Carbonic Anhydrase of Chlamydomonas: Purification and Studies on its Induction Using Antiserum against Chlamydomonas Carbonic Anhydrase

Carbonic anhydrase (EC 4.2.1.1 [EC] ; CA) was purified by affinitychromatography from cells of the unicellular green alga Chlamydomonasreinhardtii which had been grown photoautotrophically in ordinaryair. Antiserum raised in rabbit against this purified CA crossreactedwith Chlamydomonas CA but not with spinach leaf CA nor bovineerythrocyte CA. When the CO₂ concentration provided to the algalcells was decreased from 4% to the ordinary air level (0.04%),CA activity and the content of CA protein determined by theimmunodiffusion test showed parallel increases. In contrast,when the CO₂ concentration was raised from air level to 4% CO₂CA activity and its content expressed on the basis of culturevolume remained rather constant. These results indicate thatsynthesis of the CA protein is induced when the CO₂ concentrationis lowered from 4 to 0.04% during algal growth. On the otherhand, the synthesis of CA stops when CO₂ concentration is raisedfrom air level to 4%. (Received June 30, 1984; Accepted October 8, 1984)     

Immunologically Distinct Forms of Adenylate Kinase in Leaves: Comparison of Subunit Size of Adenylate Kinase from C3 and C4 Plants

Kleczkowski, L. A. and Randall, D. D. 1987. Immunologicallydistinct forms of adenylate kinase in leaves: comparison ofsubunit size of adenylate kinase from C₃ and C₄ plants.—J.exp. Bot. 38: 1440–1445. Antibodies prepared against maize leaf adenylate kinase (E.C.2.7.4.3 [EC] ) cross-reacted with the enzyme isolated from leavesof both C₃ and C₄ plants. The immunoreaction was very specificas judged by the presence of a single band on Western immunoblotscontaining proteins from leaf extracts of several species. Themolecular weight (M₁) of adenylate kinase determined by meansof the immunoblotting was 29 kD and 27 kD for C₄ and C₃ species,respectively. For both C₃ and C₄ plants, the antibodies failedto precipitate all adenylate kinase activity in leaf extracts,while they were 100% effective in pelleting the enzyme frommaize mesophyll chloroplasts. This indicated the presence ofat least two immunologically distinct forms of adenylate kinasein leaves. It is suggested that the observed differences in molecular structure(M₁s) of adenylate kinase from C₃ and C₄ species might be responsiblefor distinct catalytic and functional properties of the enzymein these two groups of plants. The irrununologically-determinedoccurrence of distinct pools of adenylate kinase in leaves supportsprevious evidence obtained by means of subcellular fractionationstudies.     

NADP-malic enzyme: immunolocalization in different tissues34 plant maize and the C3 plant wheat

In situimmunolocalization and Western blot analysis of separatedcellular and subcellular fractions, were used to determine thelocalization of different isoforms of NADP-malic enzyme in bothwheat (C₃) and maize (C₄) plants. In both techniques, an affinitypurified anti-(maize 62 kDa NADP-ME) lgG from the maize greenleaf isoform also reacted with a 72 kDa protein in tissues ofC4 plants as well as C3 plants. The light- inducible 62 kDaisofomi is located in bundle sheath chioroplasts of maize leaves.In etiolated leaves and in roots of maize there is evidencefor the occurrence of a 72 kDa isoform which co-migrates on2-D (SDS and isoelectric focusing) PAGE. The 72 kDa isoformis also present in low levels in green leaves. This form mayoccur in multiple intracellular compartments; but in situ immunolocalizationexperiments and Western blot and activity assays on fractionatedprotoplasts indicate that a significant amount of this isoformoccurs in plastids. With regards to C³ plants such as wheat,a 72 kDa isoform in leaves is largely confined to the chloroplastsbased on in situ immunolocalization and Western blots and enzymeactivity assays with fractionated protoplasts. In maize, itappears that the constitutive expression pattern of a possibleC₃ ancestral gene for NADP-malic enzyme has been maintained,and a high level expression of a light-inducible isoform locatedin bundle sheath chloroplasts (62 kDa) has been acquired duringits evolution. Key words: NADP-malic enzyme, Triticum aestivum, Zea mays     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Carbonic Anhydrase in Chlamydomonas reinhardtii II. Requirements for Carbonic Anhydrase Induction

Carbonic anhydrase (CA) activity in wild type cells of Chlamydomonasreinhardtii was low when cells were cultured under 2% CO₃ inthe light. When the gas phase was changed to air, CA activityincresaed as much as 20 fold over the next 24 hours. In contrast,CA activity did not change markedly in cells of the mutantspet 20-8 (PS II-negative), lip 10-2 (photophosphorylation-negative),and F60 (phosphoribulokinase-negative), when they were subjectedto the same induction regimen. DCMU (10^–5 M) and cydoheximide(3 µg/ml) severely inhibited the induction in wild typecells. No induction occured when CO₂ concentration was loweredin darkness. ³Present adress: Photoconversion Research Branch, Solar EnergyResearch Institute, Golden, Colorado 80401, USA. (Received June 7, 1982; Accepted December 25, 1982)     

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.     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. II. Distribution and Role in Photosynthesis

Antibody was raised against Porphyridium carbonic anhydrase(CA) which was electrophoretically recovered from the gel afterSDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) of thepartially purified enzyme. The antiserum reacted with CA ofPorphyridium, but not with that of Chlamydomonas reinhardtii.Even though the antiserum did not react with CA from P. cruentumR-l in Ouchterlony's double immunodiffusion, it blocked theenzyme activity in the presence of 1% Nonidet P-40 and 1% TritonX-100. After Western blotting and enzyme-linked immunostaining(ELIS), only one band which reacted with the antiserum was detectedin the extract of low-CO₂ cells (grown under ordinary air) ofP cruentum, while no significant band was detected in that ofhigh-CO₂ cells (grown under air enriched with 1–5% CO₂).Immunogold electron microscopy of low-CO₂ cells of P. cruentumR-l using this antibody revealed that most of the CA was localizedin the chloroplast, with some in the cytoplasm. No specificbinding of gold particles was observed in the high-CO₂ cells. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan (Received May 18, 1987; Accepted September 7, 1987)     

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Localization of superoxide dismutase in leaves of C3 and C4 plants

Chloroplasts, mitochondria and cytoplasm, isolated from pea,wheat, maize and sorghum mesophyll protoplasts, contain distinctforms of superoxide dismutase (SOD). In all species evaluated,chloroplasts exhibited a single cyanide-sensitive SOD. Thischloroplastic enzyme was the most anionic SOD observed in wholeleaf and protoplast extracts and constitutes 50–80% ofthe total soluble SOD. Pea and wheat protoplasts had only onecytoplasmic SOD, a cyanide-sensitive form of intermediate mobility;maize and sorghum had two such cytoplasmic enzymes. A singlecyanide-insensitive SOD was present in extracts from both C₃and C₄ tissues and was associated with mitochondria. Although bundle sheath cells of sorghum and maize are knownto be deficient in Photosystem II, there was no apparent differencein SOD between mesophyll and bundle sheath cells. Mesophyllprotoplasts and bundle sheath strands from these C₄ plants containedthe same forms of SOD. Levels of soluble SOD were similar, ona chlorophyll basis, in the two cell types as was distributionof activity among the various forms of the enzyme. (Received May 19, 1980; )     

Phosphorus responses of C3 and C4 species

An hypothesis was formulated that phosphorus (P) partitioningin tissues of C₄ leaves would permit C₄ plants to resist P deficiencybetter than C₃ plants. To test this hypothesis, 12 C₃, C₄, andC₃–C₄ intermediate species were grown at adequate, deficient,and severely deficient P supply in a solid-phase-buffered sandculture system to characterize photosynthetic and growth responses.Species differed considerably in response to P stress. The growthof C₃ species was more sensitive to P supply than C₄ species,but C₃ and C₄ species had similar photosynthetic P use efficiency,and C₄ species did not have low leaf P content, contrary toour hypothesis. In fact, leaf photosynthetic rates were notcorrelated with growth responses. Moncots had lower leaf P contentand better maintenance of leaf production under P stress thandicots, because of greater inhibition of branching (dicots)than of tillering (monocots). The most P efficient species inthis survey was Brachiaria, a C₄ monocot that increased rootbiomass allocation under stress while maintaining P allocationto the shoot. It is concluded that C₄ species are not inherentlymore P efficient than C₃ species, but that monocots are moreP efficient than dicots, because of contrasting P and biomassallocation under stress. Key words: Phosphorus deficiency, C₃ plants, C₄ plants, growth response     

Role of Carbonic Anhydrase and Identification of the Active Species of Inorganic Carbon Utilized for Photosynthesis in Chora corallina

Carbonic anhydrase (CA, EC. 4.2.1.1 [EC] ) activity in air-grown Characorallina was detected mainly in the intracellular fraction,most of which composed of chloroplasts and cytoplasmic gel,and not on the cell surface. Only minor levels of CA activity,on the basis of equivalent volumes, were detected in the cellsap and the cytoplasmic sol. The maximum rate of photosynthetic O₂ evolution by air-grownChara corallina at pH 6.0 was twice that at pH 7.6, while theapparent K_m for external inorganic carbon (C_i) at pH 7.6 wasabout three times that at pH 6.0. However, the apparent K_m(CO₂)was about three times larger at pH 6.0 than at pH 7.6. The K_m(C_i)-valueat pH 7.6 increased severalfold in the presence of acetazolamide(AZA), an inhibitor of CA, but no inhibition was observed atpH 6.0. The pH-dependence may be due to differences in the permeabilityof AZA at the given pH values. Fixation of ¹⁴CO₂ at 20 µMand of H¹⁴CO₃^– at 200 µM over the course of 5 swas very similar at pH 7.4. Addition of CA significantly suppressedthe photosynthetic ¹⁴CO₂-fixation but it stimulated the H¹⁴CO₃^–-fixation.This result indicates that free CO₂ is an active species ofC_i that is incorporated into the cell during photosynthesis. These results together suggest the following: (1) Free CO₂ isutilized for photosynthesis, (2) CA is mainly located insidethe cell and functions to increase the affinity for CO₂ in photosynthesisby facilitating the supply of CO₂ from the plasmalemma to thesite of CO₂-fixation. ³Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Chiba, 260 Japan. (Received December 9, 1988; Accepted March 22, 1989)     

Carbonic Anhydrase of the Alkaliphilic Cyanobacterium Microcoleus chthonoplastes

The activity of carbonic anhydrase (CA) was studied in different cell fractions of the alkaliphilic cyanobacterium Microcoleus chthonoplastes. The activity of this enzyme was found in the soluble and membrane protein fractions, as well as in intact cells and in a thick glycocalyx layer enclosing the cyanobacterium cells. The localization of CA in glycocalyx of M. chthonoplastes was shown by western blot analysis and by immunoelectron microscopy studies with antibodies to the thylakoid CA from Chlamydomonas reinhardtii (Cah3). At least one of the CA forms occurring in M. chthonoplastes CA was shown to be an -type enzyme. A possible mechanism of the involvement of the glycocalyx CA in calcification of cyanobacteria is discussed.     

Quantum Yields of Photosystem II and Photosynthesis in an Aurea Mutant of Tobacco (C3) and an Oil Yellow Mutant of Maize (C4) Which Have High Capacities for Photosynthesis Despite Low Chlorophyll Contents

The quantum yields of O₂ evolution (_o2), CO₂ fixation (_co2)and of photosystem II measured by fluorescence analysis (_PSII),were determined in a mutant of tobacco (C₃) and a mutant ofmaize (C₄) having large deficiencies in light harvesting chlorophyll,but having similar capacities for photosynthesis on a leaf areabasis as the wild-type plants. The heterozygous dominant mutantof tobacco (aurea, Su/+) having 30% chlorophyll content of thewild-type, and of maize (oil yellow, Oy/+) having 60% chlorophyllcontent of the wild-type, maintain high leaf absorptances. Themaximum _o2 on an absorptance basis, measured under limitingred light and saturating CO₂ were the same in the mutant versuswild-type plants. _PSII and _co2 decreased in a similar mannerwith increasing light intensity, while the ratio of _PSII/_co2under varying light remained reasonably constant, in mutantversus wild-type seedlings. The results indicate that theseC₃ and C₄ mutants which have high capacities for photosynthesisdespite large deficiencies in chlorophyll content, maintaina high leaf absorptance, and a balanced utilization of absorbedquanta in photochemistry. ¹Present address: Botany Dept., University of California, Davis,CA, 95616, U.S.A.     

Factors Controlling Induction of Carbonic Anhydrase and Efficiency of Photosynthesis in Chlorella vulgaris llh Cells

When Chlorella vulgaris llh cells which had been grown in airenriched with 2–4% CO₂ (high-CO₂ cells) were bubbled withair containing ca. 400 ppm CO₂, illumination at an intensityas low as the light compensation point (350 lux) was sufficientto increase the photosynthetic rate under limiting CO₂ concentrations.The same treatment induced carbonic anhydrase (CA) activity.The induction of CA activity and increase in photosyntheticrate at limiting CO₂ concentrations were observed in the presenceof 10 µM DCMU which completely inhibits photosynthesis.These results indicate that photosynthetic electron transportis not involved in CA induction in Chlorella vulgaris llh cells.The parallelism between the changes in CA activity and the rateof photosynthesis under limiting CO₂ concentrations agree withthe previous conclusion that the transport of CO₂ from outsideto the site of CO₂ fixation is facilitated by CA and hence lowersthe apparent K_m(CO₂) for photosynthesis. (Received December 24, 1982; Accepted May 10, 1983)     

Survey of pyruvate,phosphate dikinase activity of plants in relation to the C3, C4 and CAM mechanisms of CO2 assimilation

The pyruvate, phosphate dikinase activity (PPD, EC 2.7.9.1) associated with crude extracts of leaf tissue of some C₃ and C₄ plants was determined by phosphoenolpyruvate plus PPi-dependent phosphorylation of AMP. The PPD activity of all C₄ plants examined was > 15 nmol/mg protein/min. Several factors contributed to the underestimation of PPD activity in crude extracts of at least some species. Significant PPD activity (> 0.15 nmol/mg protein/min) was not detected in the majority of C₃ species but several C₃ species and the two CAM species studied exhibited activity in the range 0.4–4 nmol/mg protein/min while the C₃ species Avena sativa showed activity up to 8 nmol/mg protein/min. The oat leaf enzyme was partially purified; it exhibited properties similar to those of partially purified PPD from maize. Leaf extracts of the orchids Cymbidium canaliculatum and C. madidum contained high levels of PPD activity similar to the majority of C₄ plants. PPD activity has also been shown in other previously unstudied species.     

Distribution of enzymes related to C3 and C4 pathway of photosynthesis between mesophyll and bundle sheath cells of Panicum hians and Panicum milioides

Enzymes of the C₄, C₃ pathway and photorespiration have beenanalyzed for P. hians and P. milioides, which have chlorenchymatousbundle sheath cells in the leaves. On whole leaf extracts thelevels of PEP carboxylase are relatively low compared to C₄species, RuDP carboxylase is typical of C₃ species, and enzymesof photorespiratory metabolism appear somewhat intermediatebetween C₃ and C₄. Substantial levels of PEP carboxylase, RuDPcarboxylase, and photorespiratory enzymes were found in bothmesophyll and bundle sheath cells. Low levels of C₄-acid decarboxylatingenzymes may limit the capacity for C₄ photosynthesis in P. hiansand P. milioides. The results on enzyme activity and distributionbetween mesophyll and bundle sheath cells are consistent withCO₂ fixation via C₃ pathway in these two species. ¹ This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin, Madison; and bythe University of Wisconsin Research Committee with funds fromthe Wisconsin Alumni Research Foundation; and by the NationalScience Foundation Grant BMS 74-09611. (Received September 16, 1975; )     

Photosynthetic Capacity and Nitrogen Use Efficiency of Maize, Wheat, and Rice: A Comparison Between C3 and C4 Photosynthesis

The C₃ species wheat and rice and the C₄ species maize weregrown for 2–3 weeks in controlled environment growth chambersat 20 or 30 °C day and 15 °C night temperatures. CO₂assimilation rates (at 20 and 30 °C) and several leaf parametersincluding total nitrogen, soluble protein, and RuBP carboxylaseprotein were determined. When the assimilation rates under atmosphericCO₂ and O₂ levels were expressed on a total nitrogen basis (=nitrogen use efficiency), the C₄ species maize had a greaternitrogen use efficiency than either of the two C₃ species examined,regardless of the combination of temperatures used for growthor measurement of CO₂ assimilation. Maize is also shown to makemore efficient use of its soluble protein and RuBP carboxylaseprotein than either wheat or rice when measurements are madeat 320 parts 10^–6 CO₂ and 21% O₂. Atmospheric CO₂ enrichmentduring CO₂ assimilation measurements increased the nitrogenuse efficiency in the C₃ species. In one treatment (wheat grownand measured at 20 °C), CO₂ assimilation under saturatingCO₂ showed nitrogen, soluble protein, and RuBP carboxylase proteinuse efficiencies equal to or greater than that of the C₄ species. These data indicate that C4 species may make more efficientuse of their nitrogen, soluble protein, and RuBP carboxylaseprotein than C₃ species under atmospheric CO₂ conditions. Thismay be due in part to the C₄ cycle and CO₂-concentrating mechanismin C₄ photosynthesis.     

The Role of Carbonic Anhydrase in Blood Ion and Acid-Base Regulation

The role of carbonic anhydrase (CA) in ion transport processesof aquatic and terrestrial arthropod species is reviewed. Inboth insects and crustaceans CA is found in a variety of iontransporting tissues. The bulk of CA activity in crustaceansis concentrated in the posterior gills, which are morphologicallyand biochemically adapted for ion transport. The enzyme canbe specifically localized to gill lamellae which contain largepopulations of salt transporting chloride cells. Enzyme activityin the posterior gills of species having the ability to regulateblood ion concentrations increases when these organisms areacclimated to environmental salinities in which they ion regulate.In stenohaline, ion conforming species branchial CA activityis uniformly low, being only 5–10% that in regulatingspecies. Studies on the blue crab, Callinectes sapidus, usingthe specific CA inhibitor acetazolamide have shown that theenzyme is indeed important in blood ion regulation. Blood Na^$and Cl^– concentrations are both severely lowered in drug-treatedanimals acclimated to low salinity, while they remain virtuallyunaffected in animals acclimated to high salinity, in whichthe animal is an ion conformer. High salinity acclimated crabstreated with acetazolamide do not survive transfer to low salinity,and mortality is related to a breakdown in the ion regulatorymechanism. Branchial CA most likely functions in the hydrationof respiratory CO₂ to H^$ and HCO₃^–, which serve as counterionsfor the active uptake of Na^$ and Cl^–, respectively. Interrestrial species the role of CA is unclear and merits furtherinvestigation.