High Carbonic Anhydrase Activity Research Articles

CAH3 from Chlamydomonas reinhardtii: Unique Carbonic Anhydrase of the Thylakoid Lumen.

CAH3 is the only carbonic anhydrase (CA) present in the thylakoid lumen of the green algae Chlamydomonas reinhardtii. The monomer of the enzyme has a molecular weight of ~29.5 kDa with high CA activity. Through its dehydration activity, CAH3 can be involved either in the carbon-concentrating mechanism supplying CO2 for RuBisCO in the pyrenoid or in supporting the maximal photosynthetic activity of photosystem II (PSII) by accelerating the removal of protons from the active center of the water-oxidizing complex. Both proposed roles are considered in this review, together with a description of the enzymatic parameters of native and recombinant CAH3, the crystal structure of the protein, and the possible use of lumenal CA as a tool for increasing biomass production in higher plants. The identified involvement of lumenal CAH3 in the function of PSII is still unique among green algae and higher plants and can be used to understand the mechanism(s) of the functional interconnection between PSII and the proposed CA(s) of the thylakoid lumen in other organisms.

A leader peptide of the extracellular cyanobacterial carbonic anhydrase ensures the efficient secretion of recombinant proteins in Escherichia coli

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli.

Limitation of C4 photosynthesis by low carbonic anhydrase activity increases with temperature but does not influence mesophyll CO2 conductance.

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.

Biomass Production of Three Biofuel Energy Plants’ Use of a New Carbon Resource by Carbonic Anhydrase in Simulated Karst Soils: Mechanism and Capacity

To determine whether the bicarbonate in karst limestone soil could be used as a new carbon resource for biomass production by the catalysis of carbonic anhydrase (CA), a simulative karst drought stress experiment was designed and performed. Three plants used for biofuel energy, Orychophragmus violaceus L. (Ov), Brassica juncea L. (Bj), and Euphorbia lathyris L. (El), were grown under simulated karst drought stress. In response to drought stress, the photosynthesis of the three energy plants was inhibited, but their CA activity increased. The hypothesis was confirmed by plant physiological and stable isotope techniques. The obtained results showed that plant biomass was produced with atmospheric CO2 as well as bicarbonate under drought stress. Bicarbonate use was proportional to the CA activity of the plants. With high CA activity over a long period, El had the highest proportional bicarbonate use compared to Ov and Bj, reaching 26.95%. Additionally, a new method is proposed for the screening of plants grown for energy in karst habitats.

Effects of novel auto-inducible medium on growth, activity and CO2 capture capacity of Escherichia coli expressing carbonic anhydrase

A glucose-based auto-inducible medium (glucose-AIM) has been developed to enhance both growth and expression of lac operon-linked carbonic anhydrase (CA) expression in a recombinant strain of Escherichia coli. When the E. coli expressing CA was grown on various media, the glucose-based auto-inducible medium (glucose AIM) resulted in a CA activity of 1022mUOD600nm−1mL−1 at 24h and a specific growth rate of 0.082h−1. The CA activity was four to fourteen times higher than those by LB-IPTG. The E. coli expressing CA grown on the glucose-AIM showed highest activity at pH8.5 while it kept high stability up to 40°C and an inlet CO2 concentration of 6%. These findings indicate that the glucose-AIM would be a cost-effective medium to support high cell growth, CA activity and stability for effective CO2 capture.

Characterization of carbonic anhydrase XIII in the erythrocytes of the Burmese python, Python molurus bivittatus.

Carbonic anhydrase (CA) is one of the most abundant proteins found in vertebrate erythrocytes with the majority of species expressing a low activity CA I and high activity CA II. However, several phylogenetic gaps remain in our understanding of the expansion of cytoplasmic CA in vertebrate erythrocytes. In particular, very little is known about isoforms from reptiles. The current study sought to characterize the erythrocyte isoforms from two squamate species, Python molurus and Nerodia rhombifer, which was combined with information from recent genome projects to address this important phylogenetic gap. Obtained sequences grouped closely with CA XIII in phylogenetic analyses. CA II mRNA transcripts were also found in erythrocytes, but found at less than half the levels of CA XIII. Structural analysis suggested similar biochemical activity as the respective mammalian isoforms, with CA XIII being a low activity isoform. Biochemical characterization verified that the majority of CA activity in the erythrocytes was due to a high activity CA II-like isoform; however, titration with copper supported the presence of two CA pools. The CA II-like pool accounted for 90 % of the total activity. To assess potential disparate roles of these isoforms a feeding stress was used to up-regulate CO2 excretion pathways. Significant up-regulation of CA II and the anion exchanger was observed; CA XIII was strongly down-regulated. While these results do not provide insight into the role of CA XIII in the erythrocytes, they do suggest that the presence of two isoforms is not simply a case of physiological redundancy.

Extramitochondrial domain rich in carbonic anhydrase activity improves myocardial energetics

CO2 is produced abundantly by cardiac mitochondria. Thus an efficient means for its venting is required to support metabolism. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2 clearance by catalyzing CO2 hydration (to H(+) and HCO3(-)), thereby changing the gradient for CO2 venting. Using fluorescent dyes to measure changes in pH arising from the intracellular hydration of extracellularly supplied CO2, overall CA activity in the cytoplasm of isolated ventricular myocytes was found to be modest (2.7-fold above spontaneous kinetics). Experiments on ventricular mitochondria demonstrated negligible intramitochondrial CA activity. CA activity was also investigated in intact hearts by (13)C magnetic resonance spectroscopy from the rate of H(13)CO3(-) production from (13)CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-(13)C]pyruvate. CA activity measured upon [1-(13)C]pyruvate infusion was fourfold higher than the cytoplasm-averaged value. A fluorescent CA ligand colocalized with a mitochondrial marker, indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CA isoform CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extramitochondrial CA activity acidified the matrix (as determined by fluorescence measurements in permeabilized myocytes and isolated mitochondria), impaired cardiac energetics (indexed by the phosphocreatine-to-ATP ratio measured by (31)P magnetic resonance spectroscopy of perfused hearts), and reduced contractility (as measured from the pressure developed in perfused hearts). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2 venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity therefore may reduce the heart's energetic efficiency.

Photosynthetic response of three climber plant species to osmotic stress induced by polyethylene glycol (PEG) 6000

We examined the photosynthesis response to osmotic stress in three climber plant species, Pharbitis nil (Linn.) Choisy, Lonicera japonica Thunb, and Parthenocissus tricuspidata (Sieb.et Zucc.) Planch. All climber plants were exposed to osmotic stress induced by polyethylene glycol (PEG) 6000 at 4 levels (slight, moderate, severe osmotic and the control) for 30 days. Photosynthesis response was determined by measuring leaf photosynthesis, chlorophyll fluorescence, carbonic anhydrase activity and stable carbon isotope ratios. P. nil maintained high photosynthetic activity under long-term moderate osmotic stress due to both stable photosystem II photochemical efficiency and high carbonic anhydrase activity. L. japonica maintained high photosynthetic activity under long-term moderate stress due to high carbonic anhydrase activity rather than photosystem II photochemical efficiency. P. tricuspidata tolerated only short-term moderate osmotic stress and long-term slight osmotic stress because its response was mainly stomatal limitation, with the lowest photosynthetic activity and hardly any carbonic anhydrase activity. Carbonic anhydrase activity was inversely correlated with stable carbon isotope ratios. The regulation by carbonic anhydrase was probably the reason for P. nil and L. japonica to tolerate long-term moderate osmotic stress. The selection on the species should consider the differential adaptation mechanism to osmotic stress during the development of drought-resistant plants.

Localization and diurnal variations of carbonic anhydrase mRNA expression in the inner ear of the rainbow trout Oncorhynchus mykiss

Physiological studies have suggested that carbonic anhydrase (CA) plays a central role in otolith biomineralization via ion transport. However, the presence and exact function of CA in the inner ear have not been determined. In the present study, to investigate the localization of CA and its involvement in otolith calcification, we cloned two cDNAs encoding CAs from the rainbow trout sacculus. These two cDNAs, designated rainbow trout CAa (rtCAa) and rtCAb, both had an open reading frame encoding 260 amino acids with a sequence identity of 78%. Remarkably, rtCAb has a high degree of homology (82%) with “high activity CA” in the zebrafish, and its mRNA expression showed variation in the range 1.9–11.4 × 10 4 copies/ng total RNA in the sacculus. In contrast, rtCAa mRNA was constantly expressed at approximately 3 × 10 4 copies/ng total RNA. In situ hybridization revealed that rtCAb mRNA was strongly expressed in the distal squamous epithelial cells and transitional epithelial cells, except the mitochondria-rich cells, whereas, rtCAa was localized in extrasaccular tissue. These results suggest that the rtCAb isozyme is involved in the daily increment formation and calcification of otoliths via phase and spatial differences of the bicarbonate supply to the endolymph.

Non-catalytic role of carbonic anhydrase in rat intestinal absorption

Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO 2-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na + and Cl −, and total HCO 3 − generation (estimated as the sum of radiolabeled HCO 3 − and CO 2 produced from glucose) were measured in Ussing chambers in nominally CO 2, HCO 3 −-free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na + and Cl − absorption. In the ileum, methazolamide reduced J ms Na and J ms Cl and caused net Cl − secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na + and Cl − absorption at pH 7.6. Total HCO 3 − generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl − absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function.

Absence or reduction of carbonic anhydrase II in the red cells of the beluga whale and llama: implications for adaptation to hypoxia.

Carbonic anhydrase (CA) expression was examined in the red cells of two mammals that have adapted to low oxygen stress: the llama, which has adapted to high altitudes, and the beluga (or white) whale, which routinely dives for extended periods. Immunodiffusion analyses of their Hb-free hemolysates and partial amino acid sequencing of their HPLC-separated nonheme proteins indicate that the low-activity CA I isozyme is the major nonheme protein in erythrocytes of both the beluga whale and the llama. The high-activity CA II isozyme was not detected in the whale red cells but was present at low levels in erythrocytes of the llama. These results suggest that the absence or decrease in the expression of the high-activity CA II isozyme may be advantageous under hypoxic conditions.

Unexpected expression of carbonic anhydrase I and selenium-binding protein as the only major non-heme proteins in erythrocytes of the subterranean mole rat ( Spalax ehrenbergi)

Chromatographic separation of the non-heme proteins from the erythrocytes of the subterranean mole rat belonging to the superspecies Spalax ehrenbergi from Israel revealed two major peaks. On sequence analyses, the larger peak corresponded to a 56 kDa selenium-binding protein (SeBP) previously characterized from mouse and human liver, and the second peak to the low-activity carbonic anhydrase (CA) isozyme, CA I. There was no evidence of the high-activity CA II isozyme normally found in the red cells of all amniotes tested to date. Thus, the mole rat appears to be the first mammalian species to express both a SeBP and the low-activity CA I isozyme, as the major non-heme proteins in its red blood cells. It is possible that the absence of the high-activity CA II isozyme may be advantageous to the mole rat in adapting to the low O 2 and high CO 2 environment of its underground burrows. It is also likely that the 56 kDa SeBP may play an important adaptive role in the physiology of the red cell.

Isolation and characterization of a carbonic anhydrase homologue from the zebrafish (Danio rerio).

We have isolated a 29,000-Da carbonic anhydrase (CA) protein from the zebrafish, Danio rerio, sequenced two peptide fragments, and tentatively identified it as a high-activity CA by inhibition kinetics. We have also characterized a 1,537-bp message whose deduced sequence of 260 amino acids matches that of the isolated protein. This CA is clearly an alpha-CA based on the similarity of its sequence to that of other members of the alpha-CA gene family. A phylogenetic analysis suggested CAH-Z diverged after the branching of the CA-V and CA-VII genes and prior to the duplications that generated the CA-I, CA-II, and CA-III genes of amniotes. This marks the first characterization of the mRNA and its protein product from the CA gene of a teleost.

Carbonic anhydrase in deep-sea chemoautotrophic symbioses

Carbonic anhydrase (CA) facilitates the rapid interconversion of carbon dioxide and bicarbonate. It is ubiquitous among organisms, and participates in numerous cellular processes. In several photoautotrophic marine organisms, CA facilitates uptake of inorganic carbon by catalyzing the reversible dehydration of bicarbonate to carbon dioxide, which diffuses more rapidly across cell membranes. In algal/invertebrate symbioses, this mechanism is correlated with significantly elevated CA activity in tissues where zooxanthellae are housed. Here, we determine whether elevated CA activity is also characteristic of tissues involved in inorganic carbon uptake by chemosynthetic invertebrates. Measurements of CA activity in several chemosynthetic clam and vestimentiferan species indicate that CA facilitates inorganic carbon uptake, with high activities present in clam gill tissue, and vestimentiferan plume and trophosome tissues. However levels of CA activity among species varied widely, from very low in mytilids to extraordinarily high in some vesicomyids and vestimentiferans. High CA activity is generally correlated with other adaptations for control of internal chemical environments where symbionts are housed, such as the presence of serum-borne sulfide-binding moieties. These findings suggest that a CA-based mechanism of carbon uptake may be one of a suite of adaptations to provide symbionts with essential metabolites.

Photobiont-related differences in carbon acquisition among green-algal lichens

The photosynthetic properties of a range of lichens (eight species) containing green algal primary photobionts of either the genus Coccomyxa, Dictyochloropsis or Trebouxia were examined with the aim of obtaining a better understanding for the different CO2 acquisition strategies of lichenized green algae. Fast transients of light/dark-dependent CO2 uptake and release were measured in order to screen for the presence or absence of a photosynthetic CO2-concentrating mechanism (CCM) within the photobiont. It was found that lichens with Trebouxia photobionts (four species) were able to accumulate a small pool of inorganic carbon (DIC; 70–140 nmol per mg chlorophyll (Chl)), in the light, which theoretically may result in, at least, a two to threefold increase in the stromal CO2 concentration, as compared to that in equilibrium with ambient air. The other lichens (four species), which were tripartite associations between a fungus, a cyanobacterium (Nostoc) and a green alga (Coccomyxa or Dictyochloropsis) accumulated a much smaller pool of DIC (10–30 nmol·(mg Chl)−1). This pool is most probably associated with the previously documented CCM of Nostoc, inferred from the finding that free-living cells of Coccomyxa did not show any signs of DIC accumulation. In addition, the kinetics of fast CO2 exchange for free-living Nostoc were similar to those of intact tripartite lichens, especially in their responses to the CCM and the carbonic anhydrase (CA) inhibitor ethoxyzolamide. Trebouxia lichens had a higher photosynthetic capacity at low and limiting external CO2 concentrations, with an initial slope of the CO2-response curve of 2.6–3.9 μmol·(mg Chl)−1·h−1·Pa−1, compared to the tripartite lichens which had an initial slope of 0.5–1.1 μmol-(mg Chl)−1·h−1·-Pa−1, suggesting that the presence of a CCM in the photobiont affects the photosynthetic performance of the whole lichen. Regardless of these indications for the presence or absence of a CCM, ethoxyzolamide inhibited the steady-state rate of photosynthesis at low CO2 in all lichens, indicating a role of CA in the photosynthetic process within all of the photobionts. Measurements of CA activity in photobiont-enriched homogenates of the lichens showed that Coccomyxa had by far the highest activity, while the other photobionts displayed only traces or no activity at all. As the CCM is apparently absent in Coccomyxa, it is speculated that this alga compensates for this absence with high internal CA activity, which may function to reduce the CO2-diffusion resistance through the cell.

Extra‐ and intra‐cellular carbonic anhydrase in relation to culture age in a high‐calcifying strain of Emiliania huxleyi Lohmann

SUMMARYThe relationships between extra‐ and intra‐cellular carbonic anhydrase, calcification rate, utilization of dissolved inorganic carbon (DIG) and culture age were investigated in a high‐calcifying strain of Emiliania huxleyi. The detection of carbonic anhydrasc was dependent on culture age; neither the extra‐cellular activity of intact cells nor activity in crude homogenates was detected until the stationary phase. By the stationary phase DIC in the medium was totally depleted and the calcification rate had decreased by 60°0. Extra‐ and intra‐cellular carbonic anhydrase was stimulated by Na+but not Cl ions. The isolation of intact organelles by isopycnic gradient centrifugal ion from exponential and stationary phase cultures of high calcifying cells showed high carbonic anhydrase activity in the chloroplast fraction but carbonic anhydrase was not detected in low‐calcifying cells even after gradient centrifugation. Although 50/IM ethoxyzolamide was an effective inhibitor of carbonic anhydrase activity in vitro, photosynthetic 14CO2 fixation was only inhibited 30%, calcification rate 10%, and the internal inorganic carbon pool unaffected in intact cells. It is proposed that chloroplast carbonic anhydrase maintains the steady state flux of COj, from the chloroplast envelope to Rubisco, much of the CO2 arising by release from HCO3−in the cytosol, required to maintain cytoplasmic pH near neutrality in high‐calcifying cells.

Role of carbonic anhydrase in the supply of inorganic carbon to the giant clam?zooxanthellate symbiosis

The mechanism whereby inorganic carbon (Ci) is acquired by the symbiotic association between the giant clam (Tridacna derasa) and zooxanthellae (Symbiodinium sp.) has been investigated. Ci in the haemolymph of the clam is in equilibrium with the surrounding sea water. The photosynthesis rate exhibited by the intact clam varies as a function of the Ci concentration in the clam haemolymph. The gill tissue contains high carbonic anhydrase activity which may be important in adjusting the Ci equilibrium between haemolymph and sea water. Zooxanthellae (Symbiodinium sp.) isolated from the clam mantle prefer CO2 to HCO 3 - as a source of inorganic carbon. The zooxanthellae have low levels of carbonic anhydrase on the external surface of the cell; however, mantle extracts display high carbonic anhydrase activity. Carbonic anhydrase is absent from the mantle of aposymbiotic clams (T. gigas), indicating that this enzyme may be essential to the symbiosis. The enzyme is probably associated with the zooxanthellae tubes in the mantle. The results indicate that carbonic anhydrase plays an important role in the supply of carbon dioxide within the clam symbiosis.

Production of monoclonal antibodies against gastric parietal cell antigens.

Two mice DBA/1 were each immunized with a single injection of one million enriched parietal cells in the hind foot pads. Monoclonal antibodies to be used as research tools in studies on regulatory mechanisms in gastric parietal cells were obtained after fusion of mouse myeloma cells (SP2) with cells from the popliteal lymph nodes of the mice. Twelve hybridomas produced antibodies reactive with structures only present in parietal cells as assessed by immunohistochemistry of oxyntic mucosa sections. Three hybridomas were subcloned and the antibodies produced by them, designated as PC4, PC8, and PC117, were characterized. In an enzyme-linked immunosorbent assay, all antibodies reacted with H,K-ATPase-containing vesicles. The antibody PC8 recognized a 94 kDa protein after immunoblotting of H,K-ATPase-containing vesicles and all antibodies precipitated a 94 kDa protein from [125I]H,K-ATPase-containing vesicles. The antibodies PC4 and PC117 recognized extracellular structures with a polarized distribution in viable, purified parietal cells. The results suggest that the structure recognized by all three antibodies is the alpha-subunit of the H,K-ATPase. The antibodies produced by another hybridoma, PC43, recognized a structure present in parietal and surface epithelial cells of the oxyntic mucosa. In an enzyme-linked immunosorbent assay, they reacted with a high-activity carbonic anhydrase which had been affinity-purified from pig oxyntic mucosa and they recognized a 30 kDa protein after immunoblotting. Thus, monoclonal antibodies against both intracellular and extracellular parietal cell structures were obtained after immunization with a small number of parietal cells.

A high activity carbonic anhydrase isoenzyme (CA II) is present in mammalian spermatozoa

Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO2 produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.

C, N and P nutrition of Lemanea mamillosa Kütz. (Batrachospermales, Rhodophyta) in the Dighty Burn, Angus, U.K.

Abstract. Field measurements of the growth rate of the red freshwater macroalga Lemanca mamillosa Kutz, in the Dighty Burn, together with measurements of water velocity, [CO2], [NO3], [NH3+ NH4+] and [phosphate], have been made between February and July. This period covers the growth of the erect gametophyte and later of the carposporophyte inside the gametophyte. Hydrodynamic studies in the laboratory on benzoic acid models of the gametophyte suggest an average in situ unstirred layer some 12 μm thick. For growth of the gametophyte, this estimated boundary layer thickness, together with the measured inorganic C transport pathway within the plant, suggest that growth is not significantly restricted by CO2 transport from the bulk phase to the plastids. δ13C measurements on source CO2 and on plant organic C bear this out. Habitat choice (low temperatures: CO2 enrichment from ground‐water input: rapid water flow), plant morphology and anatomy (turbulence‐generating ‘knobbles’ on the nodes; plastids close to the outside of the plant), and plant biochemistry (high CO2 affinity of the RUBISCO carboxylase; quite high carbonic anhydrase activity) are responsible for this lack of limitation by inorganic C transport in the growing gametophyte which lacks HCO3 transport and a CO2 concentrating mechanism. Transport through the boundary layer does not significantly restrict acquisition by the plant of N (probably as NH4+, despite the preponderance of NO3 in the environment) or of P (as orthophosphate) in the field. The membrane transporters, which have high substrate affinities (K½'s about 2 mmol NH4+ m‐3 and < 2 mmol inorganic phosphate m−3), probably impose the major limitation. The development of the carposporophyte later in the season, and an increase in the thickness of the cortex of the gametophyte, result in an increased (less negative) δ13C, suggesting a significant diffusion limitation to CO2 transport. This conclusion is reinforced by consideration of the opposing effect on Δδ13 C of the decreased demand for products of phosphoenolpyruvate carboxylase activity as the N/C ratio decreases late in the growing season.