Insertional mutants of Chlamydomonas reinhardtii that require elevated CO(2) for survival.

Aquatic photosynthetic organisms live in quite variable conditions of CO(2) availability. To survive in limiting CO(2) conditions, Chlamydomonas reinhardtii and other microalgae show adaptive changes, such as induction of a CO(2)-concentrating mechanism, changes in cell organization, increased photorespiratory enzyme activity, induction of periplasmic carbonic anhydrase and specific polypeptides (mitochondrial carbonic anhydrases and putative chloroplast carrier proteins), and transient down-regulation in the synthesis of Rubisco. The signal for acclimation to limiting CO(2) in C. reinhardtii is unidentified, and it is not known how they sense a change of CO(2) level. The limiting CO(2) signals must be transduced into the changes in gene expression observed during acclimation, so mutational analyses should be helpful for investigating the signal transduction pathway for low CO(2) acclimation. Eight independently isolated mutants of C. reinhardtii that require high CO(2) for photoautotrophic growth were tested by complementation group analysis. These mutants are likely to be defective in some aspects of the acclimation to low CO(2) because they differ from wild type in their growth and in the expression patterns of five low CO(2)-inducible genes (Cah1, Mca1, Mca2, Ccp1, and Ccp2). Two of the new mutants formed a single complementation group along with the previously described mutant cia-5, which appears to be defective in the signal transduction pathway for low CO(2) acclimation. The other mutations represent six additional, independent complementation groups.

Molecular and Structural Changes in Chlamydomonas under Limiting CO2 (A Possible Mitochondrial Role in Adaptation).

When Chlamydomonas reinhardtii cells are transferred to limiting CO2, one response is the induction of a CO2-concentrating mechanism (CCM) with components that remain to be identified. Characterization of membrane-associated proteins induced by this transfer revealed that synthesis of the 21-kD protein (LIP-21) was regulated at the level of translatable message abundance and correlated well with the induction of CCM activity. Phase partitioning of LIP-21 and the previously characterized LIP-36 showed that both appeared to be peripherally associated with membranes, which limits their potential to function as transporters of inorganic carbon. Ultrastructural changes that occur when cells are transferred to limiting CO2 were also examined to help form a model for the CCM or other aspects of adaptation to limiting CO2. Changes were observed in vacuolization, starch distribution, and mitochondrial location. The mitochondria relocated from within the cup of the chloroplast to between the chloroplast envelope and the plasma membrane. In addition, immunogold labeling demonstrated that LIP-21 was localized specifically to the peripheral mitochondria. These data suggest that mitochondria, although not previously incorporated into models for the CCM, may play an important role in the cell's adaptation to limiting CO2.

Post-translational processing of the highly processed, secreted periplasmic carbonic anhydrase of Chlamydomonas is largely conserved in transgenic tobacco.

The periplasmic carbonic anhydrase (CA) gene CAH1 of Chlamydomonas reinhardtii codes for a highly processed secreted glycoprotein. The primary translation product of the CAH1 gene is targeted to the ER, where it is proteolytically processed to yield two different subunits, glycosylated, assembled into an active heterotetramer, and secreted. After replacing the target leader sequence with that from tobacco anionic peroxidase, expression of this gene in transgenic tobacco plants was investigated. SDS-PAGE gels of the purified protein from tobacco, showed that it migrated as a series of discrete bands (two large and one small) with slightly faster mobility than the comparable bands in the purified algal protein. The expressed protein in the plant was active, and staining with thymol and sulfuric acid confirmed that it was also glycosylated. The periplasmic CA1 (peri-CA1) also was found to be enriched in the intercellular fluid of transgenic tobacco, indicating it was secreted. The specific activity of the enzyme and its sensitivity to sulfonamide inhibitors were similar to that of the native algal enzyme. These results suggest that the post translational processing of Chlamydomonas peri-CA1 is largely conserved in a higher plant.

The Low CO2-Inducible 36-Kilodalton Protein Is Localized to the Chloroplast Envelope of Chlamydomonas reinhardtii.

The localization of the 36-kD polypeptide of Chlamydomonas reinhardtii induced by photoautotrophic growth on low CO2 concentrations (0.03% in air [v/v], low CO2-grown cells) has been investigated. This polypeptide was specifically localized to the chloroplast envelope membranes isolated from low CO2-grown cells and was not present in the chloroplast envelopes isolated from high (5% CO2 in air [v/v]) CO2-grown cells. The 36-kD protein does not show carbonic anhydrase activity and was not present on the plasma membranes isolated from low CO2-grown cells. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO2-grown cells of C. reinhardtii.

Translational Regulation of the Large and Small Subunits of Ribulose Bisphosphate Carboxylase/Oxygenase during Induction of the CO(2)-Concentrating Mechanism in Chlamydomonas reinhardtii.

In conditions of limiting external inorganic carbon, the unicellular alga Chlamydomonas reinhardtii induces a mechanism to actively transport and accumulate inorganic carbon within the cell. A high internal inorganic carbon concentration enables the cell to photosynthesize efficiently with little oxygen inhibition, even in conditions of limiting external inorganic carbon. A correlation between limiting inorganic carbon-induced induction of the CO(2)-concentrating mechanism and decreased synthesis of the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase has been observed. Cells that had been transferred from elevated CO(2) to limiting CO(2) exhibit transient declines of label incorporation into both subunit polypeptides. We have found that this decrease in synthesis of large and small subunits results from specific and coordinated down-regulation of translation of both subunits possibly resulting, at least in part, from modification of large and small subunit transcripts.

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 CO(2)-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO(2). Adaptation to air levels of CO(2) by Chlamydomonas involves induction of a CO(2)-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 CO(2)-enriched cells. Unlike WT, the high-CO(2)-requiring mutant, cia-5, does not respond to limiting CO(2) 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 CO(2) 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.

A 36 Kilodalton Limiting-CO(2) Induced Polypeptide of Chlamydomonas Is Distinct from the 37 Kilodalton Periplasmic Carbonic Anhydrase.

Chlamydomonas reinhardtii possesses a CO(2)-concentrating mechanism, induced by limiting CO(2), which involves active transport and accumulation of inorganic carbon within the cell. Synthesis of several proteins is induced by limiting CO(2), but, of those, only periplasmic carbonic anhydrase has an identified function in the system. No proteins involved in active transport have yet been identified, but induced, membrane-associated polypeptides, such as the 36 kilodalton polypeptide focused on in this paper, would seem to be candidates for such involvement. The 36 kilodalton polypeptide was shown to be synthesized de novo upon transfer of cells to limiting CO(2). It was purified using SDS-PAGE and used to produce polyclonal antibodies. Antibodies were used to confirm the air-specific nature of the polypeptide, its strict association with membrane fractions, and the time course of its induction. Using the antibodies, a single, 36 kilodalton polypeptide was found to be specifically immunoprecipitated from in vitro translation products of poly(A(+)) RNA from cells only after exposure to limiting CO(2). The absence of translatable mRNA for this polypeptide in CO(2)-enriched cells indicated that regulation occurs at the level of message abundance. The antibodies were also used to demonstrate the distinction between the limiting-CO(2) induced 36 kilodalton polypeptide and the similarly sized, limiting-CO(2) induced periplasmic carbonic anhydrase.

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 CO(2) concentration for photoautrophic growth in spite of the apparent induction of a functional CO(2) concentrating mechanism in air-adapted cells. In 2% O(2) the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O(2), photosynthetic O(2) evolution is severely inhibited in the mutant by preillumination in limiting CO(2), although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O(2) evolution measurements. Net CO(2) uptake is also inhibited when the cells are exposed to air (21% O(2), 0.035% CO(2), balance N(2)) for longer than a few minutes. [(14)C]Phosphoglycolate accumulates within 5 minutes of photosynthetic (14)CO(2) 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-CO(2) 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.

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO 2-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 µmol 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.

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

Photosynthetic characteristics of four high-CO(2)-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO(2) concentrations. The four mutants represent two loci involved in the CO(2)-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 CO(2) concentration, indicating that the genetic lesion in each is expressed even at elevated CO(2) concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO(2) characteristic of the induction of the CO(2)-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO(2)-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 CO(2)-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.

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

Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO(2), little O(2) inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO(2)-concentrating system. The CO(2)-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO(2) concentrations have inorganic carbon transport activity, but cells grown at 5% CO(2) 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 CO(2) concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO(2)-concentrating system activity in response to CO(2) limitation.

Effect of O2 and CO 2 on net CO 2 exchange in a high-CO 2-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 µl/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.

Imazaquin and chlorsulfuron resistance and cross resistance in mutants of Chlamydomonas reinhardtii.

Chlorsulfuron and/or imazaquin resistant mutants of Chlamydomonas reinhardtii strain CW15 have been obtained and shown to have actolactate synthase (ALS) with altered sensitivity to one or both of these herbicides. Herbicide resistance in the three mutants described is allelic, and resistance appears to result from a dominant or semidominant mutation in a single, nuclear gene. Imazaquin and chlorsulfuron resistant ALS from imazaquin and chlorsulfuron resistant mutants, together with single-gene Mendelian inheritance of these phenotypes, suggests that ALS is the sole site of action of the two herbicides in Chlamydomonas. A high degree of cross resistance between the two herbicides was found in only one mutant. This mutant (IMR-13) was selected for resistance to imazaquin and has a high level of in vitro resistance to both imazaquin (270-fold increased I50) and chlorsulfuron (900-fold increased I50). In another mutant selected for resistance to imazaquin (IMR-2), hyper-sensitivity to chlorsulfuron was found. A mutant selected for resistance to chlorsulfuron (CSR-5), had a substantial degree of resistance of chlorsulfuron (80-fold increased I50), but not to imazaquin (7-fold increased I50).

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.

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 [(14)CO2]-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. [(14)CO2]-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.

Influence of carbon dioxide concentration during growth on fluorescence induction characteristics of the Green Alga Chlamydomonas reinhardii.

Carbon dioxide concentration during growth is commonly not considered to be a factor influencing the photochemical properties of plants. It was observed that fluorescence induction in Chlamydomonas reinhardii cells grown at air levels of CO2 was both qualitatively and quantitatively different from that of cells grown at 5% CO2. In the two cell types, measured at equivalent chlorophyll and irradiance levels, the fluorescence intensity and the ratio of the levels of peak fluorescence (Fp) to that of the initial fluorescence (Fo) were much lower in the air-adapted than in the 5% CO2 adapted cells. The maximum fluorescence (Fmax) in the presence of diuron was also lower for air-adapted cells. Roughly twice the light input was required for the air-adapted cells to give a fluorescence induction transient and intensity equivalent to that of the 5% CO2-adapted cells. Similar properties were observed in several other unicellular green algae and in cyanobacteria. Chlamydomonas grown under variable CO2 concentrations exhibit significant differences in photosynthetic carbon metabolism and are presumed to have altered energy requirements. The observed variation in fluorescence induction may be due to changes in the properties of the thylakoid reactions (e.g. cyclic electron flow) of Chlamydomonas cells, which may, in turn, be due to a response to the altered energy requirements.

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 CO(2) requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO(2) concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O(2). In contrast to a CO(2) compensation concentration of near zero in wild type at all O(2) concentrations examined, the mutant exhibited a high, O(2)-stimulated CO(2) 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 (14)CO(2). At air levels of CO(2) and O(2), 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 CO(2) concentration. At ambient CO(2), 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 CO(2) 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 CO(2) concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO(2), creating a high internal CO(2) 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 CO(2) concentration.

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 CO(2) concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO(2) response of photosynthesis indicated a reduced affinity for CO(2) in the mutant. At air levels of CO(2) (0.03-0.04%), O(2) inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O(2) at saturating CO(2) concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO(2) concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO(2) concentrating system in C. reinhardii photosynthesis.

Genetic and physiological analysis of the CO2-concentrating system of Chlamydomonas reinhardii.

When grown photoautotrophically at air levels of CO2, Chlamydomonas reinhardii possesses a system involving active transport of inorganic carbon which increases the intracellular CO2 concentration considerably above ambient, thereby stimulating photosynthetic CO2 assimilation. In previous investigations, two mutant strains of this unicellular green alga deficient in some component of this CO2-concentrating system were recovered as strains requiring high levels of CO2 to support photoautotrophic growth. One of the mutants, ca-1-12-1C, is a leaky (nonstringent) CO2-requiring strain deficient in carbonic anhydrase (EC 4.2.1.1) activity, while the other, pmp-1-16-5K, is a stringent CO2-requiring strain deficient in inorganic carbon transport. In the present study a double mutant (ca pmp) was constructed to investigate the physiological and biochemical interaction of the two mutations. The two mutations are unlinked and inherited in a Mendelian fashion. The double mutant was found to have a leaky CO2-requiring phenotype, indicating that the mutation ca-1 overcomes the stringent CO2-requirement conferred by the mutation pmp-1. Several physiological characteristics of the double mutant were very similar to the carbonic-anhydrase-deficient mutant, including high CO2 compensation concentration, photosynthetic CO2 response curve, and deficiency of carbonic-anhydrase activity. However, the labeling pattern of metabolites during photosynthesis in (14)CO2 was more like that of the bicarbonatetransport-deficient mutant, and accumulation of internal inorganic carbon was intermediate between that of the two original mutants. These data indicate a previously unsuspected complexity in the Chlamydomonas CO2-concentrating system.

Quantum Requirement for Photosynthesis in Sedum praealtum during Two Phases of Crassulacean Acid Metabolism.

The quantum requirement (QR) for photosynthesis in Sedum praealtum, a Crassulacean acid metabolism plant, was compared with that of wheat, a C(3) plant, and maize, a C(4) plant, at 30 C. During the deacidification phase in S. praealtum, approximately 16 moles quanta were absorbed per mole malate consumed. This is equivalent to 16 moles quanta per mole CO(2) fixed, assuming 1 mole CO(2) is assimilated per mole malate decarboxylated. This QR for Crassulacean acid metabolism is similar to that of the C(3) or C(4) plant under atmospheric conditions, even though there are considerable differences in the biochemistry of photosynthesis. During late-afternoon C(3)-like fixation of atmospheric CO(2) in S. praealtum, the QR was relatively high with values of 41 under 21% O(2) and 19 under 2% O(2). During the deacidification phase in S. praealtum, the relatively low QR can be accounted for by the repression of photorespiration and saturation of photosynthesis from the elevated CO(2) concentration in the leaves during malate decarboxylation.