Iowa State UniversityFrom the SelectedWorks of Martin Spalding

1983

Carbonic Anhydrase-DeficientMutant of Chlamydomonasreinhardii Requires Elevated CarbonDioxide Concentration forPhotoautotrophic GrowthMartin H. Spalding, University of Illinois at Urbana-ChampaignRobert J Spreitzer, University of Illinois at Urbana-ChampaignWilliam L Ogren, University of Illinois at Urbana-Champaign

Available at: https://works.bepress.com/martin-spalding/

9/

Plant Physiol. (1983) 73, 268-2720032-0889/83/73/0268/05/$00.50/0

Carbonic Anhydrase-Deficient Mutant of Chlamydomonasreinhardii Requires Elevated Carbon Dioxide Concentration forPhotoautotrophic Growth'

Received for publication February 14, 1983 and in revised form April 11, 1983

MARTIN H. SPALDING2, ROBERT J. SPREITZER3, AND WILLIAM L. OGRENDepartment ofAgronomy, University ofIllinois, and the United States Department ofAgriculture,Agricultural Research Service (W L. 0.), Urbana, Illinois 61801

ABSTRACT

A mendelian mutant of the unicellular green alp Chlamydomonasreinhardii has been isolated which is deficient in carbonic anhydrase (EC4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locusca-i), was selected on the basis of a high C02 requirement for photoau-totrophic growth. Photosynthesis by the mutant at atmospheric C02concentration was very much reduced compared to wild type and, unlikewild type, was strongly inhibited by 02. In contrast to a CO2 compensationconcentration of near zero in wild type at all 02 concentrations examined,the mutant exhibited a high, 02-stimulated C02 compensation concentra-tion. Evidence of photorespiratory activity in the mutant but not in wildtype was obtained from the analysis of photosynthetic products in thepresence of '4CO2. At air levels of C02 and 02, the mutant synthesizedlarge amounts of glycolate, while little glycolate was synthesized by wildtype under identical conditions. Both mutant and wild type strains formedonly small amounts of glycolate at saturating CO2 concentration. Atambient C02, wild type accumulated inorgaic carbon to a concentrationseveral-fold higher than that in the suspension medium. The mutant cellsaccumulated inorganic carbon internally to a concentration 6-fold greaterthan found in wild type, yet photosynthesis was C02 limited. The mutantphenotype was mimicked by wild type cells treated with ethoxyzolamide,an inhibitor of carbonic anhydrase activity. These observations indicatea requirement for carbonic anhydrase-catalyzed dehydration of bicarbon-ate in maintaining high internal CO2 concentrations and high photosyn-thesis rates. Thus, in wild type cells, carbonic anhydrase rapidly convertsthe bicarbonate taken up to C02, creating a high internal C02 concentra-tion which stimulates photosynthesis and suppresses photorespiration.In mutant cells, bicarbonate is taken up rapidly but, because ofa carbonicanhydrase deficiency, is not dehydrated at a rate sufficiently rapid tomaintain a high internal CO2 concentration.

Chlamydomonas reinhardii cells grown photoautotrophicallyat air levels ofCO2 (0.03-0.04%) exhibit a much higher apparentaffinity for inorganic carbon in photosynthesis than do cellsgrown in air enriched to 1 to 5% CO2 or higher plants which fixCO2 by the C3 pathway (4). A low apparent Km(CO2) was also

' Supported in part by a Rockefeller Foundation postdoctoral fellow-ship to R. J. S.

2Present address: MSU/DOE Plant Research Laboratory, MichiganState University, East Lansing, MI 48824.

3 Present address: Departement de Biologie Moleculaire, Universite deGeneve, CH-1211 Geneve 4, Switzerland.

observed in other unicellular green algae (7, 1 1). The apparentKm(CO2) of air-adapted C. reinhardii was much less than that ofthe C02-assimilating enzyme, RuBP4 carboxylase (EC 4.1.1.39),isolated from the same cells (4). In addition to an increasedapparent affinity for C02, air-adapted algae also showed nosignificant 02 inhibition of photosynthesis (16). Plants whichphotosynthesize by the C4 photosynthetic pathway have certaingas exchange characteristics similar to those of the air-adaptedalgae due to the ability of C4 plants to concentrate C02 in thebundle sheath cells, where RuBP carboxylase is located (9). AC02-concentrating system operating by a totally different mech-anism than that of C4 photosynthesis appears to be responsiblefor the efficient photosynthetic characteristics of air-adaptedChlamydomonas (2) and other algae (3, 5, 12, 18, 23). Althoughnot well characterized, this C02-concentrating mechanism mayinvolve active bicarbonate transport into the algal cells, raisingthe internal inorganic carbon concentration several-fold higherthan that of the surrounding medium (2, 3, 5, 13, 18, 23).

Operation of a C02-concentrating mechanism in unicellulargreen algae would require a number of interacting components.In addition to a bicarbonate transporting protein, the operationalsystem may require an ATPase to supply energy for activetransport of bicarbonate, substantial activity of carbonic anhy-drase for dehydration of the transported bicarbonate, and resist-ance to back-diffusion of the concentrated internal CO2. Toidentify the components of the system and better understand themechanism involved in the C02-concentrating system of C.reinhardii, we have taken a genetic approach to the problem.This approach involves isolation and characterization ofmutantsof C. reinhardii which require elevated CO2 concentrations fornormal photoautotrophic growth and, therefore, may includethose which are defective in some component of the C02-concentrating mechanism. In this paper, we describe the char-acterization of the first of several high-CO2 requiring mutants,one that is deficient in carbonic anhydrase activity. The analysisof this mutant indicates that carbonic anhydrase is essential forefficient operation of the C02-concentrating system in C. rein-hardii and illustrates the utility of the approach described.

MATERIALS AND METHODS

Agal Strains and Culture Conditions. Chlamydomonas rein-hardii wild type strain 2137 mt+ and a collection of light-sensitive, acetate-requiring mutants described previously weremaintained on acetate medium in the dark (21). For analysis,

'Abbreviations: RuBP, Ribulose 1,5-bisphosphate; mt, mating type;PD, parental ditype; NPD, nonparental ditype; T, tetratype; EZA,ethoxyzolamide.

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CHLAMYDOMONAS CARBONIC ANHYDRASE MUTANT

cells were cultured photoautotrophically in the minimal mediumdescribed by Spreitzer and Mets (21) except that the Tris bufferwas replaced by 50 mm MOPS (pH 7.0) and the micronutrientswere as according to Allen (1). The carbonic anhydrase-deficientstrain was grown initially in air enriched to 5% CO2 and trans-ferred to air 2 d prior to analysis. Cells grown in light wereexposed to a quantum flux density of approximately 150 ME m 2s-1 from powergroove fluorescent lamps.Where zinc-free liquid medium was required, it was prepared

by applying the diphenylthiocarbazone (dithizone) chelationmethod of Donald et al. (6) to all stock nutrient solutions exceptthe micronutrients. The medium was then prepared from thesestocks and a micronutrient stock without ZnSO4. The zincchelator dipicolinic acid (17) was added to the growth medium(1 mM) to further reduce zinc availability.Mutant Isolation and Genetic Analysis. Photosynthesis-defi-

cient mutants were originally isolated as light-sensitive, acetate-requiring strains by Spreitzer and Mets (21) following 5-fluoro-deoxyuridine treatment and ethyl methanesulfonate mutagene-sis. Using spot tests (21), these mutants were screened for abilityto grow photoautotrophically in air enriched to 5% CO2.

Genetic analysis was performed with the centromere-markerstrain pf-2 (paralyzed flagella) mr. Gamete induction, zygotematuration, zygote germination, and tetrad analysis were per-formed as described previously (21). Tetrads were scored as PD,NPD, or T following replica-plating at 80 ME m 2 s-' on minimalmedium, minimal medium and 5% C02, and acetate medium,and on acetate medium in the dark. This information was usedto calculate gene-centromere distance (21).

Photosynthetic 02 Exchange. Cells were harvested by centrif-ugation and resuspended in 50 mm MOPS-KOH (pH 7.0) to aChl concentration of 100 to 200 gg/ml. Prior to use, the cellswere kept on a rotary shaker under atmospheric and light con-ditions equivalent to those of growth. Assays of photosynthetic02 evolution were performed at 25°C in a Hansatech 02 elec-trode. The illuminated (700 MAE m 2 s-') wild type cells wereallowed to deplete the medium of inorganic carbon (marked bycessation of02 evolution) prior to addition of known concentra-tions ofNaHCO3. Inasmuch as the mutant cells exhibited a highCO2 compensation concentration, they were centrifuged in aBeckman Microfuge B and resuspended in C02-free buffer. Thecarbonic anhydrase inhibitor EZA (12), when used, was addedafter depletion of endogenous inorganic carbon. The 02 concen-tration was maintained between 18 and 25% 02 (227-315 uM02).

Inorganic Carbon Accumulation. Uptake and accumulation ofinorganic carbon and simultaneous carbon fixation were deter-mined using silicone oil filtering centrifugation (2, 10).

'4C-Product Labeling. Cells in 2.5 ml total volume were ini-tially treated as for 02 exchange including depletion of endoge-nous inorganic carbon. After NaH'4CO3 was added to a finalconcentration of either 100 Mm or 2.5 mm, samples (750 Ml) weretaken at 1, 2.5, and 5 min and mixed immediately with an equalvolume of 8 M HCOOH in ethanol. The insoluble fraction wasrecovered by centrifugation, and the supernatant (soluble frac-tion) was dried under vacuum. The insoluble fraction was quan-titated by liquid scintillation spectroscopy. The residue from thesoluble fraction was redissolved in a minimum of water andpassed through a Dowex 50 cation exchange column to removeamino acids. After eluting anions and neutral compounds withwater, the amino acids were eluted with 2 M NH.OH, dried undervacuum, redissolved in water, and separated by cellulose TLC ina solvent system adapted from paper chromatography (19). La-beled amino acids were detected by autoradiography and quan-titated by liquid scintillation spectroscopy of cut-out spots. Thewater eluant from the Dowex 50 column was used directly forseparation of organic acids and phosphorylated compounds by

HPLC. A nonlinear phosphate gradient was used to elute theanions from a Whatman Partisil PXS 10/25 SAX column similarto the method of Giersch (8). The neutral compounds eluted inthe void volume. Radioactivity was detected by a flow-through,heterogeneous scintillation counter.CO2 Compensation Concentration Measurement and Gas Ex-

change Analysis. CO2 compensation concentrations were esti-mated in a closed system in which gas of the desired 02 concen-tration was bubbled (350 ml/min) through water and an illumi-nated (500 ME m 2 s-') algal suspension at 25°C and pH 6.5. TheCO2 concentration was allowed to come to equilibrium and wasmonitored with a UNOR-2 IR gas analyzer (Bendix Corpora-tion).

Steady state photosynthetic rate was measured in the samesystem except that it was open, with gas mixtures of knowncomposition bubbled at 350 ml/min through the illuminatedalgal suspension. The CO2 concentration of the gas stream wasmonitored before and after passing through the algal suspension.Carbonic Anhydrase and Chl. Extract preparation and assay

of carbonic anhydrase (EC 4.2.1.1) activity were performed aspreviously described (20). Chl was determined after extractioninto 96% (v/v) ethanol (22).

RESULTSMutant Recovery. The ca-1-12-lC mutant described in this

paper was recovered from a collection of acetate-requiring mu-tants based on its ability to grow photoautrophically at 5% CO2.This mendelian mutant was previously designated 12- IC andwas found to have wild type fluorescence induction kinetics, PS-II activity, and Chl content (21). In addition, the level of RuBPcarboxylase activity was found to be normal (data not shown).As the first high C02-requiring C. reinhardii mutant determinedto be deficient in carbonic anhydrase activity, we have assignedthe mutation to the ca- 1 locus.The mutant strain is phenotypically indistinguishable from

wild type when grown photoautotrophically at 5% CO2 or onacetate medium in the dark. It is a leaky acetate-requiring strain,growing slightly on minimal medium. The mutant strain isphotosensitive but survives on acetate medium at 80 ME m 2 s-'(21).

Genetic Analysis. When ca-1-12-lC mt+ was crossed with pf-2 mt-, mendelian 2:2 segregation of C02-requiring and wildtype phenotypes was observed (Table I). No segregation of ace-tate-requiring, C02-requiring, and photosensitive phenotypeswas found. Furthermore, reduced carbonic anhydrase activityand high compensation point were always associated with theC02-requiring phenotype (Table I). Tetrad analysis with regardto pf-2 (PD = 8, NPD = 4, T = 15) indicated that ca-l was 28map units from its centromere.

Photosynthetic Characteristics. The response of photosyn-thesis to bicarbonate concentration at 21% 02 is illustrated inFigure 1 for wild type, zinc-deficient wild type, ca-l mutant, andwild type in the presence of 50 Mm EZA, an inhibitor concentra-tion determined to be optimal (data not shown). The responseof photosynthesis to inorganic carbon concentration in the mu-tant was similar to that of wild type treated with EZA and inboth cases the affinity for inorganic carbon was reduced relativeto the wild type control. Zinc-deficient cells had an intermediateaffinity for inorganic carbon. The photosynthetic rates at satu-ration CO2 (5 mM NaHCO3) were similar for the four treatments,indicating similar capacities for maximum photosynthesis (TableII).The CO2 compensation concentration of wild type C. reinhar-

dii grown at air levels of CO2 was near zero and was unaffectedby 02 concentration. The ca-l mutant, in contrast, exhibited ahigh, 02-sensitive CO2 compensation concentration (Table II).In the presence of 50 Mm EZA, wild type also exhibited a high,

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Plant Physiol. Vol. 73, 1983

Table I. Tetrad Analysis ofa Cross between C. reinhardii MutantStrain ca-1-12-lC mt+ and Wild Type Strain pf-2 mt-

CO2Tetrad- Carbonic CompensationProgeny Phenoty Anydrase Concn.

at 50% 02units/mg Chl /lll CO2

5-1 wt 687 <105-2 C02R 78 2815-3 wt 803 <105-4 CO2R 99 260

6-1 wt 723 <106-2 CO2R 137 3106-3 C02R 160 3216-4 wt 574 <10

9-1 wt 1084 <109-2 wt 1135 <109-3 C02R 151 2919-4 CO2R 322 301

10-1 wt 961 <1010-2 C02R 257 27710-3 wt 687 <1010-4 CO2R 131 293

a wt, wild type; CO2R, required elevated (5%) CO2 concentration forphotoautotrophic growth.

m 200 400 60pM NaHCO3

FIG. 1. Response of C02-dependent photosynthetic 02 evolution toNaHCO3 concentration (pH 7.0) in C. reinhardii wild type (4 *4),zinc-deficient wild type (l-O), wild type plus 50 gM EZA(U-U), and ca-I mutant (A A). The EZA was added to wild-

type cells just prior to assay. Chl concentrations were: wild type, 35 ug/ml; mutant, 41 5Ag/ml; zinc-deficient wild type, 45 ug/ml.

02-sensitive compensation concentration (Table II).Carbonic anhydrase activity was found to be low in both wild

type and the mutant when grown with CO2 enrichment (TableII). However, after transfer to air for 2 d, the carbonic anhydraseactivity of the mutant showed little change whereas that of wildtype cells increased approximately 8-fold (Table II). These datasuggest that either the mutant is deficient in a distinct, inducibleisozyme of carbonic anhydrase or this enzyme is present in atleast two separate compartments.Carbonic anhydrase is a zinc-requiring enzyme, and growing

C. reinhardii on a zinc-deficient medium decreased carbonic

Table II. Photosynthetic Characteristics ofC. reinhardii Wild Typeand ca-I Mutant Strains

Assay (Growth) Wild ca- I WildConditions Type Mutant + EZA

CO2 compensation concn. (,ul/l C02)2%02 <10 15 3021%02 <10 111 4550% 02 <10 193 87

Photosynthetic ratea (350 u1/1 C02)2%02 202 62 6421% 02 202 23 3850% 02 185 3 25

Photosynthetic rateb (5 mM NaHCO3) 221 179 204Carbonic anhydrase activitycGrown at 5% CO2 89 98Two d at 350 l/1 C02 616 103a Rate measured as zmol CO2 taken up h-' (mg Chl)-' using an IR

gas analysis system.b Rate measured as umoI 02 evolved (mg Chl)-' h-' in an 02 electrode

at pH 7.0 and an 02 concentration between 227 and 315 AM.c Expressed as units (mg Chl)-' as previously defined (20).

anhydrase activity by about 60%. Even lower activity was ob-tained by the addition of dipicolinic acid, a Zn2+ chelator. Thecarbonic anhydrase activity in this treatment was 122 units (mgChl)-'. The decrease in carbonic anhydrase activity was not dueto an inhibitory effect of Zn2+ chelation on carbonic anhydrasein the in vitro assay, as addition of excess ZnSO4 (2 mM) to thecell suspension prior to sonication did not alter the observedlevel of carbonic anhydrase activity.The addition of a large excess of carbonic anhydrase to the

suspending medium of the mutant cells had no effect on photo-synthetic rate measured either as O2 evolution or CO2 uptake(data not shown). In addition, exposure of the mutant to 50 ,MEZA resulted in less than 10% inhibition of the photosyntheticrate (data not shown). The absence ofEZA inhibition of photo-synthesis indicates that the residual carbonic anhydrase activityof the mutant cells is not serving the same function as theinducible level observed in the wild type cells. It also supportsthe observation reported previously ( 12) that EZA has a negligibledirect effect on photosynthesis per se, a conclusion further sub-stantiated by the lack of inhibition by EZA of photosynthesis at5 mM NaHCO3 in wild type (Table II).

Inorganic Carbon Accumulation. Time courses for the accu-mulation of inorganic carbon, the depletion of inorganic carbonfrom the suspending medium, and CO2 fixation are shown inFigure 2 for wild type (with and without addition of 50 ,uM, EZA)ca-I mutant, zinc-deficient wild type, and wild type in the dark.As has been previously demonstrated (2), wild type C. reinhardiiaccumulated inorganic carbon to a level several-fold higher thanthat in the surrounding medium (Fig. 2A). This level eventuallydropped and CO2 fixation ceased (Fig. 2C) due to depletion ofinorganic carbon in the medium (Fig. 2B). Distribution of inor-ganic carbon by passive penetration was demonstrated by accu-mulation in wild type cells in the dark (Fig. 2, A and B). Inorganiccarbon accumulation in the mutant was approximately 5-foldhigher than that of wild type (Fig. 2A). This accumulation wassimilar to that observed in wild type when carbonic anhydrasewas inhibited. The level ofinorganic carbon accumulation in thezinc-deficient cells was approximately double that of wild type.

Products of Photosynthesis. In wild type C. reinhardii, thebulk of '4C02 incorporated during a 5-min labeling period at 100M NaHCO3 was found in the insoluble fction (prumably

starch) and in Calvin cycle intermediates (Table III). Very little

270 SPALDING ET AL.

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CHLAMYDOMONAS CARBONIC ANHYDRASE MUTANT

FIG. 2. Time course of: A, internal inorganic carbon accumulation;B, external inorganic carbon depletion; and C, CO2 fixation in C.reinhardii wild type (e ), wild type dark control (O- O),zinc-deficient wild type (O-O), wild type plus 50 Mm EZA(U -U, and ca-l mutant strain (A A). The initial externalinorganic carbon concentration was 80 gM, and the pH was 7.0. Chlconcentrations were: wild type, 32 gg/ml; mutant, 28 gg/ml; zinc-deficient wild type, 44 Mg/ml. TIC, total inorganic carbon.

label was found in metabolites indicative of photorespiratoryactivity, such as glycolate, glycine, and serine. However, in theca-l mutant and in wild type in the presence of 50 UM EZA,glycolate was the major labeled product, with a concomitantdecrease in the proportion oflabel found in the insoluble fraction(Table III). In both cases, approximately 90% of the labeledglycolate was excreted into the medium (data not shown). Atsaturating inorganic carbon concentration (2.5 mm NaHCO3),wild type and the mutant exhibited similar labeling patterns withlittle label in photorespiratory metabolites (Table III).

DISCUSSION

C. reinhardii mutants defective in photosynthesis have beenshown to have reduced carbonic anhydrase activity (14, 20).

Table III. Products ofPhotosynthetic '4C02 Fixation by C. reinhardiiWild Type and ca-I Mutant Strains

Cells photosynthesized for 5 min in NaH'4CO3 at pH 7.0, 25°C, andat an 02 concentration of between 227 and 315 MM 02. Cell density wasadjusted so that approximately 50% of the CO2 was fixed in 5 min whenthe initial NaHCO3 concentration was 100 MM.

100 Mm NaHCO3 2.5 mM NaHCO3

Fraction Wild ca-l Wild Wild ca-ltype mutant + EZA type mutant

% oflabel incorporatedInsoluble 48.6 17.8 8.7 21.3 27.7Neutral 2.2 0.8 1.2 4.9 4.9Acids

Glycolate 1.1 36.4 45.9 0.2 2.2Sugar mono-P 11.0 7.1 4.5 20.3 15.0Malate 3.4 5.1 2.4 8.3 11.5Glycerate-3-P 2.4 4.7 4.5 11.0 9.9RuBP 6.2 8.8 2.5 3.7 4.2

Amino acidsAspartate 3.3 1.2 7.5 5.5 3.7Glutamate 5.8 1.0 5.1 7.0 4.6Glycine 1.9 8.6 0.8 1.7 3.3Alanine 2.1 0.9 2.7 1.3 1.7Serine 2.5 1.6 3.3 4.5 3.5

Inorganic carbon accumulation in photosynthesis-deficientstrains F-60 and NLS 6-2A was found to be indistinguishablefrom the passive distribution found in wild type cells examinedin the dark (20), so these mutants also lack the ability to activelyaccumulate inorganic carbon. This suggests a coordinated regu-lation of carbonic anhydrase activity and bicarbonate transport.Inasmuch as the ca-l mutant possessed an active bicarbonatetransport capability and accumulated a very high level ofinternalinorganic carbon (Fig. 2A), it is distinctly different from themutants defective in photosynthesis. The reduced carbonic an-hydrase activity in the ca-l mutant is therefore not simply dueto the reduction of photosynthesis.The deficiency of carbonic anhydrase activity obtained in wild

type cells by growth at low zinc or by exposing the cells to EZAresulted in increased levels ofaccumulated inorganic carbon, butreduced photosynthetic rates. Because the maximum photosyn-thetic rate (at saturating external CO2 concentrations) was unaf-fected by these conditions, the simplest explanation for thereduced photosynthetic rate is that the carbonic anhydrase defi-ciency caused a CO2 limitation. Thus, with low carbonic anhy-drase activity, bicarbonate was transported into the cytoplasmfaster than it was dehydrated to CO2 so bicarbonate accumulatedto higher than normal levels. In spite of the very high internalbicarbonate concentration, the rate of spontaneous dehydrationof bicarbonate was not sufficiently rapid to prevent the CO2concentration from becoming rate limiting.An 02-stimulated CO2 compensation concentration was asso-

ciated with carbonic anhydrase deficiency caused by the ca-Imutation and by treatment of wild type cells with EZA (TablesI and II). The magnitude ofthe CO2 compensation concentrationin C3 plants is determined by the relative rates of glycolateoxidation to CO2 and photosynthetic CO2 fixation (15). In con-trast to higher plants, glycolate was not metabolized to CO2 butlargely excreted by the mutant and EZA-treated wild type cellsin short-term labeling experiments (Table III). If glycolate wasalso excreted during the longer term CO2 compensation meas-urements, then the biochemical basis of the CO2 compensationconcentration in these cells may be different from that in leavesof C3 plants.

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272 SPALDING ET AL.

Transport of bicarbonate in the mutant was normal, but, as inzinc-deficient and EZA-inhibited wild type cells, the transportedbicarbonate accumulated to an abnormally high level apparentlybecause of a slow rate of dehydration to C02. Even though theinternal inorganic carbon concentration at 80 uM external inor-ganic carbon was high in the mutant (Fig. 2A), the rate ofphotosynthesis was much reduced compared to wild type (Fig.2C). This observation suggests that the mutant cells are CO2limited (see also Fig. 1). Stronger support for this conclusioncomes from the distribution of 14C in the products of photosyn-thesis (Table III). Accumulation of label in glycolate in mutantbut not wild type cells is a strong indication that photosynthesisin mutant cells was C02 limited at 100 Mm NaHCO3, sinceglycolate arises from RuBP oxygenase activity, a reaction whichis competitively inhibited by C02 (15). Glycolate synthesis inmutant cells was reduced to that found in wild type by increasingthe concentration ofNaHCO3 to a level saturating photosynthesis(Table III). Inhibition ofphotosynthesis by 02 in the ca- 1 mutantat 350 ,ul/l CO2 (Table II) also indicated a limiting internal CO2concentration, since 02 inhibition of C3 photosynthesis is deter-mined by the relative C02 and 02 concentrations present at thesite of RuBP carboxylase/oxygenase (15). The photosyntheticcharacteristics of EZA-inhibited wild type cells were similar tothose of the mutant cells.We conclude that the high C02-requiring phenotype and the

associated physiological characteristics of this mutant result froma deficiency of carbonic anhydrase activity, and that the majorrole of carbonic anhydrase in Chlamydomonas photosynthesis isdehydration of transported bicarbonate. At low carbonic anhy-drase activity, caused by either mutation or chemical inhibition,the dehydration of bicarbonate becomes limiting so that thesupply of C02 for photosynthesis remains suboptimal even

though bicarbonate accumulates to very high levels internally.The simultaneous occurrence and genetic cosegregation of thehigh CO2 requirement for normal growth, deficiency in carbonicanhydrase activity, low affinity for C02, high rate of glycolatesynthesis in air, and an 02-stimulated C02 compensation con-centration indicate that all these characteristics arise from a singlemutation. The isolation and analysis of the ca-l mutant dem-onstrate that the genetic approach will be valuable in identifyingadditional requisite components involved in the operation oftheC02-concentrating mechanism of C. reinhardii.

Acknowledgments-We thank C. Critchley and A. R. Portis for helpful discus-sions, and M. E. Hageman and Phuc Tran for expert technical assistance.

Plant Physiol. Vol. 73, 1983

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