Alteration of Dark Respiration and Reduction of Phototrophic Growth in a Mitochondrial DNA Deletion Mutant of Chlamydomonas Lacking cob, nd4, and the 3' End of nd5

Correspondence to: René F. Matagne, rf.matagne{at}ulg.ac.be (E-mail), 324-3663840 (fax)

	ABSTRACT

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We describe here a new type of mitochondrial mutation (dum24; for dark uniparental minus inheritance) of the unicellular photosynthetic alga Chlamydomonas reinhardtii. The mutant fails to grow under heterotrophic conditions and displays reduced growth under both photoautotrophic and mixotrophic conditions. In reciprocal crosses between mutant and wild-type cells, the meiotic progeny only inherit the phenotype of the mating-type minus parent, indicating that the dum24 mutation exclusively affects the mitochondrial genome. Digestion with various restriction enzymes followed by DNA gel blot hybridizations with specific probes demonstrated that dum24 cells contain four types of altered mitochondrial genomes: deleted monomers lacking cob, nd4, and the 3' end of the nd5 gene; deleted monomers deprived of cob, nd4, nd5, and the 5' end of the cox1 coding sequence; and two types of dimers produced by end-to-end fusions between monomers similarly or differently deleted. Due to these mitochondrial DNA alterations, complex I activity, the cytochrome pathway of respiration, and presumably, the three phosphorylation sites associated with these enzyme activities are lacking in the mutant. The low respiratory rate of the dum24 cells results from the activities of rotenone-resistant NADH dehydrogenase, complex II, and alternative oxidase, with none of these enzymes being coupled to ATP production. To our knowledge, this type of mitochondrial mutation has never been described for photosynthetic organisms or more generally for obligate aerobes.

	INTRODUCTION

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Few mitochondrial mutations affecting components of the mitochondrial electron transport chain and causing an abnormal phenotype have been characterized in higher plants. Certain mutants of maize, designated nonchromosomal stripe (NCS), feature poor growth, decreased yield, pale striping of leaves, and strict maternal inheritance (Coe 1983 ; Newton et al. 1989 ). The mutations correspond to deletions in mitochondrial genes encoding subunit 2 of cytochrome c oxidase or subunits 4 and 7 of complex I (Lauer et al. 1990 ; Newton et al. 1990 ; Marienfeld and Newton 1994 ). All NCS mutants are heteroplasmic and still contain at least 50% of nonmutated mitochondrial DNA copies. The NCS plants segregate somatically defective sectors, and only these pale green sectors are homoplasmic for the mitochondrial mutations. More recently, cytoplasmic male-sterile mutants I and II of Nicotiana sylvestris with reduced respiration and breakdown of complex I activity have been characterized (Pla et al. 1995 ; Gutierres et al. 1997 ). Both are near homoplasmic for a deletion in the nd7 gene (subunit 7 of complex I), indicating that the lack of detectable amounts of the ND7 polypeptide is not lethal to the plant.

The unicellular green alga Chlamydomonas reinhardtii can be used as a model system to investigate mitochondrial gene function in plant cells. Its small 15.8-kb linear mitochondrial genome has been totally sequenced, and all of the genes residing in the organelle have been identified (Vahrenholz et al. 1993 ; Figure 1). Moreover, several mutations altering the mitochondrial genes encoding apocytochrome b (cob gene) or subunit 1 of cytochrome c oxidase (cox1 gene) have been characterized (Matagne et al. 1989 ; Dorthu et al. 1992 ; Randolph-Anderson et al. 1993 ; Colin et al. 1995 ). Mutant cells are homoplasmic for the mutations and do not retain any DNA copies carrying the corresponding functional gene. The mutants lack the cytochrome pathway of the mitochondrial electron transport chain, but their respiratory activity is partially maintained via the alternative pathway of respiration. Phenotypically, the mutants have lost their capacity to grow under heterotrophic conditions (darkness and the addition of acetate as a reduced carbon source), whereas their photoautotrophic growth is barely affected (reviewed in Remacle and Matagne 1998 ).

View larger version (11K): [in this window] [in a new window]   Figure 1. Functional Map of the 15.8-kb Linear Mitochondrial Genome of Chlamydomonas. Arrows indicate the direction of transcription. The eight genes encoding polypeptides include cob (apocytochrome b), cox1 (cytochrome c oxidase subunit 1), nd1, nd2, nd4, nd5, nd6 (subunits of the NADH:ubiquinone oxidoreductase), and RTL (reverse transcriptase–like protein). L1 through L8 and S1 through S4 represent modules encoding segments of rRNA (large and small subunits, respectively). W, Q, and M represent tRNAs for Try, Gln, and Met, respectively. B, C, H, S, and X represent the BamHI, ClaI, HindIII, SstI, and XbaI restriction sites, respectively. P2, P3, P4, P6, and P8 represent mitochondrial DNA fragments contained in the molecular probes. ND4-3, ND4-4, ND5-1, ND5-2, ND5-4, II, VII, IX, 747, and 749 are the primers used for PCR amplification. The dotted lines correspond to the two types of deletions (del) identified in dum24 (see the text for details).

Mutants altered in one of the nd mitochondrial genes that encode subunits of rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) have never been isolated from Chlamydomonas. Here, we describe a new type of deletion affecting the mitochondrial genome of the alga. Certain copies of organelle DNA lack cob, nd4, and the 3' end of the nd5 gene, whereas others contain a much larger deletion encompassing cob, nd4, nd5, and part of the cox1 sequence. The mutant, which grows at slow rate when cultivated in the light, lacks the cyanide-sensitive cytochrome pathway of respiration and also the activity associated with complex I. Because mitochondrial phosphorylation is coupled to complex I, III, and IV activities, this finding demonstrates that photosynthetically active Chlamydomonas cells can survive in the absence of mitochondrial ATP production associated with the activities of these complexes.

	RESULTS

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Phenotypic and Genetic Analyses
Several clones unable to grow under heterotrophic conditions (dk^- phenotype) were selected after mutagenic treatment of mating-type minus (mt^-) wild-type cells with acriflavine. One clone (hereafter called dum24 for dark uniparental minus inheritance) displaying an extreme phenotype when cultivated in the light was retained for further analyses.

Compared with both the wild-type strain and the dum19 mutant, which lacks cytochrome c oxidase activity (Colin et al. 1995 ), the dum24 strain produced very small colonies on Tris–minimal-phosphate (TMP) and Tris–acetate–phosphate (TAP) agar plates (Table 1). The slow growth of the dum24 mutant under photoautotrophic (TMP) conditions was confirmed in aerated liquid culture (data not shown).

To identify the genome carrying the dum24 mutation, we performed a genetic analysis. The dum24 mt^- cells were crossed to wild-type mt⁺ cells, and the meiotic progeny were analyzed for their ability to grow in both the dark and the light. The 80 progeny clones analyzed inherited the dk^- phenotype of the mt^- parent. A dk^- mt⁺ meiotic progeny clone was selected and crossed to mt^- wild-type cells. In this case, all of the meiotic progeny were phenotypically wild type. This exclusive transmission of the phenotype from the mt^- parent is typical of the transmission pattern observed for mitochondrial mutations (Matagne et al. 1989 ; Bennoun et al. 1991 ; Dorthu et al. 1992 ; Colin et al. 1995 ); thus, it indicates that the dum24 mutation resides in the genome of this organelle. The new mutant was named dum24.

Molecular Characterization of the dum24 Mitochondrial Genome
In Chlamydomonas, many of the mutants induced by acriflavine possess a terminal deletion encompassing the cob gene of the mitochondrial genome and lack complex III activity (Dorthu et al. 1992 ). In contrast to wild-type cells, which contain a single type of mitochondrial genome, the deletion mutants are heteroplasmons in which deleted monomers and dimers resulting from the fusion of two monomers by their deleted ends coexist (Dorthu et al. 1992 ; Colin et al. 1995 ). The deletion mutants also share the property of mitotically segregating a majority of viable cells and a few cells that divide eight or nine times to produce "minute" lethal colonies (Dorthu et al. 1992 ; Randolph-Anderson et al. 1993 ).

When cultivated phototrophically on agar plates, the dum24 mutant also produced 0.3 to 1% of the small lethal colonies, which suggests that this strain may contain deleted mitochondrial DNA molecules. DNA gel blot hybridizations with specific probes were therefore performed to analyze the mitochondrial genome of the mutant. When nonrestricted total cell DNA was probed with P2 (see Figure 1), two types of mitochondrial DNA copies were detected (Figure 2A): molecules whose lengths (~12 kb) were less than that of the wild-type mitochondrial genome (15.8 kb) and molecules of greater length (~24 kb). DNA was then digested with various restriction enzymes and hybridized with different probes to determine the exact length and location of the deletion and to identify the nature of the large-sized mitochondrial DNA molecules. After digestion with ClaI and hybridization with P3 or P4 (Figure 1), no fragment was detected in dum24, whereas a fragment of the expected length (2.8 kb) was obtained from the wild type (Table 2). A deletion including cob and at least the major part of nd4 is therefore present in the mutant. To determine whether molecules carrying cob and nd4 sequences are present in substoichiometric amounts in the mutant cells, we performed polymerase chain reaction (PCR) analyses using the primers 747 and 749 to amplify the cob region, and the primers ND4-3 and ND4-4 to amplify the nd4 region (Figure 1). No amplification product was obtained using DNA from dum24, whereas fragments of expected length were produced from the wild-type DNA (data not shown).

View larger version (41K): [in this window] [in a new window]   Figure 2. Hybridization Patterns Obtained Using the P2 Probe from Undigested Total Cell DNA and DNA Digested with ClaI. (A) Undigested total DNA. (B) DNA digested with ClaI. Lanes 1 contain wild-type DNA, and lanes 2 contain dum24 DNA. Numbers at left indicate the lengths of DNA molecular markers in kilobases.

The following analyses performed with DNA from mutant cells showed a more complex pattern of alteration than did a single deletion of the cob and nd4 genes. When DNA from dum24 was restricted with BamHI and hybridized with P2, the 10.4-kb fragment typical of the wild type (Figure 1) was not detected. Instead, two short fragments (5.2 and 7.0 kb) and two long fragments (12.2 and 14.0 kb) were obtained (Table 2), suggesting that two types of deleted monomers and two types of dimers coexist in dum24. The same conclusion could be drawn from the hybridization patterns observed after digestion with ClaI or SstI and probing with P2 (Table 2 and Figure 2B). After cleavage with SstI, the 10.3-kb fragment present in the wild type was retained in dum24, indicating that the part of the genome located to the right of the SstI site was unaltered in the mutant (cf. Table 2 and Figure 1).

From the complete set of data presented in Table 2, it can be deduced that four types of mitochondrial DNA molecules coexist in dum24. Two differently deleted monomers (~12.4 and 10.7 kb, respectively) are present: the first type of deletion (3.4 kb) encompasses the telomere, cob, nd4, and perhaps the 3' end of nd5, whereas the second (5.1 kb) extends from the telomere to the 5' end of cox1 (Figure 1). Two types of dimeric molecules are also present: symmetrical molecules (24.8 kb), resulting from the junction between the two large monomers, and asymmetrical molecules (23.1 kb), resulting from the union of the monomer carrying the 3.4-kb deletion with the monomer carrying the 5.1-kb deletion. This model was confirmed by several other hybridization experiments using the probes P6 and P8 after DNA restriction by NcoI and PstI (data not shown). However, all of the fragments corresponding to the four types of mitochondrial DNA molecules were not always detected simultaneously on the same blot, but the fragments characteristic of the asymmetrical dimer were always seen. For unknown reasons, restriction patterns compatible with the presence of a symmetrical dimer resulting from the fusion of the two smaller monomers (with a 5.1-kb deletion) were never obtained.

The presence of asymmetrical dimers was confirmed by PCR amplification: using the primers ND5-1 and IX (Figure 1 and Figure 3), we obtained a 1-kb fragment from dum24 total DNA. The junction between the two unequally deleted monomers was identified by sequencing the fragment. Figure 3 shows that the largest monomer ends 21 nucleotides upstream of the stop codon of nd5, whereas the smallest monomer has lost cob, nd4, nd5, and the first 67 nucleotides of the cox1 coding sequence. Interestingly, the junction occurs in an 11-bp segment common to the cox1 and nd5 genes.

View larger version (10K): [in this window] [in a new window]   Figure 3. Junction Sequence between nd5 and cox1 in the Asymmetrical Dimers from dum24. The nd5 gene (encoding 546 amino acids; Boer and Gray 1986 ) is deprived of its last 21 nucleotides, and the cox1 gene (encoding 505 amino acids; Vahrenholz et al. 1985 ) is deprived of its first 67 nucleotides. The numbering of the nucleotides of the nd5 and cox1 sequences starts at the adenine of the ATG initiation codon. The 11-bp segment within brackets shows the common junction between the cox1 and nd5 genes. Below, the positions of the primers (ND5-1 and IX) used to amplify the sequence are shown. The vertical line corresponds to the junction segment between the large (left) and the small (right) monomers. The two long arrows indicate the direction of transcription. (del) denotes the genes that are partially deleted.

To determine whether all mitochondrial monomers and dimers from dum24 had lost the 3' end of nd5, we performed a PCR amplification by using the ND5-1 primer and a primer (ND5-4) corresponding to the last 20 nucleotides of the nd5 coding sequence (Figure 1). No PCR product was obtained, whereas a fragment of the expected size was amplified with ND5-1 and a primer located 20 nucleotides upstream of ND5-4 (data not shown).

Presence of Mitochondrial DNA in the Minute Colonies from dum24
As mentioned above, the dum24 mutant mitotically segregates a few cells that divide eight or nine times to produce minute lethal colonies. The phenotypically similar minute mutations induced by acriflavine and ethidium bromide are deprived of mitochondrial DNA (Gillham et al. 1987 ). In contrast, the minute colonies produced by the dum1 deletion mutant (which lacks the cob gene) possess mitochondrial genomes, but their size is reduced, with the original deletion now extending at least into the adjacent nd4 gene (Randolph-Anderson et al. 1993 ).

To analyze the mitochondrial DNA of the minute lethal colonies produced from dum24, we undertook PCR amplifications by using primers II and VII (Figure 1) to amplify a 0.34-kb cox1 segment present in all types of monomers and dimers and primers ND5-1 and IX to amplify the 1-kb fragment specific to the asymmetrical dimers (Figure 3). Figure 4 shows the results of the PCR analyses performed with DNAs extracted from pools of viable and minute colonies. Amplification products were observed in all cases, indicating that the minute colonies actually contain mitochondrial genomes. Comparing the amounts of the PCR products obtained from each DNA template allowed us to draw additional conclusions. With the DNA from viable colonies, the 1-kb band characteristic of asymmetrical dimers was more intense than the 0.34-kb band, which could have been due to the longer length of the fragment and/or to different primer efficiencies under the PCR conditions used. The opposite was found for the DNA from minute colonies. From the data of Figure 4, it can be concluded that the relative amounts of asymmetrical dimers were much lower in minute lethal colonies than in viable dum24 colonies.

View larger version (59K): [in this window] [in a new window]   Figure 4. Agarose Gel Electrophoresis of PCR Products Obtained with dum24 DNA. dum24 DNA was obtained from viable colonies (3 µL; lanes 1 and 2) and minute colonies (30 µL; lanes 3 and 4) (see Methods). Primer pairs are II and VII (lanes 1 and 3) and ND5-1 and IX (lanes 2 and 4). Numbers at left indicate the lengths of DNA molecular markers in kilobases.

Respiratory Chain and Complex I Activities
In Chlamydomonas, as in many microeukaryotes and in higher plants, the oxidation of ubiquinol occurs via two specific routes (Figure 5). Aside from the classic cytochrome pathway of respiration (complexes III and IV), an alternative pathway insensitive to KCN but sensitive to salicylhydroxamic acid (SHAM) branches from the main chain at the level of the ubiquinone pool (Wiseman et al. 1977 ; Husic and Tolbert 1987 ; Matagne et al. 1989 ). Because the alternative pathway is not associated with proton translocation, the oxidation of one NADH molecule via this route generates only one ATP molecule, whereas three ATP molecules are produced when oxidation occurs via the cytochrome pathway (Figure 5). The alternative pathway of respiration corresponds to a single enzyme, the alternative oxidase. A 36-kD protein cross-reacting with a monoclonal antibody raised against the alternative oxidase from voodoo lily has recently been detected in Chlamydomonas (Derzaph and Weger 1996 ). Two nuclear genes encoding two different forms of the alternative oxidase from Chlamydomonas have been identified recently (Dinant et al. 1998 ).

View larger version (10K): [in this window] [in a new window]   Figure 5. Schematic Representation of the Chlamydomonas Respiratory Chain. I to IV indicate respiratory complexes of the main chain. The asterisks indicate coupled oxidative phosphorylations. Arrows denote electron transfer. UQ, ubiquinone.

dum24 cells were grown under mixotrophic conditions (light, plus acetate as a carbon source), and their dark respiration activity was measured in the absence or presence of respiratory inhibitors. Cells from the wild type and from the dum19 mutant deprived of cytochrome c oxidase activity (Colin et al. 1995 ) were used as controls. The total respiratory rate of dum24 cells was low and represented only 22 and 42% of that found with wild-type and dum19 cells, respectively (Table 3).

The addition of KCN (an inhibitor of cytochrome c oxidase) reduced the dark respiration of wild-type cells to 24%, whereas the respiratory activity of the two mutants was affected very little by cyanide. The further addition of SHAM reduced but did not totally abolish the oxygen consumption in any genotype. When SHAM was first added, dark respiration did not change much in the wild type but strongly decreased in dum19 and dum24. After the addition of cyanide, the sensitivity of wild-type respiration and the insensitivity of mutant respiration to that inhibitor were confirmed. Thus, similar to the previously characterized dum19 mutant (Colin et al. 1995 ), dum24 lost the cytochrome pathway of respiration but still possessed a functional SHAM-sensitive alternative pathway.

The activity of complex I in dum24 cells could be checked by using rotenone, a specific inhibitor of that complex. The addition of rotenone induced a reduction of 69 and 50% of the dark cell respiration in wild-type and dum19 strains, respectively, whereas no effect was observed for dum24 (Table 3). This indicates that in contrast to the two other strains, the dum24 mutant had lost the rotenone-sensitive activity associated with complex I. The absence of complex I activity in dum24 was confirmed by NADH dehydrogenase assays performed with membrane-enriched fractions, using duroquinone as an electron acceptor (Table 4). Despite the marked experimental variability observed for each genotype, probably due to the variable quality of the membrane preparations, measurements of enzyme activities showed that in contrast to the wild type, the dum24 mutant had lost the rotenone-sensitive NADH dehydrogenase activity associated with complex I. In the three experiments with wild-type extracts, complex I activity represented 52 to 66% of the activity measured before the addition of rotenone. Rotenone-resistant NADH dehydrogenase activities detected in both wild-type and dum24 enzyme preparations seemed to be in the same range. From the data of Table 3 and Table 4, we conclude that both complex I activity and the cytochrome pathway of respiration are absent in dum24 mutant cells.

	DISCUSSION

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In Chlamydomonas, the lack of functional cob or cox1 gene results in the loss of the cytochrome pathway of respiration and prevents cell division under heterotrophic conditions but has little effect on photoautotrophic growth (Dorthu et al. 1992 ; Colin et al. 1995 ; this study). The dum24 strain described here represents a new type of mitochondrial mutant that, in addition to being unable to grow heterotrophically, displays markedly reduced growth when cultivated in the light. The addition of acetate as a carbon source does not improve the growth capacity of this mutant.

Our molecular analyses led us to conclude that the dum24 mitochondrial genome is composed of four different types of DNA molecules, all containing deletions much larger than those detected in mitochondrial mutants previously characterized for the same organism. Indeed, the deletions described to date range from 0.7 to 1.7 kb and exclusively affect the left telomeric region and part or all of the cob sequence (Dorthu et al. 1992 ; Randolph-Anderson et al. 1993 ; Remacle and Matagne 1998 ). In dum24 cells, two types of deleted monomers have been identified: monomers with a 3.4-kb left terminal deletion, extending from the telomere to the 3' end of the nd5 coding sequence, and monomers with a 5.1-kb deletion, which encompasses the telomere, cob, nd4, nd5, and the 5' end of the cox1 coding sequence. The dum24 mutant also contains dimeric molecules that result from an end-to-end fusion of the two larger monomers (symmetrical dimers) or of two monomers differently deleted (asymmetrical dimers). Copies retaining nd4 and the 3' end of nd5 are missing. Thus, contrary to the hypothesis previously proposed by Randolph-Anderson et al. 1993 and Colin et al. 1995 , the simultaneous loss of cob, nd4, and terminal end of nd5 is not lethal to the cells.

Attempts to separate the different mitochondrial genomic forms present in dum24 by successive subclonings have failed (data not shown). The dum24 mutant is maintained as a heteroplasmon carrying both monomeric and dimeric mitochondrial DNA molecules. This property is shared by all Chlamydomonas mitochondrial deletion mutants thus far characterized (Dorthu et al. 1992 ). Such a molecular polymorphism is similar to that described for several ² mutants of yeast, in which numerous circular molecular forms corresponding to multimeric series have been identified (Lazowska and Slonimski 1976 ). It has been proposed that the coexistence of monomers and dimers in a same cell would result from illegitimate recombination events. The mechanism responsible for the joining of mitochondrial DNA segments at precisely defined sites would not require extensive sequence homology (Lazowska and Slonimski 1977 ).

The sequencing of the junction region in the asymmetrical dimer from dum24 has shown that the fusion between the two different monomers occurs in an 11-bp segment common to the nd5 and cox1 genes (Figure 3). In another deletion mutant previously described (the dum11 mutation), asymmetrical dimers resulting from the fusion of two monomers differently deleted have also been identified (Colin et al. 1995 ). In these dimers, the fusion associates the first 443 nucleotides of cob with the last 1193 nucleotides of cox1. Interestingly, analysis of the DNA sequence of the junction region in dum11 shows that the fusion takes place in a segment in which 18 of 20 successive base pairs are common to both the cob and cox1 sequences (data not shown). One can thus postulate that the formation of asymmetrical dimers in dum11 and dum24 results from recombinational events between short DNA sequences that share homology. In higher plants, recombination between very small repeats may also explain the mitochondrial gene rearrangements that cause the generation of cytoplasmic male-sterile and NCS mutants (Marienfeld and Newton 1994 ; Vedel et al. 1994 ; Newton 1995 ; Pla et al. 1995 ).

The presence of dimeric molecules with intact telomeric ends, identified in all deletion mitochondrial mutants characterized thus far in Chlamydomonas, could be related to the instability of the monomers deprived of one of their telomeric ends. Telomeric sequences from Chlamydomonas are believed to prevent end-to-end fusion and degradation of the linear mitochondrial DNA molecules (Randolph-Anderson et al. 1993 ) and to be essential for DNA replication (Vahrenholz et al. 1993 ). As mentioned in the description of the hybridization experiments, the restriction fragments characteristic of the asymmetrical dimer were always present on the blots, whereas in several cases, the fragments typical of the monomers were not detected. The monomers, however, could exist in substoichiometric amounts that are too low to be detected by hybridization. These data suggest that in dum24 cells, the different genomic forms are in unstable equilibrium and can coexist in variable amounts.

The relative instability of the dum24 mitochondrial genomes, and more generally of the mitochondrial genomes of any deletion mutant, can also account for the occasional production of cells that give rise to minute lethal colonies. As previously discussed (Dorthu et al. 1992 ), these minute nonviable colonies are similar to those induced by prolonged acriflavine or ethidium bromide treatment and deprived of mitochondrial DNA (Gillham et al. 1987 ). The minute colonies derived from dum24 were shown to contain mitochondrial DNA, with a very low amount of asymmetrical dimers relative to the entire mitochondrial genome population (Figure 4). Because the PCR amplifications were performed from pools of colonies, we could not determine whether each minute colony had a reduced amount of the asymmetrical dimers or most often lacked this type of dimeric form. Moreover, we have no information on the amount of symmetrical dimers because these DNA molecules cannot be identified by PCR. It is tempting to hypothesize, however, that the production of minute lethal colonies could be correlated with the elimination of dimeric genomes, which might be the only molecular forms able to replicate and to avoid degradation (see above). In minute colonies segregated by another deletion mutant, it was shown that the 1.7-kb deletion encompassing the cob gene had spread into the adjacent gene (Randolph-Anderson et al. 1993 ), which favors the hypothesis that linear mitochondrial DNA molecules deprived of their left telomeric ends are subject to exonuclease degradation. In this respect, the particular structure of mitochondrial telomeres (Nosek et al. 1998 ) or the presence of proteins at the termini of some linear plant mitochondrial plasmids (Kemble and Thompson 1982 ) could conceivably serve a protective function.

The data presented in Table 3 and Table 4 indicate that the absence of nd4 and the 3' end of nd5 in all dum24 mitochondrial DNA copies is correlated with the loss of complex I activity. In Neurospora crassa (Weiss et al. 1991 ), mammals (Walker 1992 ), and various higher plants (Leterme and Boutry 1993 ; Herz et al. 1994 ; Rasmusson et al. 1994 ), the mitochondrial complex I enzyme is by far the largest complex among the proton translocating enzymes of mitochondria. It comprises at least 30 different subunits, most of which are encoded by nuclear genes and imported from the cytoplasm. Despite the fact that in many organisms the primary structure of the mitochondrial DNA–encoded subunits has been determined, little is known about their functions, with the exception of the ND1 subunit, which is known to bind rotenone (Earley et al. 1987 ) and interact with ubiquinone (Friedrich et al. 1990 ).

An insight into the functional role of these subunits has come from their location within the complex I enzyme. The enzyme from N. crassa has been shown to have an L-shaped structure, with one arm buried in the inner mitochondrial membrane and the other protruding into the matrix (Hofhaus et al. 1991 ). The hydrophobic membrane arm contains all of the mitochondrial DNA–encoded subunits, whereas the matrix arm contains most of the nuclear-encoded subunits and most of the prosthetic groups involved in redox reactions between NADH and ubiquinone (Weiss et al. 1991 ). The membrane arm of the enzyme would be involved in proton translocation out of the mitochondrial matrix, thereby generating an electrochemical gradient across the inner mitochondrial membrane (Hofhaus and Attardi 1995 ).

The availability of mutations affecting mitochondrial nd genes has allowed further progress in the understanding of the functional role of mitochondrial DNA–encoded, complex I subunits. In maize, the mutant genome of the heteroplasmic NCS2 line contains a fused nd4–nd7 gene, with the fourth exon of nd4 being deleted (Marienfeld and Newton 1994 ). Transcripts from the intact nd4 and nd7 wild-type genes in the heteroplasmon are greatly reduced and could account for the decrease of complex I activity in the mutant. However, the rather modest reduction recorded in complex I activity with respect to the very low amount of normal nd4 and nd7 transcripts does not permit one to determine whether the ND4 and ND7 subunits are necessarily required for complex I function. In cytoplasmic male-sterile mutants of tobacco, deletion of the mitochondrial nd7 gene results in the lack of ND7, ND9, and the nuclear-encoded, 38-kD subunit (Pla et al. 1995 ; Gutierres et al. 1997 ). The slight rotenone inhibition of the NADH dehydrogenase activity, however, suggests that complex I partially functions in these cytoplasmic male-sterile mutants (Gutierres et al. 1997 ) and that the three above-mentioned subunits are not absolutely required for the activity of complex I.

In various homoplasmic mutants of human cell lines, the ND4 subunit is missing due to frameshift mutations in the corresponding gene (Hofhaus and Attardi 1993 , Hofhaus and Attardi 1995 ). The mutants exhibit a total loss of complex I activity and do not assemble the mitochondrial DNA–encoded subunits. Another mutant exhibiting a frameshift mutation in the nd5 gene, in a near-homoplasmic form, results in the absence of the ND5 subunit. The mutant is almost totally deficient in complex I activity; however, the capacity to assemble the mitochondrial DNA–encoded subunits is preserved (Hofhaus and Attardi 1995 ).

In the dum24 mutant described here, the loss of the nd4 gene and the deletion of the 3' end terminal sequence of nd5 coupled with the absence of rotenone-sensitive NADH dehydrogenase activity indicate that in Chlamydomonas, at least one of these genes is essential for complex I activity. Moreover, our results show that dum24 mutant cells retain some dark respiration capacity and are still capable of oxidizing NADH via a rotenone-resistant NADH dehydrogenase. As pointed out by Douce and Neuburger 1989 , Siedow 1995 , and Soole and Menz 1995 , plant mitochondria possess, in addition to complex I, at least three other NADH dehydrogenases located on the inner and outer faces of the internal mitochondrial membrane and on the outer face of the external membrane.

Because an active complex I and cytochrome pathway of respiration are missing in dum24 cells, mitochondrial electron transfer must occur through the activities of rotenone-resistant NADH dehydrogenase, complex II, and alternative oxidase (Figure 4). None of these is coupled to oxidative phosphorylation. To our knowledge, this type of mitochondrial mutation has never been described for any other obligate aerobe. The viability of dum24 cells in the light demonstrates that the energy required for growth necessarily results solely from the photosynthetic activity. The slow growth of the mutant, however, indicates that the mitochondrial functions lost in dum24 play an important role in the regulation of metabolic functions related to photosynthesis.

	METHODS

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Plant Materials, Culture Conditions, and Growth Analysis
Wild-type strains, mating-type plus (mt⁺), and mating-type minus (mt^-) were derived from strain 137c of Chlamydomonas reinhardtii. dum19 is a mutant strain that lacks cytochrome c oxidase (complex IV) activity as a consequence of a frameshift mutation in the mitochondrial cox1 gene (Colin et al. 1995 ).

The cells were grown in liquid medium or on agar plates (15 g/L Gibco agar), under cool-white fluorescent light (45 µmol m^-2 sec^-1), or in the dark at 25°C. Tris–minimal-phosphate (TMP) and Tris–acetate–phosphate (TAP) media are as described previously (Gorman and Levine 1965 ). A nitrogen-free minimal medium was also used for zygote maturation (VanWinckle-Swift, 1977).

The growth curves of cells in liquid cultures were established from counts performed with a ZF Coulter counter (Coulter Electronics Ltd., Harpenden, UK). The sizes of the colonies produced on the agar plates were measured on digital pictures taken with a 1 CCD JVC camera (Yokohama, Japan) by using the SigmaScan Pro 4 image analysis software (Jandel Scientific, San Rafael, CA).

Mutagenesis
Cells of the mt^- wild-type strain were grown for 3 days under a 12-hr-light and 12-hr-dark regime in TMP liquid medium containing 6 µg/mL acriflavine (Sigma). After washing, samples of 10³ cells were plated on TAP agar medium and incubated under continuous light. Obligate phototrophic mutants (dk^- phenotype; unable to grow in the dark) were detected as described previously (Matagne et al. 1989 ).

Genetic Analysis
Zygotes were matured for 3 to 4 days under continuous light on nitrogen-free minimal agar plates. After maturation, blocks of agar carrying ~50 zygotes were transferred to fresh TMP agar plates and treated for 30 sec with chloroform vapors. Germination was induced by exposure to light and nitrogen restoration. After 10 days of culture, 80 meiotic clones were randomly sampled, plated after dilution on TAP agar plates, and incubated in the light or in the dark to determine their phenotype.

Whole-Cell Respiration
Whole cells from a mixotrophic culture were harvested by centrifugation and resuspended in TAP medium. Their respiratory rate was measured in the dark at 25°C by using a Clark-type O₂ electrode (Hansatech, King's Lynn, UK). Measurements were made with 5 x 10⁷ cells for the wild-type strain and 10⁸ cells for the dum19 and dum24 mutant strains in a total volume of 1.5 mL. For the inhibition studies, 1.3 mM KCN, 2.6 mM salicylhydroxamic acid (SHAM), and 100 µM rotenone were used.

Measurement of NADH:Ubiquinone Oxidoreductase Activity
For Chlamydomonas, preparation of partially purified mitochondria requires the use of wall-less mutant cells as a starting material (Eriksson et al. 1995 ). Because a wall-less dum24 strain was not available for this study, NADH:ubiquinone oxidoreductase activity was assayed on membrane-enriched fractions by using duroquinone as an electron acceptor (Leterme and Boutry 1993 ). The cells were cultured under light in 300 mL of TAP medium and collected by centrifugation (700g for 10 min) at late-exponential phase. They were resuspended in 2 to 3 mL of MET buffer (280 mM mannitol, 100 µM EDTA, 10 mM Tris-HCl, and 0.1% BSA, pH 7.0), and then disrupted by sonication (2 x 30 sec; Vibra Cell Sonicator, Danbury, CT). The membrane fraction was pelleted by centrifugation (20,000g for 15 min) and resuspended in 1 mL of MET buffer. Thirty to fifty microliters of the membrane fraction was added to the assay buffer (20 mM Tris-HCl, pH 8.0, 100 µM NADH, and 100 µM duroquinone) in a final volume of 1 mL. Enzyme activity was monitored by recording NADH oxidation at 340 nm, using the extinction coefficient ₃₄₀ = 6.22 mM^-1 cm^-1. Rotenone-sensitive NADH:ubiquinone oxidoreductase activity (complex I) was determined by adding rotenone at a final concentration of 10 µM in the assay mixture.

Protein content was determined according to the method of Bradford 1976 .

Hybridization Experiments
Chlamydomonas total-cell DNA was prepared according to the rapid procedure of Newman et al. 1990 . After digestion with restriction enzymes, the DNA fragments were separated by electrophoresis on 0.8% agarose gels and then blotted to Hybond N membranes (Amersham) and hybridized using digoxigenin-labeled probes according to the procedure recommended by the manufacturer (Boehringer Mannheim). To characterize physically the mitochondrial DNA of the dum24 mutant, we used five molecular probes (Figure 1): P2, P3, P4 (Dorthu et al. 1992 ), P6 (pUC19 plasmid containing a 1.6-kb HindIII-XbaI fragment), and P8 (pUC19 plasmid containing the entire cox1 gene).

Polymerase Chain Reaction Amplifications
Amplifications by the polymerase chain reaction (PCR) were performed with 5 to 10 ng of total DNA from the wild-type or the mutant strains. PCR amplifications with 100 ng of total DNA from dum24 were also performed. The reaction mixture containing DNA, 5 pmol of each primer, 200 µM deoxynucleotide triphosphates, and 1 unit of Taq polymerase (Boehringer Mannheim) was subjected to 30 cycles (94°C for 1 min; primer annealing temperature for 1 min; 72°C for 1 min) of amplification, with a final extension step of 7 min at 72°C by using a Techne GeneE thermal cycler (Techne Ltd., Cambridge, UK). The oligonucleotide primers used for the amplifications (Figure 1) were the following: 745, 746, 747, and 749 (Colin et al. 1995 ); ND4-3 (5'-GAAAAGAAGACTGGAAAAAGAATC-3'), which primes within the nd4–nd5 intergenic region; ND4-4 (5'-TAGGCTACCAAATGAGTG-TT-3'), which primes at the 5' end of the cob sequence; ND5-1 (5'-CACTGCTGGTGTATACTTGC-3') and ND5-2 (5'-AGTAGTAT T-AT TGCTAT TGGC-3'), which prime with the nd5 sequence; ND5-4 (5'-TAAGCACGCAAAT TACCAACGC-3'), which primes with the last 20 nucleotides of the nd5 coding sequence; and IX (5'-AAAACCACCGAATAGGGC-3'), which primes with the cox1 sequence downstream of base 214.

For the PCR analysis of the minute lethal colonies, 25 minute colonies (~1 to 3 x 10² cells per colony) were picked using a dissecting microscope and pooled into a microcentrifuge tube containing 40 µL of PCR buffer. At the same time, 10 viable colonies (~10⁵ cells per colony) were pooled and suspended in 100 µL of PCR buffer. DNA from each type of colony was extracted by treating cells with proteinase K, as described previously (Randolph-Anderson et al. 1993 ). DNA (10 µL from minute colonies and 5 µL from viable colonies) was then amplified by PCR in a total volume of 50 µL by using the following pairs of primers: II (5'-ACTGGTTGGACCGCTTAT-3') and VII (5'-GGTGACCAAAGAACCAGA-3') for the cox1 fragment (Figure 1); and ND5-1 and XI for the fragment characteristic of the asymmetrical dimer (see Results and Figure 3). Thirty microliters of the PCR reaction mixture for the minute colonies and 3 µL of the PCR reaction mixture for the viable colonies were loaded onto a 0.8% agarose gel. After electrophoresis, the PCR products were stained with ethidium bromide.

Sequence Analysis
DNA fragments from PCR amplifications were cloned in the pGEM-T vector (Promega) by using the T4 DNA ligase supplied with the system. Two clones from independent amplification reactions were sequenced on both strands by using the dideoxynucleotide chain termination method of Sanger et al. 1977 , with universal and reverse primers. The –³⁵S-dATP was purchased from Amersham and the T7 sequencing kit from Pharmacia Biotechnology.

	ACKNOWLEDGMENTS

We thank Michelyne Dejace for preparing the manuscript. This research was supported by grants from the Belgian Fonds de la Recherche Fondamentale et Collective (No. 2.4527.97) and Actions de Recherches Concertées (No. 93-98/170). F.D. is a fellow of Fonds de la Recherche dans l'Industrie et l'Agriculture (Belgium).

Received August 28, 1998; accepted November 6, 1998.

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