Partitioning of CO(2) Fixation in the Colonial Cyanobacterium Microcystis aeruginosa: Mechanism Promoting Formation of Surface Scums

Constraints on inorganic carbon (C(i)) availability stimulated buoyancy in natural, photosynthetically active populations of the colonial blue-green alga (cyanobacterium) Microcystis aeruginosa. In nonmixed eutrophic river water and cultures, O(2) evolution determinations indicated C(i) limitation of photosynthesis, which was overcome either by CO(2) additions to the aqueous phase or by exposure of buoyant colonies to atmospheric CO(2). Microautoradiographs of M. aeruginosa colonies revealed partitioning of CO(2) fixation and photosynthate accumulation between peripheral and internal cells, particularly in large colonies. When illuminated colonies were suspended in the aqueous phase, peripheral cells accounted for at least 90% of the CO(2) assimilation, whereas internal cells remained unlabeled. However, when CO(2) was allowed to diffuse into colonies 15 min before illumination, a more uniform distribution of labeling was observed. Resultant differences in labeling patterns were most likely due to peripheral cells more exclusively utilizing CO(2) when ambient C(i) concentrations were low. Among colonies located at the air-water interface, internal cells showed an increased share of photosynthate production when atmospheric CO(2) was supplied. This indicated that C(i) transport was restricted in large colonies below the water surface, forcing internal cells to maintain a high degree of buoyancy, thus promoting the formation of surface scums. At the surface, C(i) restrictions were alleviated. Accordingly, scum formation appears to have an ecological function, allowing cyanobacteria access to atmospheric CO(2) when the C(i) concentration is growth limiting in the water column.     

Ethoxyzolamide Inhibition of CO(2) Uptake in the Cyanobacterium Synechococcus PCC7942 without Apparent Inhibition of Internal Carbonic Anhydrase Activity

In high inorganic carbon grown (1% CO₂ [volume/volume]) cells of the cyanobacterium Synechococcus PCC7942, the carbonic anhydrase (CA) inhibitor, ethoxyzolamide (EZ), was found to inhibit the rate of CO₂ uptake and to reduce the final internal inorganic carbon (C_i) pool size reached. The relationship between CO₂ fixation rate and internal C_i concentration in high C_i grown cells was little affected by EZ. This suggests that in intact cells internal CA activity was unaffected by EZ. High C_i grown cells readily took up CO₂ but had little or no capacity for HCO₃⁻ uptake. These cells appear to possess a CO₂ utilizing C_i pump that has a CA-like function associated with the transport step such that HCO₃⁻ is the species delivered to the cell interior. This CA-like step may be the site of inhibition by EZ. Low C_i grown cells possess both CO₂ uptake and HCO₃⁻ uptake activities and EZ inhibited both activities to a similar degree, suggesting that a common step in CO₂ and HCO₃⁻ uptake (such as the C_i pump) may have been affected. The inhibitor had no apparent effect on internal CO₂/HCO₃⁻ equilibria (internal CA function) in low C_i grown cells.     

Carbonyl sulfide: an inhibitor of inorganic carbon transport in cyanobacteria

Cells of a high CO₂-requiring mutant (E₁) and wild type of Synechococcus PCC7942 were incubated with COS in the light, then suspended in COS-free medium and their CO₂ exchange was measured using an open gas-analysis system under the conditions where photosynthetic CO₂ fixation is inhibited. When the suspension of cells untreated with COS was illuminated, the rate of CO₂ uptake was high and addition of carbonic anhydrase during illumination released a large amount of CO₂ from the medium into the gas phase. The COS treatment in the light markedly reduced the rate of CO₂ uptake by the cells and the amount of CO₂ released by carbonic anhydrase. Incubation of cells with COS in the dark had no effect on the CO₂-exchange profile. The COS concentration required for 50% inhibition of CO₂ uptake was about 25 micromolar when the concentration of inorganic carbon (C_i) in the medium was 60 micromolar; higher C_i concentrations reduced the inhibitory effect of COS. Measurement of C_i uptake in E₁ cells by a silicone oil centrifugation method also indicated marked reduction of the activities of ¹⁴CO₂ and H¹⁴CO₃⁻ uptake in the cells treated with COS in the light. The results demonstrated that COS is a potent inhibitor of C_i transport.     

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : I. The Use of Steady-State Labeling

The pathway and kinetics of photosynthate unloading in developing seeds of bean (Phaseolus vulgaris L.) were investigated using steady-state labeling with ¹⁴CO₂. The continuous assimilation of ¹⁴CO₂ at constant specific activity produced stable tracer fluxes that facilitated straightforward analyses of photosynthate import and unloading in developing seeds. The kinetics of tracer equilibration within intact seeds were compatible with a symplastic route of photosynthate unloading in the seed coat. The import and partitioning of tracer within seeds were partially disrupted by the surgical excision of the distal halves of seeds as practiced during the preparation of “empty” seed coats for perfusion.     

Isolation and Characterization of High CO(2)-Requiring-Mutants of the Cyanobacterium Synechococcus PCC7942 : Two Phenotypes that Accumulate Inorganic Carbon but Are Apparently Unable to Generate CO(2) within the Carboxysome

A total of 24 high CO₂-requiring-mutants of the cyanobacterium Synechococcus PCC7942 have been isolated and partially characterized. These chemically induced mutants are able to grow at 1% CO₂, on agar media, but are incapable of growth at air levels of CO₂. All the mutants were able to accumulate inorganic carbon (C_i) to levels similar to or higher than wild type cells, but were apparently unable to generate intracellular CO₂. On the basis of the rate of C_i release following a light (5 minutes) → dark transition two extreme phenotypes (fast and slow release mutants) and a number of `intermediate' mutants (normal release) were identified. Compared to wild-type cells, Type I mutants had the following characteristics: fast C_i release, normal internal C_i pool, normal carbonic anhydrase (CA) activity in crude extracts, reduced internal exchange of ¹⁸O from ¹⁸O-labeled CO₂, 1% CO₂ requirement for growth in liquid media, normal affinity of carboxylase for CO₂, and long, rod-like carboxysomes. Type II mutants had the following characteristics: slow C_i release, increased internal C_i pool, normal CA activity in crude extracts, normal internal ¹⁸O exchange, a 3% CO₂ requirement for growth in liquid media, high carboxylase activity, normal affinity of carboxylase for CO₂, and normal carboxysome structure but increased in numbers per cell. Both mutant phenotypes appear to have genetic lesions that result in an inability to convert intracellular HCO₃⁻ to CO₂ inside the carboxysome. The features of the type I mutants are consistent with a scenario where carboxysomal CA has been mistargeted to the cytosol. The characteristics of the type II phenotype appear to be most consistent with a scenario where CA activity is totally missing from the cell except for the fact that cell extracts have normal CA activity. Alternatively the type II mutants may have a lesion in their capacity for H⁺ import during photosynthesis.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

Sequencing and Modification of the Gene Encoding the 42-Kilodalton Protein in the Cytoplasmic Membrane of Synechococcus PCC 7942

A 42-kilodalton cytoplasmic membrane protein is synthesized when high CO₂-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) are exposed to low CO₂. The structural gene for this protein (cmpA) has been cloned and sequenced and shown to encode a 450 amino acid polypeptide with a molecular mass of 49 kilodalton. A deletion mutant lacking the 42-kilodalton protein was obtained by transformation of Synechococcus PCC 7942 following in vitro mutagenesis of the cloned gene. There were no significant differences between the mutant and wild-type cells in their growth rates under either low or high CO₂ conditions. The activity of inorganic carbon (C_i) transport in the mutant was as high as that in the wild-type strain. In both types of cells, CO₂ was the main species of C_i transported and the activities of CO₂ and HCO₃⁻ transport increased when high CO₂-grown cells were exposed to low CO₂. We conclude that the 42-kilodalton protein is not directly involved in the C_i-accumulating mechanism of Synechococcus PCC 7942.     

Ethoxyzolamide Inhibition of CO(2)-Dependent Photosynthesis in the Cyanobacterium Synechococcus PCC7942

Cells of the cyanobacterium, Synechococcus PCC7942, grown under high inorganic carbon (C_i) conditions (1% CO₂; pH 8) were found to be photosynthetically dependent on exogenous CO₂. This was judged by the fact that they had a similar photosynthetic affinity for CO₂ (K_0.5[CO₂] of 3.4-5.4 micromolar) over the pH range 7 to 9 and that the low photosynthetic affinity for C_i measured in dense cell suspensions was improved by the addition of exogenous carbonic anhydrase (CA). The CA inhibitor, ethoxyzolamide (EZ), was shown to reduce photosynthetic affinity for CO₂ in high C_i cells. The addition of 200 micromolar EZ to high C_i cells increased K_0.5(CO₂) from 4.6 micromolar to more than 155 micromolar at pH 8.0, whereas low C_i cells (grown at 30 microliters CO₂ per liter of air) were less sensitive to EZ. EZ inhibition in high and low C_i cells was largely relieved by increasing exogenous C_i up to 100 millimolar. Lipid soluble CA inhibitors such as EZ and chlorazolamide were shown to be the most effective inhibitors of CO₂ usage, whereas water soluble CA inhibitors such as methazolamide and acetazolamide had little or no effect. EZ was found to cause a small drop in photosystem II activity, but this level of inhibition was not sufficient to explain the large effect that EZ had on CO₂ usage. High C_i cells of Anabaena variabilis M3 and Synechocystis PCC6803 were also found to be sensitive to 200 micromolar EZ. We discuss the possibility that the inhibitory effect of EZ on CO₂ usage in high C_i cells of Synechococcus PCC7942 may be due to inhibition of a `CA-like' function associated with the CO₂ utilizing C_i pump or due to inhibition of an internal CA activity, thus affecting CO₂ supply to ribulose bisphosphate carboxylase-oxygenase.     

The CO2 permeability of the plasma membrane of Chlamydomonas reinhardtii: mass-spectrometric 18O-exchange measurements from 13C18O2 in suspensions of carbonic anhydrase-loaded plasma-membrane vesicles

The unicellular green alga Chlamydomonas reinhardtii possesses a CO₂-concentrating mechanism. In order to measure the CO₂ permeability coefficients of the plasma membranes (PMs), carbonic anhydrase (CA) loaded vesicles were isolated from C. reinhardtii grown either in air enriched with 50 mL CO₂ · L?1} (high-C_i cells) or in ambient air (350 μL CO₂ · L?1}; low-C_i cells). Marker-enzyme measurements indicated less than 1% contamination with thylakoid and mitochondrial membranes, and that more than 90% of the PMs from high and low-C_i cells were orientated right-side-out. The PMs appeared to be sealed as judged from the ability of vesicles to accumulate [¹⁴C]acetate along a proton gradient for at least 10 min. Carbonic anhydrase-loaded PMs from high and low-C_i cells of C. reinhardtii were used to measure the exchange of ¹⁸O between doubly labelled CO₂ (¹³C¹⁸O₂) and H₂O in stirred suspensions by mass spectrometry. Analysis of the kinetics of the ¹⁸O depletion from ¹³C¹⁸O₂ in the external medium provides a powerful tool to study CO₂ diffusion across the PM to the active site of CA which catalyses ¹⁸O exchange only inside the vesicles but not in the external medium (Silverman et al., 1976, J Biol Chem 251: 4428–4435). The activity of CA within loaded PM vesicles was sufficient to speed-up the ¹⁸O loss to H₂O to 45360–128800 times the uncatalysed rate, depending on the efficiency of CA-loading and PM isolation. From the ¹⁸O-depletion kinetics performed at pH 7.3 and 7.8, CO₂ permeability coefficients of 0.76 and 1.49·10?3} cm·s?1}, respectively, were calculated for high C_i cells. The corresponding values for low-C_i cells were 1.21 and 1.8·10?3} cm·s?1}. The implications of the similar and rather high CO₂ permeability coefficients (low CO₂ resistance) in high and low-C_i cells for the CO_i-concentrating mechanism of C. reinhardtii are discussed.     

Quantitation of Rates of Transport, Metabolic Fluxes, and Cytoplasmic Levels of Inorganic Carbon in Maize Root Tips during K Ion Uptake

Our aim was to determine whether fixation of inorganic carbon (C_i), due to phosphoenolpyruvate carboxylase activity, is limited by the availability of C_i in the cytoplasm of maize (Zea mays L.) root tips. Rates of C_i uptake and metabolism were measured during K₂SO₄ treatment, which stimulates dark C_i fixation. ¹³C_i uptake was followed by ¹³C-nuclear magnetic resonance (NMR); 5 millimolar K₂SO₄ had no significant effect on ¹³C_i influx. The contribution of respiratory CO₂ production to cytoplasmic HCO₃⁻ was measured using in vivo ¹³C-NMR and ¹H-NMR of cell extracts; K₂SO₄ treatment had no effect on respiratory CO₂ production. The concentration of cytoplasmic HCO₃⁻ was estimated to be approximately 11 millimolar, again with K₂SO₄ having no significant effect. These experiments allowed us to determine the extent to which extracellularly supplied ¹⁴C_i was diluted in the cytoplasm by respiratory CO₂ and thereby measure phosphoenolpyruvate (PEP) carboxylase activity in vivo using ¹⁴C_i. PEP carboxylase activity in root tips was enhanced approximately 70% over controls within 12 minutes of the addition of 5 millimolar K₂SO₄. The activity of carbonic anhydrase, which provides PEP carboxylase with C_i, was determined by saturation transfer ¹³C-NMR to be more than 200 times that of PEP carboxylase in vivo. The regulation of PEP carboxylase in K₂SO₄-treated roots is discussed.     

Carbonic Anhydrase Activity Associated with the Cyanobacterium Synechococcus PCC7942

Intact cells and crude homogenates of high (1% CO₂) and low dissolved inorganic carbon (C_i) (30-50 microliters per liter of CO₂) grown Synechococcus PCC7942 have carbonic anhydrase (CA)-like activity, which enables them to catalyze the exchange of ¹⁸O from CO₂ to H₂O. This activity was studied using a mass spectrometer coupled to a cuvette with a membrane inlet system. Intact high and low C_i cells were found to contain CA activity, separated from the medium by a membrane which is preferentially permeable to CO₂. This activity is most apparent in the light, where ¹⁸O-labeled CO₂ species are being taken up by the cells but the effluxing CO₂ has lost most of its label to water. In the dark, low C_i cells catalyze the depletion of the ¹⁸O enrichment of CO₂ and this activity is inhibited by both ethoxyzolamide and 2-(trifluoromethoxy)carbonyl cyanide. This may occur via a common inhibition of the C_i pump and the C_i pump is proposed as a potential site for the exchange of ¹⁸O. CA activity was measurable in homogenates of both cell types but was 5- to 10-fold higher in low C_i cells. This was inhibited by ethoxyzolamide with an I₅₀ of 50 to 100 micromolar in both low and high C_i cells. A large proportion of the internal CA activity appears to be pelletable in nature. This pelletability is increased by the presence of Mg²⁺ in a manner similar to that of ribulose bisphosphate carboxylase-oxygenase activity and chlorophyll (thylakoids) and may be the result of nonspecific aggregation. Separation of crude homogenates on sucrose gradients is consistent with the notion that CA and ribulose bisphosphate carboxylase-oxygenase activity may be associated with the same pelletable fraction. However, we cannot unequivocally establish that CA is located within the carboxysome. The sucrose gradients show the presence of separate soluble and pelletable CA activity. This may be due to the presence of separate forms of the enzyme or may arise from the same pelletable association which is unstable during extraction.     

Energy Sources for HCO3? and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 625

Light-dependent inorganic C (C_i) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO₂ or HCO₃⁻ uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular C_i pool of 20 mm, even though CO₂ fixation was completely inhibited, indicating that cyclic electron flow was involved in the C_i-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar C_i accumulation was observed, suggesting that linear electron flow could support the transport of C_i. When carbonic anhydrase was not present, initial CO₂ uptake was greatly reduced and the extracellular [CO₂] eventually increased to a level higher than equilibrium, strongly suggesting that CO₂ transport was inhibited and that C_i accumulation was the result of active HCO₃⁻ transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, C_i transport and accumulation were inhibited by inhibitors of CO₂ transport, such as COS and Na₂S, whereas Li⁺, an HCO₃⁻-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, C_i transport and accumulation were not inhibited by COS and Na₂S but were inhibited by Li⁺. These results suggest that CO₂ transport is supported by cyclic electron transport and that HCO₃⁻ transport is supported by linear electron transport.     

Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO(2)-Requiring Phenotype : Evidence for a Central Role for Carboxysomes in the CO(2) Concentrating Mechanism

Active human carbonic anhydrase II (HCAII) protein was expressed in the cyanobacterium Synechococcus PCC7942 by means of transformation with the bidirectional expression vector, pCA. This expression was driven by the bacterial Tac promoter and was regulated by the IacIQ repressor protein, which was expressed from the same plasmid. Expression levels reached values of around 0.3% of total cell protein and this protein appeared to be entirely soluble in nature and located within the cytosol of the cell. The expression of this protein has dramatic effects on the photosynthetic physiology of the cell. Induction of expression of carbonic anhydrase (CA) activity in both high dissolved inorganic carbon (C_i) and low C_i grown cells leads the creation of a high C_i requiring phenotype causing: (a) a dramatic increase in the K_0.5 (C_i) for photosynthesis, (b) a loss of the ability to accumulate internal C_i, and (c) a decrease in the lag between the initial C_i accumulation following illumination and the efflux of CO₂ from the cells. In addition, the effects of the expressed CA can largely be reversed by the carbonic anhydrase inhibitor ethoxyzolamide. As a result of the above findings, it is concluded that the CO₂ concentrating mechanism in Synechococcus PCC7942 is largely dependent on (a) the absence of CA activity from the cytosol, and (b) the specific localization of CA activity in the carboxysome. A theoretical model of photosynthesis and C_i accumulation is developed in which the carboxysome plays a central role as both the site of CO₂ generation from HCO_3⁻ and a resistance barrier to CO₂ efflux from the cell. There is good qualitative agreement between this model and the measured physiological effects of expressed cytosolic CA in Synechococcus cells.     

Allocation and Turnover of Photosynthetically Assimilated CO(2) in Leaves of Glycine max L. Clark

The allocation and turnover of photosynthetically assimilated ¹⁴CO₂ in lipid and protein fractions of soybean (Glycine max L. Clark) leaves and stem materials was measured. In whole plant labeling experiments, allocation of photosynthate from a pulse of ¹⁴CO₂ into polymeric compounds was: 25% to proteins in 4 days, 20% to metabolically inert cell wall products in 1 to 2 days, 10% to lipids in 4 days, and 4% to starch in 1 day. The amount of ¹⁴C labeled photosynthate that an actively growing leaf (leaf 4) used for its own lipid synthesis immediately following pulse labeling was about 25%. The ¹⁴C of labeled proteins turned over with half-lives of 3.8, 3.3, and 4.1 days in leaves 1, 2, and 3, respectively; and turnover of ¹⁴C in total shoot protein proceeded with a half-life of 5.2 days. Three kinetic ¹⁴C turnover patterns were observed in lipids: a rapid turnover fraction (within a day), an intermediate fraction (half-life about 5 days), and a slow turnover fraction. These results are discussed in terms of previously published accounts of translocation, carbon budgets, carbon use, and turnover in starch, lipid, protein, and cell wall materials of various plants including soybeans.     

Mechanism of c(4) photosynthesis: the size and composition of the inorganic carbon pool in bundle sheath cells

We sought to characterize the inorganic carbon pool (CO₂ plus HCO₃⁻) formed in the leaves of C₄ plants when C₄ acids derived from CO₂ assimilation in mesophyll cells are decarboxylated in bundle sheath cells. The size and kinetics of labeling of this pool was determined in six species representative of the three metabolic subgroups of C₄ plants. The kinetics of labeling of the inorganic carbon pool of leaves photosynthesizing under steady state conditions in ¹⁴CO₂ closely paralleled those for the C-4 carboxyl of C₄ acids for all species tested. The inorganic carbon pool size, determined from its ¹⁴C content at radioactivity saturation, ranged between 15 and 97 nanomoles per milligram of leaf chlorophyll, giving estimated concentrations in bundle sheath cells of between 160 and 990 micromolar. The size of the pool decreased, together with photosynthesis, as light was reduced from 900 to 95 microeinsteins per square meter per second or as external CO₂ was reduced from 400 to 98 microliters per liter. A model is developed which suggests that the inorganic carbon pool existing in the bundle sheath cells of C₄ plants during steady state photosynthesis will comprise largely of CO₂; that is, CO₂ will only partially equlibrate with bicarbonate. This predominance of CO₂ is believed to be vital for the proper functioning of the C₄ pathway.     

Stomatal and pavement cell density linked to leaf internal CO2 concentration

Background and Aims

Stomatal density (SD) generally decreases with rising atmospheric CO₂ concentration, C_a. However, SD is also affected by light, air humidity and drought, all under systemic signalling from older leaves. This makes our understanding of how C_a controls SD incomplete. This study tested the hypotheses that SD is affected by the internal CO₂ concentration of the leaf, C_i, rather than C_a, and that cotyledons, as the first plant assimilation organs, lack the systemic signal.

Methods

Sunflower (Helianthus annuus), beech (Fagus sylvatica), arabidopsis (Arabidopsis thaliana) and garden cress (Lepidium sativum) were grown under contrasting environmental conditions that affected C_i while C_a was kept constant. The SD, pavement cell density (PCD) and stomatal index (SI) responses to C_i in cotyledons and the first leaves of garden cress were compared. ¹³C abundance (δ¹³C) in leaf dry matter was used to estimate the effective C_i during leaf development. The SD was estimated from leaf imprints.

Key Results

SD correlated negatively with C_i in leaves of all four species and under three different treatments (irradiance, abscisic acid and osmotic stress). PCD in arabidopsis and garden cress responded similarly, so that SI was largely unaffected. However, SD and PCD of cotyledons were insensitive to C_i, indicating an essential role for systemic signalling.

Conclusions

It is proposed that C_i or a C_i-linked factor plays an important role in modulating SD and PCD during epidermis development and leaf expansion. The absence of a C_i–SD relationship in the cotyledons of garden cress indicates the key role of lower-insertion CO₂ assimilation organs in signal perception and its long-distance transport.     

Acclimation of photosynthesis to increasing atmospheric CO2: The gas exchange perspective

The nature of photosynthetic acclimation to elevated CO₂ is evaluated from the results of over 40 studies focusing on the effect of long-term CO₂ enrichment on the short-term response of photosynthesis to intercellular CO₂ (the A/C_i response). The effect of CO₂ enrichment on the A/C_i response was dependent on growth conditions, with plants grown in small pots (< 5 L) or low nutrients usually exhibiting a reduction of A at a given C_i, while plants grown without nutrient deficiency in large pots or in the field tended to exhibit either little reduction or an enhancement of A at a given C_i following a doubling or tripling of atmospheric CO₂ during growth. Using theoretical interpretations of A/C_i curves to assess acclimation, it was found that when pot size or nutrient deficiency was not a factor, changes in the shape of A/C_i curves which are indicative of a reallocation of resources within the photosynthetic apparatus typically were not observed. Long-term CO₂ enrichment usually had little effect or increased the value of A at all C_i. However, a minority of species grown at elevated CO₂ exhibited gas exchange responses indicative of a reduced amount of Rubisco and an enhanced capacity to metabolize photosynthetic products. This type of response was considered beneficial because it enhanced both photosynthetic capacity at high CO₂ and reduced resource investment in excessive Rubisco capacity. The ratio of intercellular to ambient CO₂ (the C_i/C_a ratio) was used to evaluate stomatal acclimation. Except under water and humidity stress, C_i/C_a exhibited no consistent change in a variety of C₃ species, indicating no stomatal acclimation. Under drought or humidity stress, C_i/C_a declined in high-CO₂ grown plants, indicating stomata will become more conservative during stress episodes in future high CO₂ environments.Abbreviations A net CO₂ assimilation rate - C_i (C_a) intercellular (ambient) partial pressure of CO₂ - operational C_i intercellular partial pressure of CO₂ at a given ambient partial pressure of CO₂ - g_s stomatal conductance - normal CO₂ current atmospheric mole fraction of CO₂ (330 to 355 mol mol^–1) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Limitation of net CO2 assimilation rate by internal resistances to CO2 transfer in the leaves of two tree species (Fagus sylvatica L. and Castanea sativa Mill.)

Using a combination of gas-exchange and chlorophyll fluorescence measurements, low apparent CO₂/O₂ specificity factors (1300 mol mol_?1) were estimated for the leaves of two deciduous tree species (Fagus sylvatica and Castanea sativa). These low values contrasted with those estimated for two herbaceous species and were ascribed to a drop in the CO₂ mole fraction between the intercellular airspace (C_i) and the catalytic site of Rubisco (C_c) due to internal resistances to CO₂ transfer. C_c. was calculated assuming a specificity of Rubisco value of 2560 mol mol^?1. The drop between C_i and C_c was used to calculate the internal conductance for CO₂ (g_i). A good correlation between mean values of net CO₂ assimilation rate (A) and g_i was observed within a set of data obtained using 13 woody plant species, including our own data. We report that the relative limitation of A, which can be ascribed to internal resistances to CO₂ transfer, was 24–30%. High internal resistances to CO₂ transfer may explain the low apparent maximal rates of carboxylation and electron transport of some woody plant species calculated from A/C_i curves.     

Is There a Role for the 42 Kilodalton Polypeptide in Inorganic Carbon Uptake by Cyanobacteria?

Cyanobacterial cells accumulate substantial amounts of a membrane-associated 42 kilodalton polypeptide during adaptation to low CO₂ conditions. The role of this polypeptide in the process of adaptation and in particular in the large increase in the ability to accumulate inorganic carbon (C_i), which accompanies this process, is not yet understood. We have isolated a mutant Synechococcus PCC7942 that does not accumulate the 42 kilodalton polypeptide. The mutant requires a high-CO₂ concentration for growth and exhibits a very low apparent photosynthetic affinity for extracellular C_i. The latter might be attributable to the observed defective ability of the mutant to utilize the intracellular C_i pool for photosynthesis. The 42 kilodalton polypeptide does not appear to participate directly in the active transport of C_i, since the difference between the observed capabilities for CO₂ and HCO₃⁻ uptake of the mutant and the wild type is not sufficient to account for their different growth and photosynthetic performance. Furthermore, high CO₂-grown wild-type cells, where we could not detect the 42 kilodalton polypeptide, transported CO₂ faster than the mutant. An analysis of the curves relating the rate of accumulation of C_i to the concentration of CO₂ or HCO₃⁻ supplied, in the presence or absence of carbonic anhydrase, indicated that under the experimental conditions used here, CO₂ was the preferred C_i species taken up by Synechococcus.     

Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration

When cells of Anacystis nidulans strain R2 grown under high CO₂ conditions (3%) were transferred to low CO₂ conditions (0.05%), their ability to accumulate inorganic carbon (C_i) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO₂ adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C_i-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C_i-accumulating capability. The incorporation of [³⁵S]sulfate into membrane proteins was greatly reduced during low CO₂ adaptation. Radioautograms of the ³⁵S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C_i transport by A. nidulans strain R2 and its synthesis under low CO₂ conditions leads to high C_i-transporting activity.