Carbonic Anhydrase-Deficient Mutant of Chlamydomonas ... - NCBI

Plant Physiol. (1983) 73, 268-272

0032-0889/83/73/0268/05/$00.50/0

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth' Received for publication February 14, 1983 and in revised form April 11, 1983

MARTIN H. SPALDING2, ROBERT J. SPREITZER3, AND WILLIAM L. OGREN Department ofAgronomy, University of Illinois, and the United States Department ofAgriculture, Agricultural Research Service (W L. 0.), Urbana, Illinois 61801 observed in other unicellular green algae (7, 1 1). The apparent

ABSTRACT

Km(CO2) of air-adapted C. reinhardii was much less than that of

A mendelian mutant of the unicellular green alp Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-i), was selected on the basis of a high C02 requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric C02 concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by 02. In contrast to a CO2 compensation concentration of near zero in wild type at all 02 concentrations examined, the mutant exhibited a high, 02-stimulated C02 compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of '4CO2. At air levels of C02 and 02, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO2 concentration. At ambient C02, wild type accumulated inorgaic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was C02 limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO2 concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to C02, creating a high internal C02 concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO2 concentration.

the C02-assimilating enzyme, RuBP4 carboxylase (EC 4.1.1.39), isolated from the same cells (4). In addition to an increased apparent affinity for C02, air-adapted algae also showed no significant 02 inhibition of photosynthesis (16). Plants which photosynthesize by the C4 photosynthetic pathway have certain gas exchange characteristics similar to those of the air-adapted algae due to the ability of C4 plants to concentrate C02 in the bundle sheath cells, where RuBP carboxylase is located (9). A C02-concentrating system operating by a totally different mechanism than that of C4 photosynthesis appears to be responsible for the efficient photosynthetic characteristics of air-adapted

Chlamydomonas (2) and other algae (3, 5, 12, 18, 23). Although

not well characterized, this C02-concentrating mechanism may involve active bicarbonate transport into the algal cells, raising the internal inorganic carbon concentration several-fold higher than that of the surrounding medium (2, 3, 5, 13, 18, 23). Operation of a C02-concentrating mechanism in unicellular green algae would require a number of interacting components. In addition to a bicarbonate transporting protein, the operational system may require an ATPase to supply energy for active transport of bicarbonate, substantial activity of carbonic anhydrase for dehydration of the transported bicarbonate, and resistance to back-diffusion of the concentrated internal CO2. To identify the components of the system and better understand the mechanism involved in the C02-concentrating system of C. reinhardii, we have taken a genetic approach to the problem. This approach involves isolation and characterization of mutants of C. reinhardii which require elevated CO2 concentrations for normal photoautotrophic growth and, therefore, may include those which are defective in some component of the C02concentrating mechanism. In this paper, we describe the characterization of the first of several high-CO2 requiring mutants, one that is deficient in carbonic anhydrase activity. The analysis of this mutant indicates that carbonic anhydrase is essential for efficient operation of the C02-concentrating system in C. reinhardii and illustrates the utility of the approach described.

Chlamydomonas reinhardii cells grown photoautotrophically

at air levels of CO2 (0.03-0.04%) exhibit a much higher apparent affinity for inorganic carbon in photosynthesis than do cells grown in air enriched to 1 to 5% CO2 or higher plants which fix CO2 by the C3 pathway (4). A low apparent Km(CO2) was also ' Supported in part by a Rockefeller Foundation postdoctoral fellowship to R. J. S. 2Present address: MSU/DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824. 3 Present address: Departement de Biologie Moleculaire, Universite de Geneve, CH-121 1 Geneve 4, Switzerland.

MATERIALS AND METHODS Agal Strains and Culture Conditions. Chlamydomonas reinhardii wild type strain 2137 mt+ and a collection of lightsensitive, acetate-requiring mutants described previously were maintained on acetate medium in the dark (21). For analysis,

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

CHLAMYDOMONAS CARBONIC ANHYDRASE MUTANT

cells were cultured photoautotrophically in the minimal medium described by Spreitzer and Mets (21) except that the Tris buffer was replaced by 50 mm MOPS (pH 7.0) and the micronutrients were as according to Allen (1). The carbonic anhydrase-deficient strain was grown initially in air enriched to 5% CO2 and transferred to air 2 d prior to analysis. Cells grown in light were exposed to a quantum flux density of approximately 150 ME m 2 s-1 from powergroove fluorescent lamps. Where zinc-free liquid medium was required, it was prepared by applying the diphenylthiocarbazone (dithizone) chelation method of Donald et al. (6) to all stock nutrient solutions except the micronutrients. The medium was then prepared from these stocks and a micronutrient stock without ZnSO4. The zinc chelator dipicolinic acid (17) was added to the growth medium (1 mM) to further reduce zinc availability. Mutant Isolation and Genetic Analysis. Photosynthesis-deficient mutants were originally isolated as light-sensitive, acetaterequiring strains by Spreitzer and Mets (21) following 5-fluorodeoxyuridine treatment and ethyl methanesulfonate mutagenesis. Using spot tests (21), these mutants were screened for ability to grow photoautotrophically in air enriched to 5% CO2. Genetic analysis was performed with the centromere-marker strain pf-2 (paralyzed flagella) mr. Gamete induction, zygote maturation, zygote germination, and tetrad analysis were performed as described previously (21). Tetrads were scored as PD, NPD, or T following replica-plating at 80 ME m 2 s-' on minimal medium, minimal medium and 5% C02, and acetate medium, and on acetate medium in the dark. This information was used to calculate gene-centromere distance (21). Photosynthetic 02 Exchange. Cells were harvested by centrifugation and resuspended in 50 mm MOPS-KOH (pH 7.0) to a Chl concentration of 100 to 200 gg/ml. Prior to use, the cells were kept on a rotary shaker under atmospheric and light conditions equivalent to those of growth. Assays of photosynthetic 02 evolution were performed at 25°C in a Hansatech 02 electrode. The illuminated (700 MAE m 2 s-') wild type cells were allowed to deplete the medium of inorganic carbon (marked by cessation of 02 evolution) prior to addition of known concentrations of NaHCO3. Inasmuch as the mutant cells exhibited a high CO2 compensation concentration, they were centrifuged in a Beckman Microfuge B and resuspended in C02-free buffer. The carbonic anhydrase inhibitor EZA (12), when used, was added after depletion of endogenous inorganic carbon. The 02 concentration was maintained between 18 and 25% 02 (227-315 uM 02).

Inorganic Carbon Accumulation. Uptake and accumulation of inorganic carbon and simultaneous carbon fixation were determined using silicone oil filtering centrifugation (2, 10). '4C-Product Labeling. Cells in 2.5 ml total volume were initially treated as for 02 exchange including depletion of endogenous inorganic carbon. After NaH'4CO3 was added to a final concentration of either 100 Mm or 2.5 mm, samples (750 Ml) were taken at 1, 2.5, and 5 min and mixed immediately with an equal volume of 8 M HCOOH in ethanol. The insoluble fraction was recovered by centrifugation, and the supernatant (soluble fraction) was dried under vacuum. The insoluble fraction was quantitated by liquid scintillation spectroscopy. The residue from the soluble fraction was redissolved in a minimum of water and

passed through a Dowex 50 cation exchange column to remove amino acids. After eluting anions and neutral compounds with water, the amino acids were eluted with 2 M NH.OH, dried under vacuum, redissolved in water, and separated by cellulose TLC in

a solvent system adapted from paper chromatography (19). La-

beled amino acids were detected by autoradiography and quantitated by liquid scintillation spectroscopy of cut-out spots. The water eluant from the Dowex 50 column was used directly for separation of organic acids and phosphorylated compounds by

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HPLC. A nonlinear phosphate gradient was used to elute the anions from a Whatman Partisil PXS 10/25 SAX column similar to the method of Giersch (8). The neutral compounds eluted in the void volume. Radioactivity was detected by a flow-through, heterogeneous scintillation counter. CO2 Compensation Concentration Measurement and Gas Exchange Analysis. CO2 compensation concentrations were estimated in a closed system in which gas of the desired 02 concentration was bubbled (350 ml/min) through water and an illuminated (500 ME m 2 s-') algal suspension at 25°C and pH 6.5. The CO2 concentration was allowed to come to equilibrium and was monitored with a UNOR-2 IR gas analyzer (Bendix Corpora-

tion). Steady state photosynthetic rate was measured in the same system except that it was open, with gas mixtures of known composition bubbled at 350 ml/min through the illuminated algal suspension. The CO2 concentration of the gas stream was monitored 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 as previously described (20). Chl was determined after extraction into 96% (v/v) ethanol (22). RESULTS Mutant Recovery. The ca-1-12-lC mutant described in this paper was recovered from a collection of acetate-requiring mutants based on its ability to grow photoautrophically at 5% CO2. This mendelian mutant was previously designated 12- IC and was found to have wild type fluorescence induction kinetics, PSII activity, and Chl content (21). In addition, the level of RuBP carboxylase activity was found to be normal (data not shown). As the first high C02-requiring C. reinhardii mutant determined to be deficient in carbonic anhydrase activity, we have assigned the mutation to the ca- 1 locus. The mutant strain is phenotypically indistinguishable from wild type when grown photoautotrophically at 5% CO2 or on acetate medium in the dark. It is a leaky acetate-requiring strain, growing slightly on minimal medium. The mutant strain is photosensitive but survives on acetate medium at 80 ME m 2 s-'

(21). Genetic Analysis. When ca-1-12-lC mt+ was crossed with pf2 mt-, mendelian 2:2 segregation of C02-requiring and wild type phenotypes was observed (Table I). No segregation of acetate-requiring, C02-requiring, and photosensitive phenotypes was found. Furthermore, reduced carbonic anhydrase activity and high compensation point were always associated with the C02-requiring phenotype (Table I). Tetrad analysis with regard to pf-2 (PD = 8, NPD = 4, T = 15) indicated that ca-l was 28 map units from its centromere. Photosynthetic Characteristics. The response of photosynthesis to bicarbonate concentration at 21% 02 is illustrated in Figure 1 for wild type, zinc-deficient wild type, ca-l mutant, and wild type in the presence of 50 Mm EZA, an inhibitor concentration determined to be optimal (data not shown). The response of photosynthesis to inorganic carbon concentration in the mutant was similar to that of wild type treated with EZA and in both cases the affinity for inorganic carbon was reduced relative to the wild type control. Zinc-deficient cells had an intermediate affinity for inorganic carbon. The photosynthetic rates at saturation CO2 (5 mM NaHCO3) were similar for the four treatments, indicating similar capacities for maximum photosynthesis (Table II). The CO2 compensation concentration of wild type C. reinhardii grown at air levels of CO2 was near zero and was unaffected by 02 concentration. The ca-l mutant, in contrast, exhibited a high, 02-sensitive CO2 compensation concentration (Table II). In the presence of 50 Mm EZA, wild type also exhibited a high,

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SPALDING ET AL.

Table I. Tetrad Analysis of a Cross between C. reinhardii Mutant Strain ca-1-12-lC mt+ and Wild Type Strain pf-2 mtCO2 TetradCarbonic Compensation Progeny Phenoty Anydrase Concn. at 50% 02 units/mg Chl /lll CO2 5-1 wt 687