Chlamydomonas Reinhardii Research Articles

Ferredoxin-glutamate synthase from Chlamydomonas reinhardii. Prosthetic groups and preliminary studies of mechanism

1. 1. Ferredoxin-glutamate synthase from the green alga Chlamydomonas reinhardii appears to contain as prosthetic groups, 1 FAD, 1 FMN and 1 |3Fe-xS | cluster, per molecule of M r = 146,000. 2. 2. The synthesis of glutamate, catalyzed by this enzyme, proceeds through the formation of an enzyme-bound free radical of flavin semiquinone. 3. 3. This enzyme may catalyze the ferredoxin-dependent reduction of 2-oxoglutarate, in the absence of glutamine.

Photosynthesis, Nitrogen Fixation and Enzyme Activities in Chlamydomonas-Azotobacter Symbioses

Summary Nitrogen-fixing associations were established by mixing Azotobacter and Chlamydomonas cells and were cultured on nitrogen and carbohydrate free media. Scanning electron micrographs show a few bacteria localized on the surface of the algal cells. The rate of the photosynthetic 02-evolution in symbioses is nearly the same as the value obtained with the control. Acetylene reduction assay showed that nitrogen fixation was detectable in associative symbioses. GS, ATP-ase, Diaphorase and IDH activities were studied by polyacrylamide gel electrophoresis in Chlamydomonas reinhardii, Azotobacter chroococcum, A. beijerinckii, A. agilis, A. vinelandii and Chlamydomonas-Azotobacter symbioses. The mobility of GS enzymes is the same in algae and bacteria, but ATP-ase migrates faster in the case of Azotobacter. The enzyme pattern of alga-bacterium symbioses is similar to that found in Chlamydomonas. IDH has no activity in Chlamydomonas, but shows high activity in Azotobacters. An enzyme with lower molecular weight is present in alga-bacterium symbioses. Diaphorase isoenzymes are different in alga and bacterium cells. In the symbioses, the enzyme are partially similar to algae, but an enzyme of lower molecular weight also exists. The results show that there is an interaction at the biochemical level between the two organisms associated.

Compartmentation of glycolysis and of the oxidative pentose-phosphate pathway in Chlamydomonas reinhardii

Glycolytic enzyme activities and enzyme activities of the oxidative pentose-phosphate pathway were measured in intact chloroplasts from Chlamydomonas reinhardii. By comparison with the total enzyme activities of intact protoplasts of the alga the data were used to locate these enzymes quantitatively in the algal chloroplast. It was found that the glycolytic chain in C. reinhardii is roughly split into a plastidic and an extraplastidic part. More than 90% of the first part of glycolysis (from fructose-6-phosphate to triose-phosphate is located in the plastid while more than 95% of the second part (from glycerate-3-phosphate to pyruvate) is outside. Around 70% of glucose-6-phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase, two key enzymes of the oxidative pentose phosphate pathway, are located in the plastid. It is concluded that in C. reinhardii the major part of hexose breakdown to triose-phosphate occurs in the chloroplast and that a tight cooperation between the plastid and the cytoplasm is required for appreciable sugar breakdown to occur in the algal cell.

Comparative kinetics and inhibition of a carbonic anhydrase from Chlamydomonas reinhardii

1. 1. A native carbonic anhydrase (CA) from Chlamydomonas reinhardii exhibited an apparent molecular weight of 165,000. Denaturation under reducing conditions yielded a single polypeptide, mol. wt 42,000, while the same treatment under nonreducing conditions yielded higher molecular weight aggregates at least two of which could be reactivated to exhibit enzyme activity. 2. 2. In CO 2 hydration the enzyme has a K m value of 3.4 mM and a relatively high turnover number. It is very sensitive to inhibition by sulfonamide and moderately sensitive to anion inhibition. 3. 3. In general, the C. reinhardii enzyme resembles many higher plant CAs in its existence as an oligomer. However, it is more like the CAs from animal species in its catalytic versatility and sensitivity to sulfonamides.

The Effects of Temperature on86Rb Uptake by Two Species ofChlamydomonas(Chlorophyta, Chlorophyceae)

Clarke, A., Leeson, E. A., and Morris, G. J. 1986. The effects of temperature on 86Rb uptake by two species of Chlamydomonas (Chlorophyta, Chlorophyceae).—J. exp. Bot. 37: 1285-1293. 86Rb uptake was examined in two species of unicellular green algae, Chlamydomonas nivalis isolated from snow, and a cell wall-less mutant of the temperate freshwater Chlamydomonas reinhardii. In C. reinhardii cells grown at 20 °C and cooled rapidly to 0 °C, 86Rb uptake was abolished. Cells cooled rapidly to — 5°C in the absence of ice accumulated 86Rb very rapidly but the time course of this uptake suggested non-selective accumulation through a damaged plasmalemma. Cells grown at 8 °C were viable, able to divide and motile; they showed no signs of cold-shock and 86Rb uptake, albeit slow, was measurable at — 5 °C in the absence of extracellular ice. Cells of C. nivalis grown at 20 °C were damaged at sub-zero temperatures although they did show an enhanced 86Rb uptake at 0°C. Cells grown at 5 °C were able to accumulate 86Rb from media undercooled to — 5 °C in the absence of extracellular ice, and again showed enhanced uptake at 0°C. The process of acclimation to low temperature appears to differ in the two species. Key words—Chlamydomonas, temperature, 86Rb uptake, membrane. Correspondence to: British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge CB3 OET, England, U.K.

Nitrate Reductase of Green Algae Is Located in the Pyrenoid

Antibodies against nitrate reductase from Monoraphidium braunii have been used to determine the antigenic relationships of nitrate reductases from different green algae. Nitrate reductases from Chlamydomonas reinhardii, Chlorella fusca, Dunaliella salina, and Scenedesmus obliquus, were inhibited by, and cross-reacted with, antibodies raised against the enzyme from Monoraphidium braunii.These antibodies were also used to determine, by immunoelectron microscopy, the intracellular location of nitrate reductase in the aforementioned green algae. In all cases, the enzyme was specifically located in the pyrenoid.

Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 45Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diffuse throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.

Isolation of chloroplast envelopes from Chlamydomonas. Lipid and polypeptide composition

A membrane fraction enriched in chloroplast envelopes was isolated from the alga Chlamydomonas reinhardii. The envelope preparations contained the envelope marker enzyme, galactosyltransferase, with a specific activity about 26- and 37-fold greater than that of the intact chloroplasts and of the starting cells, respectively. Both the envelope fraction and intact chloroplasts incorporated labelled galactose mostly into digalactosyldiglyceride (DGDG), with little incorporation into monogalactosyldiglyceride (MGDG), in marked contrast to what had been observed in higher plants. The envelope preparation was characterized in terms of its composition in polar lipids and polypeptides, and certain features were found in common with the corresponding membranes of higher plants. The ratio of galactolipids MGDG/DGDG was typically lower in the envelopes, compared to the thylakoid fraction. Analysis of the proteins by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed a polypeptide pattern that was distinct from those of the thylakoids or of the soluble fraction of the chloroplasts. The major component of the envelope fraction was a 29-hilodalton polypeptide, similar to the main envelope polypeptide found in higher plants and believed to be the phosphate translocator.

O2 Uptake in the Light in Chlamydomonas

The nature of the process responsible for the stationary O(2) uptake occurring in the light under saturating CO(2) concentration in Chlamydomonas reinhardii has been investigated. For this purpose, a mass spectrometer with a membrane inlet system was used to measure O(2) uptake and evolution in the algal suspension. First, we observed that the O(2) uptake rate was constant (about 0.5 micromoles of O(2) per milligram chlorophyll per minute) during a light to dark transition and was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Salicylhydroxamic acid had no effect on O(2) uptake in the dark or in the light, but was found to have the same inhibitory effect either in the dark or in the light when added to cyanide-treated algae. The stimulation of the O(2) uptake rate due to the uncoupling effect of carbonyl cyanide m-chlorophenylhydrazone was about the same in the dark or in the light. From these results, we conclude that mitochondrial respiration is maintained during illumination and therefore is not inhibited by high ATP levels. Another conclusion is that in conditions where photorespiration is absent, no other light-dependent O(2) uptake process occurs. If Mehler reactions are involved, in Chlamydomonas, under conditions where both photosynthetic carbon oxidation and reduction cycles cannot operate (as in cyanide-treated algae), their occurrence in photosynthesizing algae either under saturating CO(2) concentration or at the CO(2) compensation point appears very unlikely. The comparison with the situation previously reported in Scenedesmus (R. J. Radmer and B. Kok 1976 Plant Physiol 58: 336-340) suggests that different O(2) uptake processes might be present in these two algal species.

Effects of Light Intensity and Oxidized Nitrogen Sources on Hydrogen Production by Chlamydomonas reinhardii

Chlamydomonas reinhardii cells, after a period of dark anaerobic adaptation, evolve H(2) not only in the dark but also in the light. Our results show that high irradiances impair prolonged H(2) evolution, while under low irradiances or darkness H(2) evolution proceeds for more than 50 hours. NO(3) (-) and NO(2) (-) suppress H(2) evolution both in the dark or under low irradiance. Apparently the cells prefer these oxidized nitrogen sources to protons as electron acceptors, since both NO(3) (-) and NO(2) (-) become reduced to NH(4) (+), which is excreted to the culture medium in high amounts. H(2) evolution started once these oxidized anions were largely depleted from the medium. Moreover, H(2) evolution was consistently associated with NH(4) (+) excretion even if NH(4) (+) was already present in high amounts in the medium. This observation indicates that the cells utilize not only their carbohydrate but also their protein reserves as sources of reducing power for H(2) evolution. This conclusion was supported by the observation that when nitrogen-starved cells were made anaerobic in a nitrogen-free medium, they not only evolved H(2) at very high rates but excreted concomitantly NH(4) (+) up to concentrations in the millimolar range.

Synthesis and turnover of cell body-agglutinin as a pool of flagellar surface-agglutinin inChlamydomonas reinhardii gamete

Chlamydomonas reinhardii cells were broken in a French press and the soluble fraction was tested for agglutination activity. Deflagellated cell bodies ofmt+ andmt- gametes yielded soluble fractions that were able to isoagglutinate gametes of the opposite mating type. When the wild-type gametes of opposite mating types were mixed, the cell body-agglutinins were used up during flagellar agglutination and subsequent cell fusion. When thefus mt+ andmt- gametes agglutinated without successive fusion, the amount of cell body-agglutinins sharply decreased, then increased and reached the premixing level: the recovery was blocked by cycloheximide. When cells were treated with EDTA or trypsin, the cell body-agglutinins as well as flagellar surface-agglutinins were completely lost without apparent loss of motility. The EDTA extract contained the same amount of agglutinins as observed in the cell bodies before extraction, and this amount was about 100 times higher than that in the EDTA extract of isolated flagella. By the addition of trypsin inhibitor, the trypsinized gametes resynthesized the cell body-agglutinins. The process was sensitive to cycloheximide in both mating type gametes and to tunicamycin inmt+ gametes.

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.

Recombination within the inverted repeat sequences of the Chlamydomonas reinhardii chloroplast genome produces two orientation isomers

Two orientations of the Chlamydomonas reinhardii chloroplast (ct) genome are shown to be produced by recombination within the inverted repeat (IR) sequences that separate the two single copy (SC) regions. SC region 1 is bounded on its two ends by EeoRI restriction endonuclease fragments of 3.2 and 4.7 kilobase pairs (kb) (Rochaix 1978). The 3.2 kb EeoRI fragment overlaps a 51.3 kb BglII fragment spanning one of the 19.7 kb IR sequences, and the 4.7 kb EcoRI fragment overlaps a 42.1 kb BglII fragment spanning the other 19.7 kb IR sequence. We have shown by hybridization analysis that the 3.2 kb fragment also overlaps a BgIII fragment with a predicted size of 52.3 kb, and that the 4.7 kb fragment also overlaps a BglII fragment of a predicted size of 41.1 kb. The second set of BglII fragments are isomers produced by recombination localized to the IR region. The two isomers are present in approximately equimolar ratio. Knowledge of the isomeric composition of the C. reinhardii ctDNA is essential for establishing a correlation between genetic and physical maps of the ct genome.

Macromolecules released into the culture medium during the vegetative cell cycle of the unicellular green alga Chlamydomonas reinhardii.

The culture medium of growing Chlamydomonas reinhardii cells contains hydroxyproline-rich glycoproteins, which are mainly liberated during release of the zoospores from the mother-cell wall. Pulse-labelling studies with [3H]proline and [35S]methionine have been performed in order to detect the protein components released by synchronously growing cells at different stages of the cell cycle. When either [3H]proline or [35S]methionine were applied during the phase of cell growth, radioactive label appeared in the released macromolecules after a lag period of 40 min, whereas incorporation into the insoluble part of the cell wall was delayed only by 20 min. When applied at the end of the growth phase, e.g. 13 h after beginning of the illumination period, the radioactive amino acids were incorporated into the cell wall, but radioactive labelling of macromolecules released into the culture medium could not be detected before the zoospores were liberated from the mother-cell wall. Maximal incorporation of [3H]proline and [35S]methionine into the insoluble part of the cell wall was observed during cell division, but essentially no radioactively-labelled macromolecules were released into the culture medium during this time period. Analysis of the macromolecules, which were liberated during cell enlargement, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed distinct radioactive bands, which were differentially labelled with [3H]proline and [35S]methionine. Among the macromolecules released into the culture medium during cell growth, a component of an apparent Mr 35 000 was preferentially labelled with [3H]proline. This component was also detected after labelling with [35S]methionine, but components of an apparently higher Mr were more prominent after labelling with [35S]methionine. Macromolecules released during the cell-enlargement period of synchronously growing cultures in the presence of [3H]proline contained radioactively-labelled hydroxyproline in addition to proline. These results show that, during cell-wall growth, specific protein components are released into the culture medium and that at least one of these components contains large amounts of proline and hydroxyproline. At least some of these macromolecules seem to be constituents of the cell wall, because during pulse-chase experiments radioactively-labelled macromolecules appeared in the culture medium mainly during the time period when the specific radioactivity of the insoluble inner-cell-wall layer decreased.

Light-Dependent Oxygen Uptake, Glycolate, and Ammonia Release in l-Methionine Sulfoximine-Treated Chlamydomonas

Glycolate and ammonia excretion plus oxygen exchanges were measured in the light in l-methionine-dl-sulfoximine treated air-grown Chlamydomonas reinhardii. At saturating CO(2) (between 600 and 700 microliters per liter CO(2)) neither glycolate nor ammonia were excreted, whereas at the CO(2) compensation concentration (<10 microliters per liter CO(2)) treated algae excreted both glycolate and ammonia at rates of 37 and 59 nanomoles per minute per milligram chlorophyll, respectively. From the excretion values we calculate the amount of O(2) consumed through the glycolate pathway. The calculated value was not significantly different from the component of O(2) uptake sensitive to CO(2) obtained from the difference between O(2) uptake of the CO(2) compensation point and at saturating CO(2). This component was about 40% of stationary O(2) uptake measured at the CO(2) compensation point. From these data we conclude that glyoxylate decarboxylation in air-grown Chlamydomonas represents a minor pathway of metabolism even in conditions where amino donors are deficient and that processes other than glycolate pathway are responsible for the O(2) uptake insensitive to CO(2).

–SH and S–S involvement in Chlamydomonas mating

AbstractReagents that block or cross‐link sulfhydryl (–SH) groups and those that reduce disulfide (S–S) bonds have been tested for their effects on mating in Chlamydomonas reinhardii. Wild‐type (wt) gametes of mating type + (mt+) and mt−, and a fusion‐defective mt− mutant, gam‐11, were studied. Differential sensitivities of mt+ vs mt− and of wt mt− vs gam‐11 mt− were analyzed. Concentrations of reagents that did not disrupt flagellar agglutination, the first stage of the mating reaction, were generally used. Pretreatment of mt+ gametes with the membrane permeable –SH reducing agent dithiothreitol (DTT) inhibits flagellar sexual signaling at concentrations that do not inhibit any part of the mating reaction of mt− gametes. Wt mt− is more sensitive than wt mt+ to inhibition by low concentrations of p‐chloromercuribenzoate sulfonate (pCMBS), an organic mercurial. The membrane‐impermeable reducing agent, reduced glutathione (GSH), also preferentially inhibits wt mt−. Gam‐11 mt−, a fusion‐defective mutant, which has been used to study the sensitivity of the adhesion of the plasma membrane‐associated mating structures, is less sensitive to GSH and pCMBS inhibition that is wt mt−. DDT and pCMBS cause an increase in mating structure adhesion in pretreated gam‐11. The differential inhibition of pair and group formation during gam‐11 × wt mt+ matings has suggested a possible mechanism for mating structure adhesion.

A Chlamydomonas reinhardii mutant with catalytically and structurally altered ribulose-5-phosphate kinase

The biochemical lesion in a light-sensitive, acetate-requiring Chlamydomonas mutant was identified. This strain, designated rpk, exhibited photosynthetic rates less than 3% of the wild-type. Analysis of photosynthetic products by high-performance liquid chromatography demonstrated an accumulation of (14)C label in pentose and hexose monophosphates. After 1 min of photosynthesis in (14)CO2 these intermediates comprised 27.5% of the label in the mutant compared with 8% in the wild-type. The mutant pheno-type was caused by a 20-fold reduction in ribulose-5-phosphate (Ru5P)-kinase (EC 2.7.1.19) activity. The mutant exhibited wild-type levels of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) and transketolase (EC 2.2.1.1) indicating that the mutation specifically affected Ru5P kinase. In a cross of the mutant with the wild-type, tetrad progeny segregated in a Mendelian fashion (1:1) and light-sensitivity cosegregated with reduced Ru5P-kinase activity and an acetate requirement for growth. Almost normal levels of Ru5P-kinase protein were detected in the mutant by probing nitrocellulose replicas of sodium dodecylsulfate-polyacrylamide gels with anti-Ru5P-kinase antibody. The subunit size of the mutant enzyme, 42 kDa, was identical to that of the wild-type. Isoelectric focusing of the native protein determined that the mutant protein was altered, exhibiting a more acidic isoelectric point than the wild-type protein. Thus, the molecular basis for the lesion affecting Ru5P-kinase activity in mutant rpk is a charge alteration which results in a partially impaired enzyme.

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.

Chlamydomonas reinhardii gene for the 32 000 mol. wt. protein of photosystem II contains four large introns and is located entirely within the chloroplast inverted repeat

The chloroplast psbA gene from the green unicellular alga Chlamydomonas reinhardii has been localized, cloned and sequenced. This gene codes for the rapidly-labeled 32-kd protein of photosystem II, also identified as as herbicide-binding protein. Unlike psbA in higher plants which is found in the large single copy region of the chloroplast genome and is uninterrupted, psbA in C. reinhardii is located entirely within the inverted repeat, hence present in two identical copies per circular chloroplast genome, and contains four large introns. These introns range from 1.1 to 1.8 kb in size and fall into the category of Group I introns. Two of the introns contain open reading frames which are in-frame with the preceding exon sequences. We present the nucleotide sequence for the C. reinhardii psbA 5'-and 3' -flanking sequences, the coding region contained in five exons and the deduced amino acid sequence. The algal gene codes for a protein of 352 amino acid residues which is 95% homologous, excluding the last eight amino acid residues, with the higher plant protein.

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.