Adaptation of Chlamydomonas reinhardtii High-CO(2)-Requiring Mutants to Limiting CO(2)

Photosynthetic characteristics of four high-CO₂-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO₂ concentrations. The four mutants represent two loci involved in the CO₂-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO₂ concentration, indicating that the genetic lesion in each is expressed even at elevated CO₂ concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO₂ characteristic of the induction of the CO₂-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO₂-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO₂-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.     

Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO₂ concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO₂ response of photosynthesis indicated a reduced affinity for CO₂ in the mutant. At air levels of CO₂ (0.03-0.04%), O₂ inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O₂ at saturating CO₂ concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO₂ concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO₂ concentrating system in C. reinhardii photosynthesis.     

LCIB in the Chlamydomonas CO2-concentrating mechanism

The CO₂-concentrating mechanism confers microalgae a versatile and efficient strategy for adapting to a wide range of environmental CO₂ concentrations. LCIB, which has been demonstrated as a key player in the eukaryotic algal CO₂-concentrating mechanism (CCM), is a novel protein in Chlamydomonas lacking any recognizable domain or motif, and its exact function in the CCM has not been clearly defined. The unique air-dier growth phenotype and photosynthetic characteristics in the LCIB mutants, and re-localization of LCIB between different subcellular locations in response to different levels of CO₂, have indicated that the function of LCIB is closely associated with a distinct low CO₂ acclimation state. Here, we review physiological and molecular evidence linking LCIB with inorganic carbon accumulation in the CCM and discuss the proposed function of LCIB in several inorganic carbon uptake/accumulation pathways. Several new molecular characteristics of LCIB also are presented.     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga 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-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ 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 ¹⁴CO₂. At air levels of CO₂ and O₂, 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 CO₂, wild type accumulated inorganic 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 CO₂ 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 CO₂, creating a high internal CO₂ 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.     

Changes in Photorespiratory Enzyme Activity in Response to Limiting CO(2) in Chlamydomonas reinhardtii

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO₂-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO₂. Adaptation to air levels of CO₂ by Chlamydomonas involves induction of a CO₂-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO₂-enriched cells. Unlike WT, the high-CO₂-requiring mutant, cia-5, does not respond to limiting CO₂ conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO₂ which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.     

Membrane-Associated Polypeptides Induced in Chlamydomonas by Limiting CO(2) Concentrations

Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO₂, little O₂ inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO₂-concentrating system. The CO₂-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO₂ concentrations have inorganic carbon transport activity, but cells grown at 5% CO₂ do not. Four membrane-associated polypeptides (M_r 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO₂ concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO₂-concentrating system activity in response to CO₂ limitation.     

CO2 exchange characteristics during dark-light transitions in wild-type and mutant Chlamydomonas reinhardii cells

A burst of net CO₂ uptake was observed during the first 3–4 min after the onset of illumination in both wild-type Chlamydomonas reinhardii in which carbonic anhydrase was chemically inhibited with ethoxyzolamide and in a mutant of C. reinhardii (ca-1-12-1C) deficient in carbonic anhydrase activity. The burst was followed by a rapid decrease in the CO₂ uptake rate so that net evolution often occurred. After a 2–3 min period of CO₂ evolution, net CO₂ uptake again increased and ultimately reached a steady-state, positive rate. From [¹⁴CO₂]-tracer studies it was determined that CO₂ fixation proceeded at a nearly linear rate throughout the period of illumination. Thus, prior to reaching a steady state, there was a rapid accumulation of inorganic carbon inside the cells which apparently reached a supercritical concentration and the excess was excreted, causing a subsequent efflux of CO₂. A post illumination burst of net CO₂ efflux was also observed in ethoxyzolamide-inhibited wild type and ca-1 mutant cells, but not in the unihibited wild type. [¹⁴CO₂]-tracer experiments revealed that this burst was the result of a collapse of a large internal inorganic carbon pool at the onset of darkness rather than a photorespiratory post-illumination burst. These results indicate that upon illumination, chemical or genetic inhibition of carbonic anhydrase initially causes an accumulation of excess inroganic carbon in C. reinhardii cells, and that unknown regulatory mechanisms correct for this imbalance by first excreting the excess inorganic carbon and then, after several dampened oscillations, achieving an equilibrium between bicarbonate uptake, bicarbonate dehydration, and CO₂ fixation.     

Cloning and Inactivation of a Gene Essential to Inorganic Carbon Transport of Synechocystis PCC6803

A clone (HP-1) which transforms the high CO₂-requiring mutant (RKb) of Synechocystis PCC6803 defective in inorganic carbon transport to the wild-type (WT) phenotype was isolated from a WT genomic library. The clone contained a 5.4 kilobase-pair DNA insert. Complementation tests with subclones derived from HP-1 allowed the mutation in RKb to be located within 141 base-pair nucleotides. Sequencing of nucleotides in this region revealed an open reading frame encoding a hydrophobic protein consists of 80 amino acids. A defined mutant (M9) constructed by inactivating this putative inorganic carbon transport gene, designated ictA, was unable to transport CO₂ and HCO₃⁻ into the intracellular inorganic carbon pool. Cloning and sequence analysis of the respective RKb gene revealed a base substitution which generates a stop codon in the middle of ictA.     

Photosynthetic Carbon Metabolism in Photoautotrophic Cell Suspension Cultures Grown at Low and High CO(2)

Photosynthetic carbon metabolism was characterized in four photoautotrophic cell suspension cultures. There was no apparent difference between two soybean (Glycine max) and one cotton (Gossypium hirsutum) cell line which required 5% CO₂ for growth, and a unique cotton cell line that grows at ambient CO₂ (660 microliters per liter). Photosynthetic characteristics in all four lines were more like C₃ mesophyll leaf cells than the cell suspension cultures previously studied. The pattern of ¹⁴C-labeling reflected the high ratio of ribulosebisphosphate carboxylase to phosphoenolpyruvate carboxylase activity and showed that CO₂ fixation occurred primarily by the C₃ pathway. Photorespiration occurred at 330 microliters per liter CO₂, 21% O₂ as indicated by the synthesis of high levels of ¹⁴C-labeled glycine and serine in a pulse-chase experiment and by oxygen inhibition of CO₂ fixation. Short-term CO₂ fixation in the presence and absence of carbonic anhydrase showed CO₂, not HCO₃⁻, to be the main source of inorganic carbon taken up by the low CO₂-requiring cotton cells. The cells did not have a CO₂-concentrating mechanism as indicated by silicone oil centrifugation experiments. Carbonic anhydrase was absent in the low CO₂-requiring cotton cells, present in the high CO₂-requiring soybean cell lines, and absent in other high CO₂ cell lines examined. Thus, the presence of carbonic anhydrase is not an essential requirement for photoautotrophy in cell suspension cultures which grow at either high or low CO₂ concentrations.     

Isolation of a Putative Carboxysomal Carbonic Anhydrase Gene from the Cyanobacterium Synechococcus PCC7942

The Type II mutants of the cyanobacterium Synechococcus PCC7942 (G.D. Price, M.R. Badger [1989] Plant Physiol 91: 514-525) are able to accumulate a large pool of inorganic carbon inside the cell, but are unable to utilize it for CO₂ fixation, resulting in a high CO₂-requiring phenotype. We have isolated a 3.5-kb BamHI clone (pT2) that complements the Type II mutants, and complementation analysis with DNA subclones indicated that the complementing region was located in the 0.75-kb XhoI-Bg/II fragment. This same region hybridized to the chloroplastic carbonic anhydrase (CA) gene from spinach on Southern blots and to a mRNA of approximate 1 kb on northern blots. Restriction mapping and sequence analysis revealed that pT2 is the same as a genomic clone (pBM3.8) that complements another high CO₂-requiring (temperature sensitive) mutant, C3P-O (E. Suzuki, H. Fukuzawa, S. Miyachi [1991] Mol Gen Genet 226: 401-408). Recently, a 272-amino acid open reading frame showing 22% homology with pea and spinach chloroplast CA genes was identified in clone pBM3.8 (H. Fukuzawa, E. Suzuki, Y. Komukal, S. Miyachi [1992] Proc Natl Acad Sci USA 89: 4437-4441). CA activity was detected in Escherichia coli cells transformed with subclones of pT2 (pT2-A and pT2-A1) containing the HindIII-Bg/II fragment, and the expressed CA has properties similar to those of the CA activity associated with carboxysomes purified from Synechococcus PCC7942 (G.D. Price, J.R. Coleman, M.R. Badger [1992] Plant Physiol 100: 784-793). Therefore, it is reasonable to conclude that the HindIII-Bg/II fragment codes for the carboxysomal CA gene product. The result is discussed in the context of the role that carboxysomal CA plays in the operation of the CO₂-concentrating mechanism in cyanobacteria.     

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO₂ assimilation in Chlamydomonas has been developed in order to evaluate whether a CO₂-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO₂, low photorespiration, little O₂ inhibition of photosynthesis, and low CO₂ compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO₂-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO₂, and active transport of HCO ₃ ^- . Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO₂ diffusion or reversal of HCO ₃ ^- transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO₂-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO₂-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO₂-concentrating system. These calculations indicate that the system may be no more energy-costly than C₄ photosynthesis.Abbreviations Chl chlorophyll - RuBPC/O ribulose 1,5-bisphosphate carboxylase-oxygenase - CA carbonic anhydrase     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

CO2 uptake and transport in leaf mesophyll cells

Abstract The acquisition of inorganic carbon for photosynthetic assimilation by leaf mesophyll cells and chloroplasts is discussed with particular reference to membrane permeation of CO₂ and HCO^?₃. Experimental evidence indicates that at the apoplast pH normally experienced by leaf mesophyll cells (pH 6–7) CO₂ is the principal species of inorganic carbon taken up. Uptake of HCO^?₃ may also occur under certain circumstances (i.e. pH 8.5), but its contribution to the net flux of inorganic carbon is small and HCO^?₃ uptake does not function as a CO₂-concentrating mechanism. Similarly, CO₂ rather than HCO^?₃ appears to be the species of inorganic carbon which permeates the chloroplast envelope. In contrast to many C₃ aquatic plants and C₄ plants, C₃ terrestrial plants lack specialized mechanisms for the acquisition and transport of inorganic carbon from the intercellular environment to the site of photosynthetic carboxylation, but rely upon the diffusive uptake of CO₂.     

Effect of O2 and CO2 on net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii during dark-light-dark transitions

Net CO₂ exchange was monitored through a dark-light-dark transition, under 2% and 21% O₂ in the presence and absence of CO₂, in Chlamydomonas reinhardtii wild type and the high-CO₂-requiring mutant ca-1-12-1C. Upon illumination at 350 l/l CO₂, ca-1-12-1C cell exhibited a large decrease in net CO₂ uptake following an initial surge of CO₂ uptake. Net CO₂ uptake subsequently attained a steady-state rate substantially lower than the maximum. A large, O₂-enchanced post-illumination burst of CO₂ efflux was observed after a 10-min illumination period, corresponding to a minimum in the net CO₂ uptake rate. A smaller, but O₂-insensitive post-illumination burst was observed following a 30-min illumination period, when net CO₂ uptake was at a steady-state rate. These post-illumination bursts appeared to reflect the release of an intracellular pool of inorganic carbon, which was much larger following the initial surge of net CO₂ uptake than during the subsequent steady-state CO₂ uptake period.With the mutant in CO₂-free gas, O₂-stimulated, net CO₂ efflux was observed in the light, and a small, O₂-dependent post-illumination burst was observed. With wild-type cells no CO₂ efflux was observed in the light in CO₂-free gas under either 2% or 21% O₂, but a small, O₂-dependent post-illumination burst was observed. These results were interpreted as indicating that photorespiratory rates were similar in the mutant and wild-type cells in the absence of CO₂, but that the wild-type cells were better able to scavenge the photorespiratory CO₂.     

A pyrenoid-based carbon-concentrating mechanism is present in terrestrial bryophytes of the class Anthocerotae

It has been widely accepted that carbon assimilation in bryophytes is exclusively based on the conventional C₃ photosynthetic pathway. The occurrence of biochemical CO₂-concentrating mechanisms (C₄ or Crassulacean acid metabolism), which have developed in plants in the last 20–100 million years, has been discounted for bryophytes from studies of the carbon isotope composition (¹³C) of organic material. In contrast cyanobacteria and many algae show active accumulation of dissolved inorganic carbon via biophysical CO₂-concentrating mechanisms which are also found in the photobiont partners in certain lichens. The presence of a pyrenoid, a granular particle within the chloroplast, has been linked with CO₂-concentrating mechanism activity in green algae and lichens and we now show that such a mechanism is categorically associated with the occurrence of a pyrenoid in bryophytes belonging to the class of Anthocerotae. These observations have significant evolutionary implications for the development of terrestrial photosynthesis during the colonisation of the land, raising the intriguing question of why the pyrenoid-based CO₂-concentrating mechanism did not persist in the terrestrial environment.Abbreviations and Symbols CCM carbon-concentrating mechanism - DIG dissolved inorganic carbon (CO₂+HCO ₃ ^- +CO ₂ ^- ) - DW dry weight - K_0.5 external concentration of CO₂ at which half-maximal rates of CO₂ assimilation are reached - Rubisco ribulose-l,5-bisphosphate carboxylase-oxygenase - carbon isotope discrimination (%) - ¹³C carbon isotope ratio (%) This work was supported by the Natural Environment Research Council (GR3/8813) and the Leverhulme Trust. We thank Prof. A. Roy Perry (National Museum of Wales, Cardiff), Dr. B. Coppins and Mr. D. Long (Royal Botanic Garden Edinburgh) for access to herbarium specimens and Mr. M. Fletcher for providing living bryophytes.     

Isolation and characterization of the ccmM gene required by the cyanobacterium Synechocystis PCC6803 for inorganic carbon utilization

A high CO₂-requiring mutant of Synechocystis PCC6803 (G3) capable of C_i transport but unable to utilize the intracellular C_i pool for photosynthesis was constructed. A DNA clone of 6.1 kbp that transforms the G3 mutant to the wild-type phenotype was isolated from a Synechocystis PCC6803 genomic library. Complementation test with subclones allocated the mutation site within a DNA fragment of 674 bp nucleotides. Sequencing analysis of the mutation region elucidated an open reading frame encoding a 534 amino-acid protein with a significant sequence homology to the protein coded by the ccmN gene of Synechococcus PCC7942. The ccmM-like gene product of Synechocystis PCC6803 contains four internal repeats with a week similarity to the rbcS gene product. An open reading frame homologous to the ccmN gene of Synechococcus PCC7942 was found downstream to the ccmM-like gene. As opposed to the Synechococcus PCC7942 ccmM and ccmN genes located 2 kbp upstream to, and oriented in the same direction as, the rbc operon, the ccm-like genes in Synechocystis PCC6803 are not located within 22 kbp upstream to the rbcL gene of the Rubisco operon. Thus, despite the resemblance in clustering of the ccmM and ccmN genes in both cyanobacterial species, the difference in their genomic location relative to the rbc genes demonstrates variability in structural organization of the genes involved in inorganic carbon acquisition.Abbreviations CCM CO₂-concentrating mechanism - C_i inorganic carbon - HCR high CO₂-requiring - kbp kilobase pair - ORF open reading frame - Rubisco ribulose 1,5-bisphosphate carboxylase-oxygenase gene - SSC sodium chloride and sodium citrate - WT wild-type     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

A Photorespiratory Mutant of Chlamydomonas reinhardtii

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO₂ concentration for photoautrophic growth in spite of the apparent induction of a functional CO₂ concentrating mechanism in air-adapted cells. In 2% O₂ the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O₂, photosynthetic O₂ evolution is severely inhibited in the mutant by preillumination in limiting CO₂, although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O₂ evolution measurements. Net CO₂ uptake is also inhibited when the cells are exposed to air (21% O₂, 0.035% CO₂, balance N₂) for longer than a few minutes. [¹⁴C]Phosphoglycolate accumulates within 5 minutes of photosynthetic ¹⁴CO₂ fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO₂ requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.