristic chlorophyll fluorescence parameters demonstrated that no limitation in electron transport downstream of PSII was detected in mixotrophic cells. Up-regulation of photosynthetic activity was associated with high total ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylase activity and expression level of phosphoribulokinase (PRK). After 3 days, photosynthetic O 2 evolution of mixotrophic cells that went to the stationary phase, was strongly reduced, with reduced photochemical efficiency and reorganization of the PSII complex. Simultaneously, enzymatic activity for Rubisco carboxylase and mRNA levels of Rubisco and PRK diminished. Importantly, there was almost no non-photochemical quenching for mixotrophic cells, whether grown in log or stationary phase. A decline in the quantum efficiency of PSII and an oxidized plastoquinone pool (PQ pool) was observed under N-depleted conditions during mixotrophic growth. These results demonstrate that photosynthesis is regulated differently in mixotrophically cultured C. sorokiniana cells than in cells grown under photoautotrophic conditions, with a particularly strong impact by nitrogen levels in the cells.

Summary 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 b6f 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.

The CO2 concentrating mechanism (CCM) in C4 plants requires a complex coordination of both leaf anatomical and biochemical traits. While there are key traits common across the 60 plus C4 lineages, there is also significant structural and biochemical variation. Traditionally, C4 plants are described as one of three biochemical subtypes based on the primary enzyme used for C4 acid decarboxylation: NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PCK). However, there may be biochemical flexibility and overlap between these subtypes. C4 plants typically rely on Kranz-type anatomy that partitions the C4 cycle into the mesophyll (M) cells and the majority of C3 cycle into the bundle-sheath (BS) cells. However, within the succulent Chenopods some NAD-ME type C4 plants use one of two single-cell arrangements to partition and compartmentalize the C4 and C3 cycles. Here we discuss key leaf anatomical traits at the tissue, cellular, and sub-cellular level that influence the efficiency and effectiveness of C4 photosynthesis. Specifically, we discuss preconditioning of leaf traits that increase the evolvability of C4 photosynthesis, the evolutionary transition of organelles from C3 to a C4 leaf, gas and metabolite movement within the leaf, the positioning and maintenance of organelles in M and BS cells, and the movement of M chloroplasts.

Highlight Enzyme kinetic measurements and positive selection analysis show that C4 species in Suaedoideae have PEPC and Rubisco kinetics similar to other C4 species despite different amino acid convergence., 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.

Photosynthesis in different organs of Cleome was analysed in four species known to have differences in leaf photosynthesis: Cleome africana Botsch. (C3), Cleome paradoxa R.Br. (C3-C4 intermediate), Cleome angustifolia Forssk. and Cleome gynandra L. (C4). The chlorophyll content, carbon isotope composition, stomatal densities, anatomy, levels and compartmentation of some key photosynthetic enzymes, and the form and function of photosynthesis were determined in different organs of these species. In the three xerophytes, C. africana, C. paradoxa, and C. angustifolia, multiple organs contribute to photosynthesis (cotyledons, leaves, petioles, stems and pods) which is considered important for their survival under arid conditions. In C. africana, all photosynthetic organs have C3 photosynthesis. In C. paradoxa, cotyledons, leaves, stems and petioles have C3-C4 type features. In C. angustifolia, the pods have C3 photosynthesis, whereas all other organs have C4 photosynthesis with Kranz anatomy formed by a continuous, dual layer of chlorenchyma cells. In the subtropical C4 species C. gynandra, cotyledons, leaves, and pods develop C4 photosynthesis, with Kranz anatomy around individual veins; but not in stems and petioles which have limited function of photosynthesis. The diversity in forms and the capacity of photosynthesis in organs of these species to contribute to their carbon economy is discussed.

Photosynthesis captures light energy to produce ATP and NADPH. These molecules are consumed in the conversion of CO2 to sugar, photorespiration, and NO3(-) assimilation. The production and consumption of ATP and NADPH must be balanced to prevent photoinhibition or photodamage. This balancing may occur via cyclic electron flow around photosystem I (CEF), which increases ATP/NADPH production during photosynthetic electron transport; however, it is not clear under what conditions CEF changes with ATP/NADPH demand. Measurements of chlorophyll fluorescence and dark interval relaxation kinetics were used to determine the contribution of CEF in balancing ATP/NADPH in hydroponically grown Arabidopsis (Arabidopsis thaliana) supplied different forms of nitrogen (nitrate versus ammonium) under changes in atmospheric CO2 and oxygen. Measurements of CEF were made under low and high light and compared with ATP/NADPH demand estimated from CO2 gas exchange. Under low light, contributions of CEF did not shift despite an up to 17% change in modeled ATP/NADPH demand. Under high light, CEF increased under photorespiratory conditions (high oxygen and low CO2), consistent with a primary role in energy balancing. However, nitrogen form had little impact on rates of CEF under high or low light. We conclude that, according to modeled ATP/NADPH demand, CEF responded to energy demand under high light but not low light. These findings suggest that other mechanisms, such as the malate valve and the Mehler reaction, were able to maintain energy balance when electron flow was low but that CEF was required under higher flow.

The low cell growth rate of Haematococcus pluvialis is a major barrier to achieve low-cost production of the biomass. Our previous study demonstrated that ribose could significantly enhance the growth rate and cell activity of H. pluvialis under mixotrophic conditions. This study showed the photosynthetic rate of the algal cells cultured with ribose was two-fold higher than that of phototrophic after 2-day cultivation. The increased photosynthetic activity of the cells with mixotrophic culture using ribose occurred earlier than that with sodium acetate. Similar photosynthetic fluorescence characteristics was observed for cells grown in phototrophic and ribose-fed culture. Photosystem (PS) I of ribose-fed cells was enlarged relative to PS II after two days. The difference in rubisco activity and subunit gene (rbcL and rbcS) expression however, was not significant between phototrophic and mixotrophic cultivation. The photosynthetic rate of H. pluvialis doubled with ribose mainly due to the high reaction of the Calvin cycle. The results suggested that metabolites derived from ribose could provide additional substrates for the Calvin cycle. This study provides the evidence about unique ribose utilization patterns by H. pluvialis during photosynthesis processes in mixotrophic culture mode. By understanding the mechanism of ribose metabolism in H. pluvialis under mixotrophic cultivation, it offers a promising route for improvement of biomass accumulation.

The CO2 compensation point in the absence of day respiration (Γ*) is a key parameter for modelling leaf CO2 exchange. Γ* links the kinetics of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) with the stoichiometry of CO2 released per Rubisco oxygenation from photorespiration (α), two essential components of biochemical models of photosynthesis. There are two main gas-exchange methods for measuring Γ*: (i) the Laisk method, which requires estimates of mesophyll conductance to CO2 (g m) and (ii) measurements of O2 isotope exchange, which assume constant values of α and a fixed stoichiometry between O2 uptake and Rubisco oxygenation. In this study, the temperature response of Γ* measured using the Laisk and O2-exchange methods was compared under ambient (25 °C) and elevated (35 °C) temperatures to determine whether both methods yielded similar results. Previously published temperature responses of Γ* estimated with the Laisk and O2-exchange methods in Nicotiana tabacum demonstrated that the Laisk-derived model of Γ* was more sensitive to temperature compared with the O2-exchange model. Measurements in Arabidopsis thaliana indicated that the Laisk and O2-exchange methods produced similar Γ* at 25 °C; however, Γ* values from O2 exchange were lower at 35 °C compared with the Laisk method. Compared with a photorespiratory mutant (pmdh1pmdh2hpr) with increased α, wild-type (WT) plants had lower Laisk values of Γ* at 25 °C but were not significantly different at 35 °C. These differences between Laisk and O2 exchange values of Γ* at 35 °C could be explained by temperature sensitivity of α in WT and/or errors in the assumptions of O2 exchange. The differences between Γ* measured using the Laisk and O2-exchange method with temperature demonstrate that assumptions used to measure Γ*, and possibly the species-specific validity of these assumptions, need to be considered when modelling the temperature response of photosynthesis.

Objective: To determine the effect of oleic acid and linoleic acid on the production and secretion of specific diabetic retinopathy- (DR-) related cytokines: vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) by human retinal glial cells, retinal endothelial cells, and retinal pigment epithelial cells. These expression profiles will be compared to those obtained by treatment of the same cell types with elevated D-glucose, a diabetes-relevant stimulus often used in retinal cell culture experiments. Methods: Primary cultures of human retinal Muller cells, astrocytes, and microvascular endothelial cells (RMEC) and a human retinal pigment epithelial cell line (ARPE-19) were treated with oleic acid, linoleic acid, elevated Dglucose, or L-glucose as an osmotic control. VEGF, IL-6, and IL-8 concentrations in conditioned media were determined by colorimetric ELISA and normalized to total cellular protein. Results: In the conditioned medium of human Muller cells, linoleic and oleic acid increased VEGF production by 6.4-fold and 9.9-fold, respectively. Linoleic acid also significantly increased IL-6 by 2.9-fold and IL-8 by 5.7-fold. Lglucose and D-glucose both increased VEGF by 3.1-fold in Muller cell conditioned medium. Linoleic acid increased IL-8 concentrations by 56% in human RMEC conditioned medium. Human retinal astrocytes and ARPE-19 were unaffected by all stimuli. Conclusions: Linoleic and oleic acid induce inflammatory mediators believed to be involved in the pathogenesis of diabetic retinopathy (DR). In culture, the free fatty acid insults, particularly linoleic acid, significantly increased cytokine production by Muller cells. In summary, these data identified Muller cells as the primary producer of these inflammatory mediators when treated with unsaturated fatty acids. This study also demonstrates that elevated glucose is an inadequate stimulus for assessing the production of inflammatory mediators. Therefore this study provides a novel in vitro model system of the dyslipidemia-induced inflammation occurring in DR.

The efficiency of C(4) photosynthesis in Zea mays, Miscanthus x giganteus and Flaveria bidentis in response to light was determined using measurements of gas exchange, (13) CO(2) photosynthetic discrimination, metabolite pools and spectroscopic assays, with models of C(4) photosynthesis and leaf (13) CO(2) discrimination. Spectroscopic and metabolite assays suggested constant energy partitioning between the C(4) and C(3) cycles across photosynthetically active radiation (PAR). Leakiness (φ), modelled using C(4) light-limited photosynthesis equations (φ(mod)), matched values from the isotope method without simplifications (φ(is)) and increased slightly from high to low PAR in all species. However, simplifications of bundle-sheath [CO(2)] and respiratory fractionation lead to large overestimations of φ at low PAR with the isotope method. These species used different strategies to maintain similar φ. For example, Z. mays had large rates of the C(4) cycle and low bundle-sheath cells CO(2 ) conductance (g(bs)). While F. bidentis had larger g(bs) but lower respiration rates and M. giganteus had less C(4) cycle capacity but low g(bs), which resulted in similar φ. This demonstrates that low g(bs) is important for efficient C(4) photosynthesis but it is not the only factor determining φ. Additionally, these C(4) species are able to optimize photosynthesis and minimize φ over a range of PARs, including low light.

Photosynthetic carbon gain in plants using the C(3) photosynthetic pathway is substantially inhibited by photorespiration in warm environments, particularly in atmospheres with low CO(2) concentrations. Unlike C(4) plants, C(3) plants are thought to lack any mechanism to compensate for the loss of photosynthetic productivity caused by photorespiration. Here, for the first time, we demonstrate that the C(3) plants rice and wheat employ a specific mechanism to trap and reassimilate photorespired CO(2) . A continuous layer of chloroplasts covering the portion of the mesophyll cell periphery that is exposed to the intercellular air space creates a diffusion barrier for CO(2) exiting the cell. This facilitates the capture and reassimilation of photorespired CO(2) in the chloroplast stroma. In both species, 24-38% of photorespired and respired CO(2) were reassimilated within the cell, thereby boosting photosynthesis by 8-11% at ambient atmospheric CO(2) concentration and 17-33% at a CO(2) concentration of 200 µmol mol(-1) . Widespread use of this mechanism in tropical and subtropical C(3) plants could explain why the diversity of the world's C(3) flora, and dominance of terrestrial net primary productivity, was maintained during the Pleistocene, when atmospheric CO(2) concentrations fell below 200 µmol mol(-1) .

The CO2 concentration in Earth's atmosphere may double during this century. Plant responses to such an increase depend strongly on their nitrogen status, but the reasons have been uncertain. Here, we assessed shoot nitrate assimilation into amino acids via the shift in shoot CO2 and O2 fluxes when plants received nitrate instead of ammonium as a nitrogen source (deltaAQ). Shoot nitrate assimilation became negligible with increasing CO2 in a taxonomically diverse group of eight C3 plant species, was relatively insensitive to CO2 in three C4 species, and showed an intermediate sensitivity in two C3-C4 intermediate species. We then examined the influence of CO2 level and ammonium vs. nitrate nutrition on growth, assessed in terms of changes in fresh mass, of several C3 species and a Crassulacean acid metabolism (CAM) species. Elevated CO2 (720 micromol CO2/mol of all gases present) stimulated growth or had no effect in the five C3 species tested when they received ammonium as a nitrogen source but inhibited growth or had no effect if they received nitrate. Under nitrate, two C3 species grew faster at sub-ambient (approximately 310 micromol/mol) than elevated CO2. A CAM species grew faster at ambient than elevated or sub-ambient CO2 under either ammonium or nitrate nutrition. This study establishes that CO2 enrichment inhibits shoot nitrate assimilation in a wide variety of C3 plants and that this phenomenon can have a profound effect on their growth. This indicates that shoot nitrate assimilation provides an important contribution to the nitrate assimilation of an entire C3 plant. Thus, rising CO2 and its effects on shoot nitrate assimilation may influence the distribution of C3 plant species.

Carbon and oxygen isotope discrimination of CO(2) during photosynthesis (Δ(13)C(obs) and Δ(18)O(obs)) were measured along a monocot leaf, triticale (Triticum × Secale). Both Δ(13)C(obs) and Δ(18)O(obs) increased towards the leaf tip. While this was expected for Δ(18)O(obs) , because of progressive enrichment of leaf water associated with the Peclet effect, the result was surprising for Δ(13) C(obs). To explore parameters determining this pattern, we measured activities of key photosynthetic enzymes [ribulose bis-phosphate carboxylase-oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase) as well as maximum carboxylation and electron transport rates (V(cmax) and J(max)) along the leaf. Patterns in leaf internal anatomy along the leaf were also quantified. Mesophyll conductance (g(m)) is known to have a strong influence on Δ(13)C(obs) , so we used three commonly used estimation methods to quantify variation in g(m) along the leaf. Variation in Δ(13)C(obs) was correlated with g(m) and chloroplast surface area facing the intercellular air space, but unrelated to photosynthetic enzyme activity. The observed variation could cause errors at higher scales if the appropriate portion of a leaf is not chosen for leaf-level measurements and model parameterization. Our study shows that one-third of the way from the base of the leaf represents the most appropriate portion to enclose in the leaf chamber.

Nitrate for Me, Ammonium for YouThe interdependence of plant nitrogen uptake and plant responses to carbon dioxide is well established, but the influence of inorganic nitrogen form—i.e., whether nitrate or ammonium—has been largely ignored.Bloomet al.(p.899) present evidence from five independent methods in both a monocot and dicot species that carbon dioxide inhibition of nitrate assimilation is a major determinant of plant responses to rising atmospheric concentrations of carbon dioxide. This finding explains several phenomena, including carbon dioxide acclimation and decline in food quality. The large variation in these phenomena among species, locations, or years derives from the large variation in the relative dependence of plants on nitrate and ammonium as nitrogen sources among species, locations, or years. The relative importance of ammonium and nitrate for plant N nutrition in future cropping systems will be critical for quantity and quality of food.

International audience; The photosynthetic assimilation of CO2 in C-4 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 C-4 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 C-4 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 degrees C to 40 degrees C using crude leaf extracts from the C-4 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 C-3 species and the C-4 plant S. viridis.

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.

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.

In C4 plants, carbonic anhydrase (CA) facilitates both the chemical and isotopic equilibration of atmospheric CO2 and bicarbonate (HCO3−) in the mesophyll cytoplasm. The CA-catalyzed reaction is essential for C4 photosynthesis, and the model of carbon isotope discrimination (Δ13C) in C4 plants predicts that changes in CA activity will influence Δ13C. However, experimentally, the influence of CA on Δ13C has not been demonstrated in C4 plants. Here, we compared measurements of Δ13C during C4 photosynthesis in Flaveria bidentis wild-type plants with F. bidentis plants with reduced levels of CA due to the expression of antisense constructs targeted to a putative mesophyll cytosolic CA. Plants with reduced CA activity had greater Δ13C, which was also evident in the leaf dry matter carbon isotope composition (δ13C). Contrary to the isotope measurements, photosynthetic rates were not affected until CA activity was less than 20% of wild type. Measurements of Δ13C, δ13C of leaf dry matter, and rates of net CO2 assimilation were all dramatically altered when CA activity was less than 5% of wild type. CA activity in wild-type F. bidentis is sufficient to maintain net CO2 assimilation; however, reducing leaf CA activity has a relatively large influence on Δ13C, often without changes in net CO2 assimilation. Our data indicate that the extent of CA activity in C4 leaves needs to be taken into account when using Δ13C and/or δ13C to model the response of C4 photosynthesis to changing environmental conditions.

Measurements of net fluxes of CO2 and O2 from leaves and chlorophyll a fluorescence were used to determine the role of mitochondrial respiration during nitrate (NO3–) assimilation in both a C3 (wheat) and a C4 (maize) plant. Changes in the assimilatory quotient (net CO2 consumed over net O2 evolved) when the nitrogen source was shifted from NO3– to NH4+ (ΔAQ) provided a measure of shoot NO3– assimilation. According to this measure, elevated CO2 inhibited NO3– assimilation in wheat but not maize. Net O2 exchange under ambient CO2 concentrations increased in wheat plants receiving NO3– instead of NH4+, but gross O2 evolution from the photosynthetic apparatus (JO2) was insensitive to nitrogen source. Therefore, O2 consumption within wheat photosynthetic tissue (ΔΟ2), the difference between JO2 and net O2 exchange, decreased during NO3– assimilation. In maize, NO3– assimilation was insensitive to changes in intercellular CO2 concentration (Ci); nonetheless, ΔΟ2 at low Ci values was significantly higher in NO3–-fed than in NH4+-fed plants. Changes in O2 consumption during NO3– assimilation may involve one or more of the following processes: (a) Mehler ascorbate peroxidase (MAP) reactions; (b) photorespiration; or (c) mitochondrial respiration. The data presented here indicates that in wheat, the last process, mitochondrial respiration, is decreased during NO3– assimilation. In maize, NO3– assimilation appears to stimulate mitochondrial respiration when photosynthetic rates are limiting.

Measurements of CO 2 and O 2 gas exchange and chlorophyll a fluorescence were used to test the hypothesis that elevated atmospheric CO 2 inhibits nitrate (NO 3 ‐ ) photo-assimilation in the C 4 plant, maize ( Zea mays L.). The assimilatory quotient ( AQ ), the ratio of net CO 2 assimilation to net O 2 evolution, decreases as NO 3 ‐ photo-assimilation increases so that the difference in AQ between the ammonium- and nitrate-fed plants ( D D D AQ ) provided an in planta estimate of NO 3 ‐ photo-assimilation. In fully expanded maize leaves, NO 3 ‐ photo-assimilation was detectable only under high light and was not affected by CO 2 treatments. Furthermore, CO 2 assimilation and O 2 evolution were higher under NO 3 ‐ than ammonia (NH 4 + + + ) regardless of CO 2 levels. In conclusion, NO 3 ‐ photo-assimilation in maize primarily occurred at high light when reducing equivalents were presumably not limiting. Nitrate photo-assimilation enhanced C 4 photosynthesis, and in contrast to C 3 plants, elevated CO 2 did not inhibit foliar NO 3 ‐ photo-assimilation.

The present study was carried out to test the hypothesis that elevated atmospheric CO 2 (Ca) will alleviate over-excitation of the C 4 photosynthetic apparatus and decrease non-photochemical quenching (NPQ) during periods of limited water availability. Chlorophyll a fluorescence was monitored in Sorghum bicolor plants grown under a freeair carbon-dioxide enrichment (FACE) by water-stress (Dry) experiment. Under Dry conditions elevated Ca increased the quantum yield of photosystem II ( f PSII) throughout the day through increases in both photochemical quenching coefficient ( q p ) and the efficiency with which absorbed quanta are transferred to open PSII reaction centres ( F v ¢¢¢ ¢ / F m ¢¢ ). However, in the well-watered plants (Wets) FACE enhanced f PSII only at midday and was entirely attributed to changes in F v ¢¢¢ ¢ / F m ¢¢ . Under field conditions, decreases in f f f PSII under Dry treatments and ambient Ca corresponded to increases in NPQ but the de-epoxidation state of the xanthophyll pool (DPS) showed no effects. Water-stress did not lead to long-term damage to the photosynthetic apparatus as indicated by f PSII and carbon assimilation measured after removal of stress conditions. We conclude that elevated Ca enhances photochemical light energy usage in C 4 photosynthesis during drought and/ or midday conditions. Additionally, NPQ protects against photo-inhibition and photodamage. However, NPQ and the xanthophyll cycle were affected differently by elevated Ca and water-stress.

Summary We demonstrated that mitochondrial respiration contributes to energy balancing between nitrogen and carbon metabolism by competing for reductant with de novo NO3 – assimilation and avoiding chloroplastic excess reductant., Under high light, the rates of photosynthetic CO2 assimilation can be influenced by reductant consumed by both foliar nitrate assimilation and mitochondrial alternative electron transport (mAET). Additionally, nitrate assimilation is dependent on reductant and carbon skeletons generated from both the chloroplast and mitochondria. However, it remains unclear how nitrate assimilation and mAET coordinate and contribute to photosynthesis. Here, hydroponically grown Arabidopsis thaliana T-DNA insertional mutants for alternative oxidase (AOX1A) and uncoupling protein (UCP1) fed either NO3 – or NH4 + were used to determine (i) the response of NO3 – uptake and assimilation to the disruption of mAET, and (ii) the interaction of N source (NO3 – versus NH4 +) and mAET on photosynthetic CO2 assimilation and electron transport. The results showed that foliar NO3 – assimilation was enhanced in both aox1a and ucp1 compared with the wild-type, suggesting that foliar NO3 – assimilation is probably driven by a decreased capacity of mAET and an increase in reductant within the cytosol. Wild-type plants had also higher rates of net CO2 assimilation (A net) and quantum yield of PSII (ϕPSII) under NO3 – feeding compared with NH4 + feeding. Additionally, under NO3 – feeding, A net and ϕPSII were decreased in aox1a and ucp1 compared with the wild type; however, under NH4 + they were not significantly different between genotypes. This indicates that NO3 – assimilation and mAET are both important to maintain optimal rates of photosynthesis, probably in regulating reductant accumulation and over-reduction of the chloroplastic electron transport chain. These results highlight the importance of mAET in partitioning energy between foliar nitrogen and carbon assimilation.

○ The interactive effects of atmospheric CO 2 concentration and soil-water content on grain sorghum (Sorghum bicolor) are reported here. ○ Sorghum plants were exposed to ambient (control) and free-air CO 2 enrichment (FACE; ambient + 200 μmol mol -1 ), under ample (wet, 100% replacement of evapotranspiration) and reduced (dry, postplanting and mid-season irrigations) water supply over two growing seasons. ○ FACE reduced seasonal average stomatal conductance (g s ) by 0.17 mol (H 2 O) m -2 s -1 (32% and 37% for dry and wet, respectively) compared with control; this was similar to the difference between dry and wet treatments. FACE increased net assimilation rate (A) by 4.77 μmol (CO 2 ) m -2 s -1 (23% and 9% for dry and wet, respectively), whereas dry decreased A by 10.50 μmol (CO 2 ) m -2 s -1 (26%) compared with wet. Total plant water potential (ψ w ) was 0.16 MPa (9%) and 0.04 MPa (3%) less negative in FACE than in the control treatment for dry and wet, respectively. Under dry, FACE stimulated final shoot biomass by 15%. ○ By ameliorating the adverse effects of drought, elevated atmospheric CO 2 improved plant water status, which indirectly caused an increase in carbon gain.

Summary • Sorghum (Sorghum bicolor) was grown for two consecutive seasons at Maricopa, AZ, USA, using the free-air CO2 enrichment (FACE) approach to investigate evapotranspiration of this C4 plant at ample and limited water supplies. • Crop evapotranspiration (ET) was measured using two CO2 concentrations (control, c. 370 µmol mol−1; FACE, ambient +200 µmol mol−1) and two irrigation treatments (well watered and water-limited). Volumetric soil water content was measured before and after each irrigation using neutron scattering techniques. • Averaged over both years, elevated CO2 reduced cumulative ET by 10% when plants were given ample water and by 4% under severe drought stress. Water-use efficiency based on grain yield (WUE-G) increased, due to CO2 enrichment, by 9% and 19% in wet and dry plots, respectively; based on total biomass, water-use efficiency (WUE-B) increased by 16% and 17% in wet and dry plots, respectively. • These data suggest that in the future high-CO2 environment, water requirements for irrigated sorghum will be lower than at present, while dry-land productivity will increase, provided global warming is minimal.

Summary • To determine the response of C4 plants to elevated CO2 it is necessary to establish whether young leaves have a fully developed C4 photosynthetic apparatus, and whether photosynthesis in these leaves is responsive to elevated CO2. • The effect of free-air CO2 enrichment (FACE) on the photosynthetic development of the C4 crop Sorghum bicolor was monitored. Simultaneous measurements of chlorophyll a fluorescence and carbon assimilation were made to determine energy utilization, quantum yields of carbon fixation (φCO2) and photosystem II (φPSII), as well as photorespiration. • Assimilation in the second leaf of FACE plants was 37% higher than in control plants and lower apparent rates of photorespiration at growth CO2 concentrations were exhibited. In these leaves, φPSII : φCO2 was high at low atmospheric CO2 concentration (Ca) due to overcycling of the C4 pump and increased leakiness. As Ca increased, φPSII : φCO2 decreased as a greater proportion of energy derived from linear electron transfer was used by the C3 cycle. • The stimulation of C4 photosynthesis at elevated Ca in young leaves was partially due to suppressed photorespiration. Additionally, elevated Ca enhanced energy-use efficiency in young leaves, possibly by decreasing CO2 leakage from bundle sheath cells, and by decreasing overcycling of the C4 pump.

In many bioartificial liver systems currently being designed and evaluated for use in fulminant hepatic failure, direct contact is required between the patient's blood and the liver cells in the device. The efficacy of such devices will be influenced by the interaction of fulminant hepatic failure (FHF) patient serum with the cells. We have found that FHF serum inhibits the growth rate and the synthesis of DNA, RNA, and protein; disturbs glutathione homeostasis; and induces morphological changes in cultured human Hep G2 cells. These interactions should influence the design of bioartificial liver devices based on proliferating cell lines and indicate the requirement to pretreat FHF patient plasma to reduce the toxin load.

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.