Induction and Regulation of Expression of a Low-CO2-Induced Mitochondrial Carbonic Anhydrase in Chlamydomonas reinhardtii

The time course of and the influence of light intensity and light quality on the induction of a mitochondrial carbonic anhydrase (CA) in the unicellular green alga Chlamydomonas reinhardtii was characterized using western and northern blots. This CA was expressed only under low-CO₂ conditions (ambient air). In asynchronously grown cells, the mRNA was detected 15 min after transfer from air containing 5% CO₂ to ambient air, and the 21-kD polypeptide was detected on western blots after 1 h. When transferred back to air containing 5% CO₂, the mRNA disappeared within 1 h and the polypeptide was degraded within 3 d. Photosynthesis was required for the induction in asynchronous cultures. The induction increased with light up to 500 μmol m⁻² s⁻¹, where saturation occurred. In cells grown synchronously, however, expression of the mitochondrial CA was also detected in darkness. Under such conditions the expression followed a circadian rhythm, with mRNA appearing in the dark 30 min before the light was turned on. Algae left in darkness continued this rhythm for several days.     

Expressing an RbcS Antisense Gene in Transgenic Flaveria bidentis Leads to an Increased Quantum Requirement for CO2 Fixed in Photosystems I and II

It was previously shown with concurrent measurements of gas exchange and carbon isotope discrimination that the reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase by an antisense gene construct in transgenic Flaveria bidentis (a C4 species) leads to reduced CO2 assimilation rates, increased bundle-sheath CO2 concentration, and leakiness (defined as the ratio of CO2 leakage to the rate of C4 acid decarboxylation; S. von Caemmerer, A. Millegate, G.D. Farquhar, R.T. Furbank [1997] Plant Physiol 113: 469-477). Increased leakiness in the transformants should result in an increased ATP requirement per mole of CO2 fixed and a change in the ATP-to-NADPH demand. To investigate this, we compared measurements of the quantum yield of photosystem I and II ([phi]PSI and [phi]PSII) with the quantum yield of CO2 fixation ([phi]CO2) in control and transgenic F. bidentis plants in various conditions. Both [phi]PSI/[phi]CO2 and [phi]PSII/[phi]CO2 increased with a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase content, confirming an increase in leakiness. In the wild type the ratio of [phi]PSI to [phi]PSII was constant at different irradiances but increased with irradiance in the transformants, suggesting that cyclic electron transport may be higher in the transformants. To evaluate the relative contribution of cyclic or linear electron transport to extra ATP generation, we developed a model that links leakiness, ATP/NADP requirements, and quantum yields. Despite some uncertainties in the light distribution between photosystem I and II, we conclude from the increase of [phi]PSII/[phi]CO2 in the transformants that cyclic electron transport is not solely responsible for ATP generation without NADPH production.     

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C4 Carbonic Anhydrase in Flaveria

C₄ photosynthesis has evolved multiple times from ancestral C₃ species. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO₂ and is involved in both C₃ and C₄ photosynthesis; however, its roles and the intercellular and intracellular locations of the majority of its activity differ between C₃ and C₄ plants. To understand the molecular changes underlying the evolution of the C₄ pathway, three cDNAs encoding distinct β-CAs (CA1, CA2, and CA3) were isolated from the leaves of the C₃ plant Flaveria pringlei. The phylogenetic relationship of the F. pringlei proteins with other embryophyte β-CAs was reconstructed. Gene expression and protein localization patterns showed that CA1 and CA3 demonstrate high expression in leaves and their products localize to the chloroplast, while CA2 expression is low in all organs examined and encodes a cytosolic enzyme. The roles of the F. pringlei enzymes were considered in light of these results, other angiosperm β-CAs, and Arabidopsis (Arabidopsis thaliana) “omics” data. All three F. pringlei CAs have orthologs in the closely related C₄ plant Flaveria bidentis, and comparisons of ortholog sequences, expression patterns, and intracellular locations of their products indicated that CA1 and CA2 have maintained their ancestral role in C₄ plants, whereas modifications to the C₃ CA3 gene led to the evolution of the CA isoform that catalyzes the first step in the C₄ photosynthetic pathway. These changes included the loss of the chloroplast transit peptide and an increase in gene expression, which resulted in the high levels of CA activity seen in the cytosol of C₄ mesophyll cells.     

Antisense Reduction of NADP-Malic Enzyme in Flaveria bidentis Reduces Flow of CO2 through the C4 Cycle

An antisense construct targeting the C₄ isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO₂ to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (α-NADP-ME) exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ). In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO₂ assimilation rates at high intercellular CO₂ were significantly reduced, whereas the in vitro activities of both phosphoenolpyruvate carboxylase and Rubisco were increased. Δ measured concurrently with gas exchange in these plants showed a lower Δ and thus a lower calculated leakiness of CO₂ (the ratio of CO₂ leak rate from the bundle sheath to the rate of CO₂ supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of increased Δ and leakiness. We use these measurements to estimate the C₄ cycle rate, bundle sheath leak rate, and bundle sheath CO₂ concentration. The comparison of α-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of the C₃ and C₄ cycles that exist during environmental perturbations by light and CO₂ can be disrupted through transgenic manipulations. Furthermore, our results suggest that the efficiency of the C₄ pathway could potentially be improved through a reduction in C₄ cycle activity or increased C₃ cycle activity.In the leaves of a range of plants including maize (Zea mays), sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), and millet (Pennisetum americanum), a biochemical pathway known as C₄ photosynthesis has evolved to concentrate CO₂ at the site of Rubisco such that Rubisco can operate at close to its maximal activity and photorespiration is reduced, enhancing the rate of photosynthesis in air (Hatch, 1987; Sage, 2004). In most C₄ plants, CO₂ is fixed by phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells into four-carbon acids, which diffuse to an inner ring of bundle sheath cells, where they are decarboxylated and the CO₂ is refixed by Rubisco. Plants using the C₄ photosynthetic mechanism have been subdivided into three primary subtypes, the NADP-malic enzyme (ME), NAD-ME, and phosphoenolpyruvate carboxykinase types, according to the decarboxylating enzyme used to generate CO₂ from C₄ acids in the bundle sheath cells (Hatch, 1987). Flaveria bidentis is a typical NADP-ME dicot in which malate and Asp contribute equally in the transfer of CO₂ to bundle sheath cells (Meister et al., 1996). Presumably, in most C₄ plants, the reactions that facilitate the appropriation, transformation, transport, and eventual concentration of CO₂ in the bundle sheath cell chloroplasts (C₄ cycle) are balanced with the reactions that incorporate CO₂ into usable carbon compounds for energy (C₃/Calvin cycle) such that energy is not lost or wasted as environmental conditions fluctuate. This process is important in maintaining the efficiency of the CO₂-concentrating mechanism and of C₄ photosynthesis overall. The nature of the controlling mechanisms for balance and coordination between the C₃ and C₄ cycles is still unclear, however, and concrete evidence for the coordinated regulation of primary carboxylation in the mesophyll and decarboxylation of C₄ acids in the bundle sheath has not been forthcoming. A key approach to revealing these mechanisms has been the use of antisense RNA in the C₄ dicot F. bidentis to reduce levels of key photosynthetic enzymes, including Rubisco (Furbank et al., 1996), NADP-malate dehydrogenase and pyruvate phosphate dikinase (Furbank et al., 1997), Rubisco activase (von Caemmerer et al., 2005), carbonic anhydrase (Cousins et al., 2006), and PEPC protein kinase (Furumoto et al., 2007). This has proven to be a valuable method to help gain insight into enzyme function and regulation during C₄ photosynthesis and to potentially alter the balance between the C₃ and C₄ cycles.In this study, we targeted the gene encoding the chloroplastic C₄ isozyme of NADP-ME in F. bidentis (Marshall et al., 1996) with an antisense construct designed to reduce its activity in vivo. This isoform is thought to catalyze the decarboxylation of l-malate to pyruvate and CO₂ and of NADP to NADPH in bundle sheath chloroplasts during C₄ photosynthesis (Ashton, 1997; Drincovich et al., 2001), allowing the CO₂ to be fixed into the C₃ cycle by Rubisco and pyruvate to return back to mesophyll cells to be recycled into PEP. These antisense lines were generated for two purposes. First, these plants could be used to confirm the identity of the gene encoding the NADP-ME isozyme involved in C₄ photosynthesis. Several other functioning isoforms of NADP-ME have also been identified within Flaveria spp.: a chloroplastic but potentially nonphotosynthetic NADP-ME form and a cytosolic NADP-ME (Marshall et al., 1996; Drincovich et al., 1998; Lai et al., 2002). The specific role and regulation of a C₄ NADP-ME isozyme in F. bidentis is of interest in relation to the “transfer” or “generation” of a functioning C₄ cycle to C₃ plants (Sheehy et al., 2007; Furbank et al., 2009). A greater understanding of the balance and interactions between this enzyme and others in the C₄ and C₃ cycles will aid in deciding the expression locations and levels needed for C₃ plants to gain a functional CO₂-concentrating mechanism.The second use of these antisense plants was to investigate the degree of coordination between the C₄/C₃ cycles in F. bidentis and the possibility of manipulation to improve photosynthetic efficiency. As mentioned above, the mechanisms of regulation (if any) of the C₃ pathway enzymes such as Rubisco in response to the activity and CO₂ supply rate of the C₄ cycle are unknown. It is similarly unclear how much the reactions of the C₃ cycle affect the rates of the initial CO₂-fixing reactions (carbonic anhydrase and PEPC). Leakiness (ϕ), defined as the ratio of CO₂ leak rate from the bundle sheath to the rate of CO₂ supply, reflects the coordination of the C₄ and C₃ cycles by describing the amount of overcycling of the C₄ cycle that has to occur to support a given rate of net CO₂ assimilation (Furbank et al., 1990; von Caemmerer and Furbank, 1999). As a major C₄ enzyme functioning within the bundle sheath, a reduction in NADP-ME should affect both the C₄ cycle rate and the bundle sheath CO₂ concentration (C_s), possibly disrupting the enzymatic balance and coordination in F. bidentis. Here, we have designed experiments to simultaneously look at in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ), facilitating estimates of ϕ, C₄ cycle rate, and the possible range of C_s within transgenic α-NADP-ME and antisense Rubisco small subunit (α-SSu) F. bidentis plants (Furbank et al., 1996). These measurements aim to show the impact of our perturbations of the C₃/C₄ balance, highlighting possible communication pathways between the cycles and also other possible targets for future genetic manipulation to improve the rate and/or efficiency of photosynthesis in C₄ plants.     

CO(2) Concentrating Mechanism of C(4) Photosynthesis: Permeability of Isolated Bundle Sheath Cells to Inorganic Carbon

Diffusion of inorganic carbon into isolated bundle sheath cells from a variety of C₄ species was characterized by coupling inward diffusion of CO₂ to photosynthetic carbon assimilation. The average permeability coefficient for CO₂ (P_CO2) for five representatives from the three decarboxylation types was approximately 20 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. The average value for the NAD-ME species Panicum miliaceum (10 determinations) was 26 with a standard deviation of 6 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. A P_CO2 of at least 500 micromoles per minute per milligram chlorophyll per millimolar was determined for cells isolated from the C₃ plant Xanthium strumarium. It is concluded that bundle sheath cells are one to two orders of magnitude less permeable to CO₂ than C₃ photosynthetic cells. These data also suggest that CO₂ diffusion in bundle sheath cells may be made up of two components, one involving an apoplastic path and the other a symplastic (plasmodesmatal) path, each contributing approximately equally.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.     

Oxygen Requirement and Inhibition of C4 Photosynthesis : An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O₂ sensitivity of C₄ photosynthesis was evaluated using a C₄-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C₃-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C₄-cycle-limited mutant showed that atmospheric levels of O₂ (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O₂ partial pressure for photosynthesis was reduced from approximately 5 kPa O₂ to 1 to 2 kPa O₂, becoming similar to that of C₃ plants. Therefore, the higher O₂ requirement for optimal C₄ photosynthesis is specifically associated with the C₄ function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O₂ than in the wild type. When CO₂ fixation by Rubisco is limited, an increase in the CO₂ concentration in bundle-sheath cells via the C₄ cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O₂ while increasing CO₂ leakage and overcycling of the C₄ pathway. These results indicate that in C₄ plants the investment in the C₃ and C₄ cycles must be balanced for maximum efficiency.     

Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Antisense RNA in the C4 Plant Flaveria bidentis Leads to Reduced Assimilation Rates and Increased Carbon Isotope Discrimination

Transgenic Flaveria bidentis (a C4 species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO2 assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO2 assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H2O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO2 partial pressures. Carbon isotope discrimination was measured concurrently with CO2 and H2O exchange on leaves of the control plants and T1 progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C4 species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO2 concentration and CO2 leakage from the bundle sheath; however, some down-regulation of the C4 cycle also occurred.     

Localization of Carbonic Anhydrase in Mesophyll Cells of Terrestrial C3 Plants in Relation to CO2 Assimilation

The activity and intracellular compartmentation of carbonicanhydrase was examined in mesophyll protoplasts of several C₃terrestrial species including wheat, since this enzyme may facilitatediffusion of inorganic carbon in solution by converting CO₂to bicarbonate. Carbonic anhydrase was located in the mesophyllchloroplast with little or no activity in the cytosolic fraction.In wheat, carbonic anhydrase was absent in etiolated leavesand increased in the light during greening. Thus the enzymemay have a role in photosynthesis in the chloroplast but notin the cytosol of mesophyll cells of higher C₃ plants. The amount of CO₂ required for half maximum rates of photosynthesis(under low O₂) was about two-fold higher for isolated protoplaststhan with isolated chloroplasts of wheat. The form of inorganiccarbon taken up by protoplasts, like that of chloroplasts, isCO₂. The results are discussed in relation to a possible resistanceto CO₂ transfer in the cytosol of mesophyll cells. (Received February 25, 1985; Accepted May 7, 1985)     

Analysis of Promoter Activity for the Gene Encoding Pyruvate Orthophosphate Dikinase in Stably Transformed C4 Flaveria Species

The C₄ enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C₄ plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C₄ photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C₄ cycle. We fused 1.5 kb of a 5′ flanking region from the Pdk gene, including the entire 5′ untranslated region, to the uidA reporter gene and stably transformed the closely related C₄ species Flaveria bidentis. β-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C₄ form in mesophyll cells and the chloroplast form in other cells and organs are co-located within the same 5′ region of the Pdk gene.     

C4 Photosynthesis (The Effects of Leaf Development on the CO2-Concentrating Mechanism and Photorespiration in Maize)

The effect of O2 on photosynthesis was determined in maize (Zea mays) leaves at different developmental stages. The optimum level of O2 for maximum photosynthetic rates was lower in young and senescing tissues (2-5 kPa) than in mature tissue (9 kPa). Inhibition of photosynthesis by suboptimal levels of O2 may be due to a requirement for functional mitochondria or to cyclic/pseudocyclic photophosphorylation in chloroplasts; inhibition by supraoptimal levels of O2 is considered to be due to photorespiration. Analysis of a range of developmental stages (along the leaf blade and at different leaf ages and positions) showed that the degree of inhibition of photosynthesis by supraoptimal levels of O2 increased rapidly once the ribulose-1,5-bisphosphate carboxylase/oxygenase and chlorophyll contents were below a critical level and was similar to that of C3 plants. Tissue having a high sensitivity of photosynthesis to O2 may be less effective in concentrating CO2 in the bundle sheath cells due either to limited function of the C4 cycle or to higher bundle sheath conductance to CO2. An analysis based on the kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase was used to predict the maximum CO2 level concentrated in bundle sheath cells at a given degree of inhibition of photosynthesis by supraoptimal levels of O2.     

Inorganic Carbon Diffusion between C(4) Mesophyll and Bundle Sheath Cells: Direct Bundle Sheath CO(2) Assimilation in Intact Leaves in the Presence of an Inhibitor of the C(4) Pathway

Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO₂ assimilation or oxygen evolution, over a wide range of CO₂ concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO₂ near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO₂ concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HCO₃⁻, indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of ¹⁴C-incorporation from ¹⁴CO₂ into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO₂ occurs predominantly by direct CO₂ fixation by ribulose 1,5-bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO₂ concentrations, permeability coefficients for CO₂ flux into bundle sheath cells were determined for a range of C₄ species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C₃ plants. These results support the concept that a CO₂ permeability barrier exists to allow the development of high CO₂ concentrations in bundle sheath cells during C₄ photosynthesis.     

Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: II. Metabolism of Products of CO(2) Fixation after Different Illumination Times

The metabolism of fixed ¹⁴CO₂ and the utilization of the C-4 carboxyl of malate and aspartate were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. Pulse/chase experiments indicated that both malate and aspartate appeared to function directly in the C₄ cycle at all times during the induction period (examined after 30 seconds, 5 minutes and 20 minutes illumination). However, the rate of loss of ¹⁴C-label from the C-4 position of malate plus aspartate was relatively slow after 30 seconds of illumination, compared to treatments after 5 or 20 minutes of illumination. Similarly, the appearance of label in other photosynthetic products (e.g. 3-phosphoglycerate, sugar phosphates, alanine) during the chase periods was generally slower after only 30 seconds of leaf illumination, compared to that after 5 of 20 minutes illumination. This may be due to the lower rate of photosynthesis after 30 seconds illumination. The appearance of label in carbons 1→3 of each C₄ acid during the chase periods was relatively slow after either 30 seconds or 5 minutes illumination, while there was a relatively rapid accumulation of label in carbons 1→3 of both C₄ acids after 20 minutes illumination. Thus, while the turnover rate of the ¹⁴C-4 label in both C₄ acids increased only during the first 5 minutes of the induction period, only later during induction is there an increased rate of appearance of label in other carbon atoms of the C₄ acids. The implied source of ¹⁴C for labeling of the 1→3 positions of the C₄ acids is an apparent carbon flux from 3-phosphoglycerate of the reductive pentose phosphate pathway to phosphoenolpyruvate of the C₄ cycle.     

Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: I. Initial Products of CO(2) Assimilation and Levels of Whole Leaf C(4) Metabolites

Labeling patterns from ¹⁴CO₂ pulses to leaves and whole leaf metabolite contents were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. During the first one to two minutes of illumination, malate was the primary initial product of ¹⁴CO₂ assimiltion (about 77% of total ¹⁴C incorporated). After about 5 minutes of illumination, the proportion of initial label to aspartate increased from 16 to 66%, and then gradually declined during the following 7 to 10 minutes of illumination. Nutrition experiments showed that the increase in ¹⁴CO₂ partitioning to aspartate was delayed about 2.5 minutes in plants grown with limiting N, and was highly dampened in plants previously treated 10 to 12 days with ammonia as the sole N source. Measurements of C₄ leaf metabolites revealed several transients in metabolite pools during the first few minutes of illumination, and subsequently, more gradual adjustments in pool sizes. These include a large initial decrease in malate (about 1.6 micromoles per milligram chlorophyll) and a small initial decrease in pyruvate. There was a transient increase in alanine levels after 1 minute of illumination, which was followed by a gradual, prolonged decrease during the remainder of the induction period. Total leaf aspartate decreased initially, but temporarily doubled in amount between 5 and 10 minutes of illumination (after its surge as a primary product). These results are discussed in terms of a plausible sequence of metabolic events which lead to the formation of the intercellular metabolite gradients required in C₄ photosynthesis.     

Photosynthetic Gas Exchange and Discrimination against 13CO2 and C18O16O in Tobacco Plants Modified by an Antisense Construct to Have Low Chloroplastic Carbonic Anhydrase

The physiological role of chloroplastic carbonic anhydrase (CA) was examined by antisense suppression of chloroplastic CA (on average 8% of wild type) in Nicotiana tabacum. Photosynthetic gas-exchange characteristics of low-CA and wild-type plants were measured concurrently with short-term, on-line stable isotope discrimination at varying vapor pressure deficit (VPD) and light intensity. Low-CA and wild-type plants were indistinguishable in the responses of assimilation, transpiration, stomatal conductance, and intercellular CO2 concentration to changing VPD or light intensity. At saturating light intensity, low-CA plants had lower discrimination against 13CO2 than wild-type plants by 1.2 to 1.8[per mille (thousand) sign]. Consequently, tissue of the low-CA plants was higher in 13C than the control plants. It was calculated that low-CA plants had chloroplast CO2 concentrations 13 to 22 [mu]mol mol-1 lower than wild-type plants. Discrimination against C18O16O in low-CA plants was 20% of that of the wild type, confirming a role of chloroplastic CA in the mechanism of discrimination against C18O16O ([delta]C18O16O). As VPD increased, stomatal closure caused a reduction in chloroplastic C02 concentration, and since VPD and chloroplastic CO2 concentration act in opposing directions on [delta]C18O16O, no effect of VPD was seen on [delta]C18O16O.     

13CO2 labeling kinetics in maize reveal impaired efficiency of C4 photosynthesis under low irradiance

C₄ photosynthesis allows faster photosynthetic rates and higher water and nitrogen use efficiency than C₃ photosynthesis, but at the cost of lower quantum yield due to the energy requirement of its biochemical carbon concentration mechanism. It has also been suspected that its operation may be impaired in low irradiance. To investigate fluxes under moderate and low irradiance, maize (Zea mays) was grown at 550 µmol photons m⁻² s^−l and ¹³CO₂ pulse-labeling was performed at growth irradiance or several hours after transfer to 160 µmol photons m⁻² s⁻¹. Analysis by liquid chromatography/tandem mass spectrometry or gas chromatography/mass spectrometry provided information about pool size and labeling kinetics for 32 metabolites and allowed estimation of flux at many steps in C₄ photosynthesis. The results highlighted several sources of inefficiency in low light. These included excess flux at phosphoenolpyruvate carboxylase, restriction of decarboxylation by NADP-malic enzyme, and a shift to increased CO₂ incorporation into aspartate, less effective use of metabolite pools to drive intercellular shuttles, and higher relative and absolute rates of photorespiration. The latter provides evidence for a lower bundle sheath CO₂ concentration in low irradiance, implying that operation of the CO₂ concentration mechanism is impaired in this condition. The analyses also revealed rapid exchange of carbon between the Calvin–Benson cycle and the CO₂-concentration shuttle, which allows rapid adjustment of the balance between CO₂ concentration and assimilation, and accumulation of large amounts of photorespiratory intermediates in low light that provides a major carbon reservoir to build up C₄ metabolite pools when irradiance increases.

Analysis of metabolite pools, sizes, and fluxes reveals that multiple interlocking factors decrease the efficiency of photosynthesis in low irradiance in maize.     

Cryptococcus neoformans Infection in Mice Lacking Type I Interferon Signaling Leads to Increased Fungal Clearance and IL-4-Dependent Mucin Production in the Lungs

Type I interferons (IFNs) are secreted by many cell types upon stimulation via pattern recognition receptors and bind to IFN-α/β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2. Although type I IFNs are well known as anti-viral cytokines, limited information is available on their role during fungal infection. In the present study, we addressed this issue by examining the effect of IFNAR1 defects on the host defense response to Cryptococcus neoformans. In IFNAR1KO mice, the number of live colonies was lower and the host immune response mediated not only by Th1 but also by Th2 and Th17-related cytokines was more accelerated in the infected lungs than in WT mice. In addition, mucin production by bronchoepithelial cells and expression of MUC5AC, a major core protein of mucin in the lungs, were significantly higher in IFNAR1KO mice than in WT mice. This increase in mucin and MUC5AC production was significantly inhibited by treatment with neutralizing anti-IL-4 mAb. In contrast, administration of recombinant IFN-αA/D significantly suppressed the production of IL-4, but not of IFN-γ and IL-17A, in the lungs of WT mice after cryptococcal infection. These results indicate that defects of IFNAR1 led to improved clearance of infection with C. neoformans and enhanced synthesis of IFN-γ and the IL-4-dependent production of mucin. They also suggest that type I IFNs may be involved in the negative regulation of early host defense to this infection.     

Acclimation of Photosynthesis to Elevated CO2 under Low-Nitrogen Nutrition Is Affected by the Capacity for Assimilate Utilization. Perennial Ryegrass under Free-Air CO2 Enrichment

Acclimation of photosynthesis to elevated CO₂ has previously been shown to be more pronounced when N supply is poor. Is this a direct effect of N or an indirect effect of N by limiting the development of sinks for photoassimilate? This question was tested by growing a perennial ryegrass (Lolium perenne) in the field under elevated (60 Pa) and current (36 Pa) partial pressures of CO₂ (pCO2) at low and high levels of N fertilization. Cutting of this herbage crop at 4- to 8-week intervals removed about 80% of the canopy, therefore decreasing the ratio of photosynthetic area to sinks for photoassimilate. Leaf photosynthesis, in vivo carboxylation capacity, carbohydrate, N, ribulose-1,5-bisphosphate carboxylase/oxygenase, sedoheptulose-1,7-bisphosphatase, and chloroplastic fructose-1,6-bisphosphatase levels were determined for mature lamina during two consecutive summers. Just before the cut, when the canopy was relatively large, growth at elevated pCO₂ and low N resulted in significant decreases in carboxylation capacity and the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase protein. In high N there were no significant decreases in carboxylation capacity or proteins, but chloroplastic fructose-1,6-bisphosphatase protein levels increased significantly. Elevated pCO₂ resulted in a marked and significant increase in leaf carbohydrate content at low N, but had no effect at high N. This acclimation at low N was absent after the harvest, when the canopy size was small. These results suggest that acclimation under low N is caused by limitation of sink development rather than being a direct effect of N supply on photosynthesis.