Carbonic Anhydrase Activity and Localization in Some Plant Species

The aim of this work concerned the study of the differences in the carbonic anhydrase activity and localization between plant species, the photosynthesis of which is carried out according to the C₃ and C₄ pathways respectively. The measurement of enzymatic activity was made with a titrimetric evaluation of the rate of the reaction CO₂+ H₂O ? H⁺+ HCO^?₃. The C₃ plant species showed higher activities than the C₄ species. The localization of carbonic anhydrase was carried out with a histochemical method. The carbonic anhydrase appeared in the chloroplasts both in the mesophyll and the bundle sheath without any difference between C₃ and C₄ plants.     

Physiological and biochemical aspects and molecular mechanisms of plant adaptation to the elevated concentration of atmospheric CO<Subscript>2</Subscript>

The review of publications concerning the impact of increasing CO₂ concentration in the Earths atmosphere (C_a) on higher terrestrial plants. The physiological changes in plants induced by increasing C_a, including growth and biochemical composition, the characteristics of photosynthesis and respiration, as well as the molecular mechanisms of the regulation of the activity of most important biosynthetic enzymes at early and late stages of the exposure to elevated C_a are under consideration. Various concepts of metabolic regulation during acclimation to increasing CO₂ concentration are critically reviewed. The pathways of possible involvement of carbonic anhydrase-mediated systems of CO₂ transport and concentration during C₃ photosynthesis of higher plants, the metabolic and signal mechanisms of photosynthesis inhibition by carbohydrates and the role of ethylene at elevated C_a are presented. The effect of elevated C_a on plant development and source-sink relations, as well as its interaction with other environmental factors, such as mineral, primarily nitrogen nutrition, light, temperature, and water regime, are discussed in with the context of potential forecasting of the consequences of increase in C_a and temperature for the activities of various higher plant forms in the rapidly changing climate.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 129–145.Original Russian Text Copyright © 2005 by Romanova.     

A comparative immunocytochemical localization study of phosphoenolpyruvate carboxylase in leaves of higher plants

The intracellular localization of phosphoenolpyruvate (PEP) carboxylase in plants belonging to the C₄, Crassulacean acid metabolism (CAM) and C₃ types was invetigated using an immunocytochemical method with an immune serum raised against the sorghum leaf enzyme. The plants studied were sorghum, maize (C₄ type), kalanchoe (CAM type), french bean, and spinach (C₃ type). In the green leaves of C₄ plants, it was shown that the carboxylase was located in the mesophyll and stomatic cells, being largely cytosolic in the mesophyll cells. Similarly, in CAM plants, the enzyme was found mainly outside the chloroplasts. In contrast, in C₃ plants, the PEP carboxylase appeared to be distributed between the cytosol and the chloroplasts of foliar parenchyma. Examination of sections from etiolated leaves showed fluorescence emission from etioplasts and cytosol for the parenchyma of french bean as well as for the bundle sheath and mesophyll of sorghum leaves. This data indicated that during the greening process photoregulation and evolution of PEP carboxylase is dependent on the tissue and on the metabolic type of the plant considered.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate     

Patterns of phosphoenolpyruvate carboxylase activity and cytosolic pH during light activation and dark deactivation in C3 and C4 plants

The rate and extent of light activation of PEPC may be used as another criterion to distinguish C₃ and C₄ plants. Light stimulated phosphoenolypyruvate carboxylase (PEPC) in leaf discs of C₄ plants, the activity being three times greater than that in the dark but stimulation of PEPC was limited about 30% over the dark-control in C₃ species. The light activation of PEPC in leaves of C₃ plants was complete within 10 min, while maximum activation in C₄ plants required illumination for more than 20 min, indicating that the relative pace of PEPC activation was slower in C₄ plants than in C₃ plants. Similarly, the dark-deactivation of the enzyme was also slower in leaves of C₄ than in C₃ species. The extent of PEPC stimulation in the alkaline pH range indicated that the dark-adapted form of the C₄ enzyme is very sensitive to changes in pH. The pH of cytosol-enriched cell sap extracted from illuminated leaves of C₄ plants was more alkaline than that of dark-adapted leaves. The extent of such light-dependent alkalization of cell sap was three times higher in C₄ leaves than in C₃ plants. The course of light-induced alkalization and dark-acidification of cytosol-enriched cell sap was markedly similar to the pattern of light activation and dark-deactivation of PEPC in Alternanthera pungens, a C₄ plant. Our report provides preliminary evidence that the photoactivation of PEPC in C₄ plants may be mediated at least partially by the modulation of cytosolic pH.Abbreviations CAM Crassulacean acid metabolism - G-6-P glucose-6-phosphate - PMSF phenylmethylsulfonyl fluoride - PEPC phosphoenolpyruvate carboxylase - PEPC-PK phosphoenolpyruvate ca carboxylase-protein kinase     

Carbonic anhydrases in plants and algae

Carbonic anhydrases catalyse the reversible hydration of CO₂, increasing the interconversion between CO₂ and HCO₃⁻ + H⁺ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C₃ dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.     

Le vie fotosintetiche C3, C4 e CAM nel contesto ecologico

Abstract

Ecological aspects of C₃, C₄ and CAM photosynthetic pathways. - Three different photosynthetic CO₂ fixation pathways are known to occur in higher plants. However all three pathways ultimately depend on the Calvin-Benson cycle for carbon reduction. The oxygenase activity of RuBP carboxilase is responsible for photorespiratory CO₂ release. Both C₄ and CAM pathways behave as a CO₂ concentrating mechanism which prevent photorespiration. The CO₂-concentrating mechanism in C₄ plants is based on intracellular symplastic transport of C₄ dicarboxylic acids from mesophyll-cells to the adjacent bundle-sheath cells. On the contrary in CAM plants the CO₂-concentrating mechanism is based on the intracellular transport of malic acid into and out of the vacuole.

The C₄ photosynthetic pathway as compared to the C₃ pathway permits higher rates of CO₂ fixation in high light and high temperature environments at low costs in terms of water loss, given the stability of the photosynthetic apparatus under such conditions.

CAM is interpreted as an adaptation to arid environments because it enables carbon assimilation to take place at very low water costs during the night when the evaporative demand is low. Nevertheless many aquatic species of Isoetes and some relatives are CAM, suggesting the adaptive role of CAM to environments which become depleted in CO₂.

The photosynthetic carbon fixation pathway certainly contributes to the ecological success of plants in different environments. However the distribution of plants may also reflect their biological history. On the other hand plants with different photosynthetic pathways coexist in many communities and tend to share resources in time. In any case some generalizations are possible: C₄ plants enjoy an ecological advantage in hot, moist, high light regions while the majority of species in desert environments are C₃; CAM plants are more frequent in semiarid regions with seasonal rainfall, coastal fog deserts, and in epiphytic habitats in tropical rain forests.     

Arabidopsis thaliana carbonic anhydrase: cDNA sequence and effect of CO2 on mRNA levels

A full-length cDNA clone encoding carbonic anhydrase was isolated from an Arabidopsis thaliana (Columbia) leaf library. Comparison of the derived amino acid sequence obtained from this clone with those of pea and spinach reveals a considerable degree of identity. The carbonic anhydrase cDNA was used to probe the level of RNA encoding this protein in the leaves of plants grown in elevated CO₂ (660 ppm). We have found that under these conditions the steady-state level of carbonic anhydrase mRNA was increased in comparison with control plants grown in normal atmospheric concentrations of CO₂ (330 ppm). This raises the intruiging possibility that there exists in higher plants a mechanism for perceiving and responding to changes in environmental CO₂ concentrations at the genetic level.     

Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes

The photosynthetic characteristics of four transgenic rice lines over-expressing rice NADP-malic enzyme (ME), and maize phosphoenolpyruvate carboxylase (PC), pyruvate,orthophosphate dikinase (PK), and PC+PK (CK) were investigated using outdoor-grown plants. Relative to untransformed wild-type (WT) rice, PC transgenic rice exhibited high PC activity (25-fold increase) and enhanced activity of carbonic anhydrase (more than two-fold increase), while the activity of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) and its kinetic property were not significantly altered. The PC transgenic plants also showed a higher light intensity for saturation of photosynthesis, higher photosynthetic CO₂ uptake rate and carboxylation efficiency, and slightly reduced CO₂ compensation point. In addition, chlorophyll a fluorescence analysis indicates that PC transgenic plants are more tolerant to photo-oxidative stress, due to a higher capacity to quench excess light energy via photochemical and non-photochemical means. Furthermore, PC and CK transgenic rice produced 22–24% more grains than WT plants. Taken together, these results suggest that expression of maize C₄ photosynthesis enzymes in rice, a C₃ plant, can improve its photosynthetic capacity with enhanced tolerance to photo-oxidation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation

Cyanobacteria and some chemoautotrophic bacteria are able to grow in environments with limiting CO₂ concentrations by employing a CO₂-concentrating mechanism (CCM) that allows them to accumulate inorganic carbon in their cytoplasm to concentrations several orders of magnitude higher than that on the outside. The final step of this process takes place in polyhedral protein microcompartments known as carboxysomes, which contain the majority of the CO₂-fixing enzyme, RubisCO. The efficiency of CO₂ fixation by the sequestered RubisCO is enhanced by co-localization with a specialized carbonic anhydrase that catalyzes dehydration of the cytoplasmic bicarbonate and ensures saturation of RubisCO with its substrate, CO₂. There are two genetically distinct carboxysome types that differ in their protein composition and in the carbonic anhydrase(s) they employ. Here we review the existing information concerning the genomics, structure and enzymology of these uniquely adapted carbonic anhydrases, which are of fundamental importance in the global carbon cycle.     

Growth,photosynthesis, and metabolism of sugar beet at an early stage of exposure to elevated CO<Subscript>2</Subscript>

The effects of CO₂ concentration (C _a) on growth, photosynthesis, and the activity of enzymes associated with the translocation and assimilation of CO₂ were studied in sugar beet (Beta vulgaris L. subsp. saccharifera, cv. Ramonskaya) plants. The plants were grown in controlled-climate chamber to the stage of 3–4 leaves and then used in experiments. Experimental plants were exposed in boxes to doubled C _a (700 µl/l, 2C plants), whereas control plants were kept in a chamber with ambient atmosphere (350 µl/l, 1C plants). As compared with 1C plants, in 3 and 8 days, the leaf area of 2C plants increased by 14 and 26%, respectively. The rate of their photosynthesis (P _n) measured in 3, 6, and 8 days increased by 85, 47, and 52%, respectively, whereas in normal air, the values of P _n in 2C plants were by 12, 19, and 15% lower than in 1C plants. After 8-day growth, the content of soluble carbohydrates in the leaves of 2C plants attained 7.2%, being by 80% greater than in 1C plants; the content of starch did not exceed 3%. The total content of chlorophylls a and b in the leaves of 2C plants was by 14% greater than in 1C plants, but their ratio was essentially the same. The level of protein in 2C plants was by 13.4% lower than in 1C plants. The activity and content of Rubisco in 1C and 2C plants were similar. As compared with 1C plants, in 2C plants the activity of soluble carbonic anhydrase (sCA) was lower by 34% in 3 days and by 18% in 8 days; the activity of carbonic anhydrase of membrane preparations (mCA), was lower by 24 and 77%, respectively. Catalase activity in 2C plants became by 8% lower than in 1C plants only after 8 days. A reduction in the photosynthetic ability of 2C plants in ambient atmosphere, a decrease in activity of sCA and, especially, of mCA observed together with invariable activity and content of Rubisco in the leaf extracts are interpreted as early symptoms of acclimation of young plants of sugar beet to elevated CO₂.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 184–190.Original Russian Text Copyright © 2005 by Ignatova, Novichkova, Mudrik, Lyubimov, Ivanov, Romanova.This revised version was published online in April 2005 with a corrected cover date.     

Carbonic anhydrase activity in leaves and its role in the first step of c(4) photosynthesis

In C₄ plants carbonic anhydrase catalyzes the critical first step of C₄ photosynthesis, the hydration of CO₂ to bicarbonate. The maximum activity of this enzyme in C₄ leaf extracts, measured by H⁺ production with saturating CO₂ and extrapolated to 25°C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C₃ leaf extracts. However, the calculated effective activity of this enzyme at in vivo CO₂ concentrations was apparently just sufficient to prevent the rate of conversion of CO₂ to HCO₃⁻ from limiting C₄ photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities.     

Quo vadis C4? An ecophysiological perspective on global change and the future of C4 plants

C₄ plants are directly affected by all major global change parameters, often in a manner that is distinct from that of C₃ plants. Rising CO₂ generally stimulates C₃ photosynthesis more than C₄, but C₄ species still exhibit positive responses, particularly at elevated temperature and arid conditions where they are currently common. Acclimation of photosynthesis to high CO₂ occurs in both C₃ and C₄ plants, most notably in nutrient-limited situations. High CO₂ aggravates nitrogen limitations and in doing so may favor C₄ species, which have greater photosynthetic nitrogen use efficiency. C₄ photosynthesis is favored by high temperature, but global warming will not necessarily favor C₄ over C₃ plants because the timing of warming could be more critical than the warming itself. C₃ species will likely be favored where harsh winter climates are moderated, particularly where hot summers also become drier and less favorable to C₄ plant growth. Eutrophication of soils by nitrogen deposition generally favors C₃ species by offsetting the superior nitrogen use efficiency of C₄ species; this should allow C₃ species to expand at the expense of C₄ plants. Land-use change and biotic invasions are also important global change factors that affect the future of C₄ plants. Human exploitation of forested landscapes favors C₄ species at low latitude by removing woody competitors and opening gaps in which C₄ grasses can establish. Invasive C₄ grasses are causing widespread forest loss in Asia, the Americas and Oceania by accelerating fire cycles and reducing soil nutrient status. Once established, weedy C₄ grasses can prevent woodland establishment, and thus arrest ecological succession. In sum, in the future, certain C₄ plants will prosper at the expense of C₃ species, and should be able to adjust to the changes the future brings. This revised version was published online in August 2006 with corrections to the Cover Date.     

The roles of carbonic anhydrases in photosynthetic CO2 concentrating mechanisms

Cyanobacteria, algae, aquatic angiosperms and higher plants have all developed their own unique versions of photosynthetic CO₂ concentrating mechanisms (CCMs) to aid Rubisco in efficient CO₂ capture. An important aspect of all CCMs is the critical roles that the specialised location and function that various carbonic anhydrase enzymes play in the overall process, participating the interconversion of CO₂ and HCO₃ ⁻ species both inside and outside the cell. This review examines what we currently understand about the nature of the carbonic anhydrase enzymes, their localisation and roles in the various CCMs that have been studied in detail. This revised version was published online in August 2006 with corrections to the Cover Date.     

贵州喀斯特区C4植物物种组成与水分生态型划分

该研究通过查阅文献、核对贵州大学林学院标本库及现场群落调查与标本采集,并运用碳同位素比值法研究典型C_4植物水分利用特性,探索C_4植物在喀斯特植被恢复中的地位,进而揭示贵州喀斯特地区C_4植物资源的基本特征。结果表明:贵州喀斯特区共有C_4植物141种,隶属于74属15科,分别占全国科属种的62.50%、46.25%、24.48%,以禾本科(Gramineae)和莎草科(Cyperaceae)为主;区内C_4植物种均为一年生或多年生草本,多年生植物种略多于一年生植物种;水分生态型整体偏旱生,旱生和中生植物分别占总数的24.82%和31.21%;喀斯特区C_4植物具有高水分利用效率,但不同水分生态型间差异不显著;贵州喀斯特区C_4植物资源具有资源丰富、利用途径广泛、能长期利用、竞争力强、能大面积分布、偏旱生且水分利用幅度广的基本特征,适合喀斯特区生境,自然状态下多为恢复早期物种,有利于喀斯特区生态恢复。在贵州喀斯特恶劣生态环境下C_4植物有较好的生态适应性,并表现出较高的药用、食用、饲用、景观应用等价值,对其开发利用对贵州经济、社会发展及生态恢复有重要意义。     

Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C(i) Cells of Chlamydomonas: Studies Using O Exchange with C/O Labeled Bicarbonate

By measuring ¹⁸O exchange from doubly labeled CO₂ (¹³C¹⁸O¹⁸O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [C_i]) or air enriched with 5% CO₂ (high C_i). Intact low C_i protoplasts had a 10-fold higher carbonic anhydrase activity than did high C_i protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of ¹⁸O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low C_i cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low C_i than in high C_i preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment.     

Relationship between allocation of absorbed light energy in PSII and photosynthetic rates of C3 and C4 plants

Two C₃ dicotyledonous crops and five C₄ monocotyledons treated with three levels of nitrogen were used to evaluate quantitatively the relationship between the allocation of absorbed light energy in PSII and photosynthetic rates (P _N) in a warm condition (25–26°C) at four to five levels [200, 400, 800, 1,200 (both C₃ and C₄) and 2,000 (C₄ only) μmol m⁻² s⁻¹] of photosynthetic photon flux density (PPFD). For plants of the same type (C₃ or C₄), there was a linear positive correlation between the fraction of absorbed light energy that was utilized in PSII photochemistry (P) and P _N, regardless of the broad range of their photosynthetic rates due to species-specific effect and/or nitrogen application; meanwhile, the fraction of absorbed light energy that was dissipated through non-photochemical quenching (D) showed a negative linear regression with P _N for each level of PPFD. The intercept of regression lines between P and P _N of C₃ and C₄ plants decreased, and that between D and P _N increased with increasing PPFD. With P and D as the main components of energy dissipation and complementary to each other, the fraction of excess absorbed light energy (E) was unchanged by P _N under the same level of PPFD. At the same level of P _N, C₄ plants had lower P and higher D than C₃ plants, due to the fact that C₄ plants with little or no photorespiration is considered a limited energy sink for electrons. Nevertheless there was a significant negative linear correlation between D and P when data from both C₃ and C₄ plants at varied PPFD levels was merged. The slope of regression lines between P and D was 0.85, indicating that in plants of both types, most of the unnecessary absorbed energy (ca. 85%) could dissipate through non-photochemical quenching, when P was inhibited by low P _N due to species-specific effect and nitrogen limitation at all levels of illumination used in the experiment.     

Expression of photosynthetic genes from the C4 plant, maize, in tobacco.

Summary Previous results from this laboratory have demonstrated the presence of genes for phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase in C₃ plants. The structure and light-enhanced expression of these genes is very similar to that of the genes found in the C₄ plant, maize. In order to investigate whether or not the regulation of these genes is similar in C₃ and C₄ plants, we have constructed chimeric genes using -glucuronidase as a reporter gene under the control of the maize promoters of the genes for phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose bisphosphate carboxylase (RuBisCO). The chimeric genes were introduced into tobacco, a C₃ plant. These genes were expressed primarily in leaf and stem tissue and the expression was enhanced by light. Thus, as in C₄ plants, the genes are expressed in a tissue-specific and light-inducible manner in the C₃ plant. Since the expression of these genes is restricted to specific cells in leaf tissue of C₄ plants, we also investigated the spatial pattern of expression of the chimeric genes using histochemical analysis of -glucuronidase activity. High level expression of all of these genes was found in mesophyll cells. This included the small subunit of RuBisCO, which is not expressed in mesophyll cells but in bundle sheath cells in C₄ plants. This report describes similarities between C₃ and C₄ plants in regulating the expression of these genes.     

Photosynthesis pathways,ecological characteristics,and the geographical distribution of the Cyperaceae in Japan

Summary The nature of the photosynthetic pathways of Cyperaceae found in Japan were investigated on the basis of Kranz anatomy, the CO₂ compensation concentration and previously reported data. Among 301 species (96% of all cyperaceous species recorded in the region), 58 species were classified as being C₄ plants. These C₄ species were scattered among the tribes Fimbristylideae, Lipocarpheae, Cypereae and Rhynchosporeae in the subfamily Cyperoideae. The genera Cyperus, Eleocharis and Rhynchospora included, in Japan, both C₃ and C₄ species within a single genus. Using these data, an analysis was made of the ecological characteristics and geographical distribution of the C₃ and C₄ species in Japan. Although cyperaceous species grow in markedly different environments, the majority were found in wet and aquatic areas (61%) or shaded areas, such as forest floors (20%). Most of the C₃ species were also hygrophytes (58%) and forest-living species (25%), and C₃ species growing in mesic and dry areas were relatively rare. The C₄ species inhabited wet and aquatic (75%), mesic (13%) and dry areas (6%) and showed marked ecological characteristics with respect to soil-moisture conditions, unlike other C₄ plants, although they were absent from shaded habitats. In order to determine the climatic factors that influence the relative floristic abundance of C₃ and C₄ members of the Cyperaceae in Japan, the ratios of number of C₄ species to the total number of members of Cyperaceae (C₄ percentage) in 16 representative locales were examined in terms of various climatic variables. There were strong positive correlations between the C₄ percentage and temperature. Among the C₃ groups of three subfamilies, there were different distributional trends for various temperature regimes. The C₃ subfamily Caricoideae increased its relative contribution to the cyperaceous flora with a decrease in mean annual temperature, while the C₃ subfamily Sclerioideae exhibited the opposite pattern. The C₃ group of the subfamily Cyperoideae did not show any marked change in pattern along temperature gradients, unlike the two other C₃ subfamilies, and seemed to be heterogeneous in terms of its response to temperature. The relationships between the C₄ biochemical subtypes and ecological characteristics are also discussed.     

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 M, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO₂-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 M CO₂) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO₂-dependent O₂evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO₂-dependent O₂evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C₃-plants are discussed.