Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production.

Molecular hydrogen (H2) is an ideal fuel characterized by high enthalpy change and lack of greenhouse effects. This biofuel can be released by microalgae via reduction of protons to molecular hydrogen catalyzed by hydrogenases. The main competitor for the reducing power required by the hydrogenases is the Calvin cycle, and rubisco plays a key role therein. Engineered Chlamydomonas with reduced rubisco levels, activity and stability was used as the basis of this research effort aimed at increasing hydrogen production. Biochemical monitoring in such metabolically engineered mutant cells proceeded in Tris/acetate/phosphate culture medium with S-depletion or repletion, both under hypoxia. Photosynthetic activity, maximum photochemical efficiency, chlorophyll and protein levels were all measured. In addition, expression of rubisco, hydrogenase, D1 and Lhcb were investigated, and H2 was quantified. At the beginning of the experiments, rubisco increased followed by intense degradation. Lhcb proteins exhibited monomeric isoforms during the first 24 to 48 h, and D1 displayed sensitivity under S-depletion. Rubisco mutants exhibited a significant decrease in O2 evolution compared with the control. Although the S-depleted medium was much more suitable than its complete counterpart for H2 production, hydrogen release was observed also in sealed S-repleted cultures of rubisco mutated cells under low-moderate light conditions. In particular, the rubisco mutant Y67A accounted for 10-15-fold higher hydrogen production than the wild type under the same conditions and also displayed divergent metabolic parameters. These results indicate that rubisco is a promising target for improving hydrogen production rates in engineered microalgae.

An assessment of arsenate selection as a method for obtaining nonphotosynthetic mutants of chlamydomonas.

It has been proposed that the absence of photosynthesis in Chlamydomonas reinhardii produces a relative arsenate resistance and that selection for arsenate resistance therefore serves as an enrichment for nonphotosynthetic mutants (Togasaki and Hudock 1972; Harris, Boynton and Gillham 1974). We have found that: 1. mutants selected for arsenate resistance are not substantially enriched for acetate-requiring mutants as compared with unselected cells; 2. none of the acetate-requiring mutants we obtained without arsenate selection are arsenate resistant; 3. the acetate-requiring mutants obtained following arsenate selection are all capable of CO(2) fixation, suggesting that they do not have a major lesion in primary photosynthetic processes; 4. in most acetate-requiring mutants selected on arsenate medium, the arsenate-resistant and acetate-requiring characters segregate from one another during meiosis, indicating that these two characters arose independently. We conclude that if any enrichment is provided by selecting for arsenate resistance, it is for only a subclass of acetate requirers that are not obviously defective in photosynthesis.

Nuclear Mutation Inhibits Expression of the Chloroplast Gene That Encodes the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

Chlamydomonas reinhardtii mutant 76-5EN was recovered as a light-sensitive, acetate-requiring strain that failed to complement a chloroplast structural gene mutant of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Further genetic analysis revealed that the new mutation was inherited in a mendelian pattern, indicating that it resides within the nucleus. The 76-5EN mutant lacks Rubisco holoenzyme but has wild-type levels of whole-chain electron transport activity and chlorophyll. During a 1-min pulse labeling with 35SO42-, little or no Rubisco large-subunit synthesis occurred in the mutant. Nuclear-encoded small subunits were synthesized to a normal level and were subsequently degraded. When analyzed by northern hybridization, the 76-5EN mutant was found to have a decreased level of large-subunit mRNA. Large-subunit mRNA synthesis also appeared to be reduced during a 10-min pulse labeling with [32P]orthophosphate, but the labeled mRNA was stable during a 1-h chase. These results indicate that a nuclear gene mutation specifically disrupts the accumulation of large-subunit mRNA within the chloroplast. A deeper understanding of the nature of the 76-5EN gene may be useful for manipulating the expression of the agronomically important Rubisco enzyme.

Thermal Instability of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from a Temperature-Conditional Chloroplast Mutant of Chlamydomonas reinhardtii.

Mutant 68-4PP of Chlamydomonas reinhardtii has only 10% of the normal level of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme when grown at 35[deg]C. However, when grown at 25[deg]C, the amount of holoenzyme is greater than 35% of the wild-type level, and the purified enzyme has a reduced CO2/O2 specificity factor. These mutant characteristics result from a chloroplast mutation that causes leucine-290 to be replaced by phenylalanine within the Rubisco large-subunit protein. A nuclear mutation (named S52-2B) was previously identified that can suppress both the in vivo instability and reduced CO2/O2 specificity of the mutant enzyme. However, the effect of this nuclear mutation on the in vitro stability of the holoenzyme was not resolved. In the present study, purified Rubisco from mutant 68-4PP was found to be less thermally stable than the wild-type enzyme, and it had maximal carboxylase activity at a lower temperature. When incubated at 35[deg]C, the mutant enzyme lost carboxylase activity at a much faster rate than the wild-type enzyme. However, the nuclear S52-2B suppresor mutation improved the thermal stability of the mutant enzyme in all cases. These results indicate that structural changes in mutant 68-4PP Rubisco can account for its observed inactivation in vitro and degradation in vivo. Such structural alterations are alleviated by the function of a nuclear gene.

Rapid recovery of chloroplast mutations affecting ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii.

Based on the unique ability of chloroplast genes to recombine in Chlamydomonas reinhardtii, a collection of acetate-requiring mutants was screened for recombination with a mutation affecting ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu-1,5-P(2) carboxylase/oxygenase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39]. This chloroplast mutation, rcl-u-1-10-6C, causes the absence of Rbu-1,5-P(2) carboxylase/oxygenase activities and alters the isoelectric point of the larger subunit. Several mutants that displayed little or no recombination with 10-6C were recovered, and two lacked carboxylase activity. These new chloroplast mutants lack both large and small Rbu-1,5-P(2) carboxylase/oxygenase subunits. The approach demonstrated here permits the routine recovery of chloroplast mutations affecting this enzyme. Multiple mutations in the Rbu-1,5-P(2) carboxylase/oxygenase large-subunit gene can be used to investigate the function and regulation of this enzyme and the regulation of chloroplast genes in general.

Nuclear Suppressors of the Photosensitivity Associated with Defective Photosynthesis in Chlamydomonas reinhardii.

Several nuclear mutations were recovered that suppress the photosensitivity associated with the Chamydomonas reinhardii chloroplast mutant rcl-u-1-10-6C, which is defective in ribulose-1,5-bisphosphate carboxylase/oxygenase. Two of the suppressor mutations affect other components of photosynthesis. These results show that suppressors of photosensitivity are sufficiently common to permit the recovery of photosensitive, photosynthesis-deficient mutants in bright light, and indicate that photosynthesis-deficient mutants selected and maintained in the light may accumulate suppressors which can confuse the biochemical analysis of lesions in photosynthesis. One of the suppressor mutations inhibits photosystem II activity, indicating that photosensitivity can be mediated by partial reactions of the photosynthetic electron transport chain.

Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii.

Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from the green alga Chlamydomonas reinhardtii. Incubation of either CO2/Mg(2+) -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO2/O2 specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The K m values for RuBP were not significantly changed for the mutant enzymes, but the V max values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO2/O2 specificity of Rubisco.

How various factors influence the CO2/O 2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO2/O2 specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO2/O2 specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O2 has a higher free energy of activation than the corresponding reaction of this substrate with CO2. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO2/O2 specificity that is observed when activator Mg(2+) is replaced by Mn(2+) may be due to Mg(2+) being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn(2+) is a transition-metal ion that can overcome the triplet character of O2 to promote the oxygenation reaction.

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) is the key photosynthetic enzyme that catalyzes the first step of CO2 fixation. The chloroplast-localized holoenzyme of plants and green algae contains eight nuclear-encoded small subunits and eight chloroplast-encoded large subunits. Although much has been learned about the enzyme active site that resides within each large subunit, it has been difficult to assess the role of eukaryotic small subunits in holoenzyme function and expression. Small subunits are coded by a family of genes, precluding genetic screening or nuclear transformation approaches for the recovery of small-subunit mutants. In this study, the two small-subunit mutants. In this study, the two small-subunit genes of the green alga Chlamydomonas reinhardtii were eliminated during random insertional mutagenesis. The photosynthesis-deficient deletion mutant can be complemented with either of the two wild-type small-subunit genes or with a chimeric gene that contains features of both. Thus, either small subunit is sufficient for holoenzyme assembly and function. In the absence of small subunits, expression of chloroplast-encoded large subunits appears to be inhibited at the level of translation.

Complementing substitutions at the bottom of the barrel influence catalysis and stability of ribulose-bisphosphate carboxylase/oxygenase.

The temperature-conditional photosynthesis-deficient mutant 68-4PP of Chlamydomonas reinhardtii results from a Leu-290 to Phe substitution in the chloroplast-encoded large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Although this substitution occurs relatively far from the active site, the mutant enzyme has a reduced ratio of carboxylation to oxygenation in addition to reduced thermal stability in vivo and in vitro. In an attempt to understand the role of this region in catalysis, photosynthesis-competent revertants were selected. Two revertants, named R96-4C and R96-8E, were found to arise from second-site mutations that cause V262L and A222T substitutions, respectively. These intragenic suppressor mutations increase the CO2/O2 specificity and carboxylation Vmax back to wild-type values. Based on the crystal structure of the spinach holoenzyme, Leu-290 is not in van der Waals contact with either Val-262 or Ala-222. However, all three residues are located at the bottom of the alpha/beta-barrel active site and may interact with residues of the nuclear encoded small subunits. It appears that amino acid residues at the interface of large and small subunits can influence both stability and catalysis.

Directed mutagenesis of chloroplast ribulosebisphosphate carboxylase/oxygenase. Substitutions at large subunit asparagine 123 and serine 379 decrease CO2/O2 specificity.

Chloroplast-encoded large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) are insoluble when separated from the holoenzyme or expressed in Escherichia coli, limiting directed mutagenesis to prokaryotic enzymes. In the present study, we performed directed mutagenesis and chloroplast transformation with the large subunit gene of Chlamydomonas reinhardtii. Two separate mutations were created that are known to influence the CO2/O2 specificity of prokaryotic enzymes. The asparagine 123 to glycine and serine 379 to alanine substitutions gave rise to photosynthesis-deficient mutants that synthesize normal levels of holoenzyme. The Vmax for carboxylation was reduced more than 95% and the Km(CO2) was increased more than 3-fold for both mutant enzymes. Km (O2) was slightly reduced for the glycine 123 enzyme, but increased more than 5-fold for the alanine 379 enzyme. CO2/O2 specificity factors for both enzymes are decreased by more than 70%. Km values for ribulose 1,5-bisphosphate are not significantly affected, but binding affinities for the transition-state analog 2-carboxy-D-arabinitol 1,5-bisphosphate are reduced. The changes brought about by these substitutions in the eukaryotic large subunit are different from the changes observed in prokaryotic enzymes.

Suppressor mutations in the chloroplast-encoded large subunit improve the thermal stability of wild-type ribulose-1,5-bisphosphate carboxylase/oxygenase.

A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ). Rubisco purified from mutant cells grown at 25 degrees C has a reduction in CO(2)/O(2) specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35 degrees C restrictive temperature. These intragenic suppressors improve the CO(2)/O(2) specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO(2)/O(2) specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme.

Chloroplast intragenic suppression enhances the low CO2/O2 specificity of mutant ribulose-bisphosphate carboxylase/oxygenase.

The competition between CO2 and O2 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase limits net CO2 fixation in photosynthesis. In the green alga Chlamydomonas reinhardtii, a mutation in the chloroplast large-subunit gene reduces the CO2/O2 specificity of the enzyme by 37% and causes valine-331 to be replaced by alanine. Revertant selection identified an intragenic suppressor mutation that increases the CO2/O2 specificity of the mutant enzyme by 33%. This second-site mutation causes threonine-342 to be replaced by isoleucine. The complementing amino acid substitutions flank a catalytically essential lysyl residue at position 334. It thus appears that a number of amino acid residues can influence the CO2/O2 specificity of this bifunctional enzyme. The well defined chloroplast genetics of C. reinhardtii allows the interactions of these residues to be investigated.

Chloroplast heteroplasmicity is stabilized by an amber-suppressor tryptophan tRNA(CUA).

Photosynthesis-deficient mutants of the green alga Chlamydomonas reinhardtii were previously shown to arise from nonsense mutations within the chloroplast rbcL gene, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Photosynthesis-competent revertants of these nonsense mutants have often been found to be stably heteroplasmic, giving rise to both mutant and revertant cells during mitotic or meiotic divisions under nonselective growth conditions. It was proposed that the stable heteroplasmicity might arise from a balanced polymorphism of suppressor and wild-type alleles of a tRNA gene maintained within the polyploid chloroplast genome. In the present study, we have focused on determining the molecular basis for the heteroplasmicity of one such revertant, named R13-3C, which was recovered from the 18-7G amber (UAG) mutant. Restriction-enzyme analysis and DNA sequencing showed that the amber mutation is still present in the rbcL gene of the revertant strain. In contrast, DNA sequencing of the suspected tRNA(Trp) gene of the revertant revealed a mutation that would change its CCA anticodon to amber-specific CUA. This mutation was found to be heteroplasmic, being present in only 70% of the tRNA(Trp) gene copies. Under nonselective conditions, the suppressor mutation was lost from cells that also lost the revertant phenotype. We conclude that stable heteroplasmicity can arise as a balanced polymorphism of organellar alleles. This observation suggests that additional tRNA suppressors may be identified due to their heteroplasmic nature within polyploid genomes.

Directed mutagenesis of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Loop 6 substitutions complement for structural stability but decrease catalytic efficiency.

The structure of active-site loop 6 plays a role in determining the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39). Rubisco from the green alga Chlamydomonas reinhardtii differs from higher plant Rubisco within the loop 6 region, and the C. reinhardtii enzyme has a CO2/O2 specificity 25% lower than that of higher plant enzymes. To examine whether differences in sequence may account for differences in catalytic efficiency, we focused on a conserved pair of residues that are in van der Waals contact at the base of loop 6. C. reinhardtii Rubisco contains Leu-326 and Met-349, whereas higher plant enzymes contain Ile-326 and Leu-349. By employing in vitro mutagenesis and chloroplast transformation, L326I and M349L substitutions were created within the Rubisco large subunit of C. reinhardtii. M349L had little effect, but L326I destabilized the holoenzyme in vivo and in vitro. When present together, the M349L substitution partially alleviated the instability resulting from the L326I substitution, but caused a 21% decrease in CO2/O2 specificity and a 74% decrease in the Vmax of carboxylation. Interactions between loop 6 and other structural regions are likely to be responsible for both holoenzyme stability and catalytic efficiency in higher plant Rubisco enzymes.

Photosynthesis-deficient Mutants of Chlamydomonas reinhardii with Associated Light-sensitive Phenotypes.

A series of non-photoautotrophic mutants of Chlamydomonas reinhardii was isolated by replica-plating mutagenized cells which had been grown in the dark. Many of these acetate-requiring mutants are photosensitive, showing poor growth on acetate medium in the light, but normal growth in the dark. Biochemical characterization showed that the photosensitive mutants all had specific lesions in photosynthesis or photosynthetic pigment accumulation. The acetate-requiring mutants which were not photosensitive were all able to fix CO(2). Among the light-sensitive mutants are 15 which show uniparental inheritance. These include six with specific lesions in photosystem II and one with an altered large subunit of ribulose-1,5-bisphosphate carboxylase. Since these two classes of uniparental mutants have been rare or not previously reported, it seems likely that photosensitivity is an important factor which limited their detection in previous mutant isolation experiments.

C172S substitution in the chloroplast-encoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Previous work has indicated that the turnover of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1. 39) may be controlled by the redox state of certain cysteine residues. To test this hypothesis, directed mutagenesis and chloroplast transformation were employed to create a C172S substitution in the Rubisco large subunit of the green alga Chlamydomonas reinhardtii. The C172S mutant strain was not substantially different from the wild type with respect to growth rate, and the purified mutant enzyme had a normal circular dichroism spectrum. However, the mutant enzyme was inactivated faster than the wild-type enzyme at 40 and 50 degrees C. In contrast, C172S mutant Rubisco was more resistant to sodium arsenite, which reacts with vicinal dithiols. The effect of arsenite may be directed to the cysteine 172/192 pair that is present in the wild-type enzyme, but absent in the mutant enzyme. The mutant enzyme was also more resistant to proteinase K in vitro at low redox potential. Furthermore, oxidative (hydrogen peroxide) or osmotic (mannitol) stress-induced degradation of Rubisco in vivo was delayed in C172S mutant cells relative to wild-type cells. Thus, cysteine residues could play a role in regulating the degradation of Rubisco under in vivo stress conditions.

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.

Chloroplast gene suppression of defective ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas reinhardii: evidence for stable heteroplasmic genes.

The rcl-u-1-18-5B chloroplast mutation results in the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme in the green alga Chlamydomonas reinhardii. The 18-5B mutant strain lacks photosynthesis and displays alight-sensitive, acetate-requiring phenotype. In the present investigations, revertants of 18-5B were recovered that regained photosynthetic competence. These revertants have decreased levels of Rubisco holoenzyme relative to wild type and display heteroplasmicity, segregating wild-type (revertant) and acetate-requiring phenotypes during vegetative growth or through meiosis. One of these revertants, R10-I, was studied further. The heteroplasmicity associated with photoautotrophically-grown R10-I was found to be stable through subcloning and heritable through several crosses. During growth in acetate medium in the dark, where photosynthesis provides no selective advantage, the wild-type phenotype was lost. Acetate-requiring segregants became homoplasmic but wild-type segregants did not. Organellar intergenic-suppression is discussed in light of the observed stable heteroplasmicity.