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We have isolated very high light resistant nuclear mutants (VHL R) in Chlamydomonas reinhardtii, that grow in 1500–2000 μmol photons m−2 s−1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHL R mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L *) from which another class of VHL R mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHL R mutants was independent of CO2 availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 μmol photons m−2 s−1) was strongly depressed when all genotypes were grown in 5% CO2. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO2, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid ΔpH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO2 at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L * and VHL R mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO2, possibly because they were unable to maintain sufficiently high ΔpH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHL R phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHL R mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.

In this paper, we examine the effects of mutations in the 5'UTR of the chloroplast rps7 transcript of Chlamydomonas reinhardtii that reduce the stability of the mRNA. Five point mutants in the rps7 5'UTR were selected on the basis of their failure to accumulate reporter mRNA in Escherichia coli. Each of these mutations produces alterations in the predicted higher-order structures of the rps7 5'UTR that destabilize the mRNA. Cis-acting suppressors of these mutations have been selected in E. coli and in the C. reinhardtii chloroplast that restore message stability and function. No differences in RNA melting and reannealing profiles have been observed between wild type, original mutant, and suppressor 5'UTRs transcribed in vitro. Proteins of 32 kDa and 47 kDa that bind to the wild-type rps7 5'UTR are not detected by UV cross-linking assays performed with any of the mutant rps7 5'UTRs. However, binding of the 32-kDa protein is restored in the six suppressor mutants examined. This suggests that the 32-kDa protein may be involved in protecting the rps7 5'UTR and the attached coding region from digestion by ribonucleases. Alternatively, the binding site for the 32-kDa protein may be independently lost in the rearranged tertiary structure of the mutant 5'UTR that exposes the RNA to degradation and is restored in the suppressor mutants.

The green alga Chlamydomonas reinhardtii can grow photoautotrophically utilizing CO2, heterotrophically utilizing acetate, and mixotrophically utilizing both carbon sources. Growth of cells in increasing concentrations of acetate plus 5% CO2 in liquid culture progressively reduced photosynthetic CO2 fixation and net O2 evolution without effects on respiration, photosystem II efficiency (as measured by chlorophyll fluorescence), or growth. Using the technique of on-line oxygen isotope ratio mass spectrometry, we found that mixotrophic growth in acetate is not associated with activation of the cyanide-insensitive alternative oxidase pathway. The fraction of carbon biomass resulting from photosynthesis, determined by stable carbon isotope ratio mass spectrometry, declined dramatically (about 50%) in cells grown in acetate with saturating light and CO2. Under these conditions, photosynthetic CO2 fixation and O2 evolution were also reduced by about 50%. Some growth conditions (e.g. limiting light, high acetate, solid medium in air) virtually abolished photosynthetic carbon gain. These effects of acetate were exacerbated in mutants with slowed electron transfer through the D1 reaction center protein of photosystem II or impaired chloroplast protein synthesis. Therefore, in mixotrophically grown cells of C. reinhardtii, interpretations of the effects of environmental or genetic manipulations of photosynthesis are likely to be confounded by acetate in the medium.

A collection of very high-light-resistant, VHL R , mutants that grow in 5% CO 2 under 1500 to 2000 μmol photons m −2 s −1 has been isolated from both the CC-125 wild-type strain of Chlamydomonas reinhardtii and from the chloroplast psbA mutant A251L * with an Ala to Leu substitution in the D1 protein. Neither of the parental strains survives long-term exposure to very high light intensities (VHL; above 1500 μmol photons m −2 s −1 ). This paper characterizes the genetic, physiological and biochemical properties of two nuclear mutants isolated from wild-type ( S4 and S9 ), and two derived from A251L * ( L5 and L30 ). All four mutants grow photoautotrophically in 5% CO 2 at rates similar to their respective parental strains under high light (HL; 600 μmol photons m −2 s −1 ) and maintain high growth rates under VHL. Total chlorophyll per biomass, or per cell, declines in both mutant and parental strains grown in HL compared with LL, and decreases further in mutants grown under VHL. All four VHL R mutants have lower light and CO 2 -saturated rates of photosynthetic O 2 evolution per total chlorophyll than wild-type or A251L * under low light (LL; 70 μmol photons m −2 s −1 ) or HL. The VHL R mutants S4 and S9 show greater nonphotochemical fluorescence quenching than wild-type when grown and measured under HL or VHL. In contrast, A251L * , L5 and L30 exhibit low nonphotochemical quenching under all conditions. Although xanthophyll pigments, including lutein, are elevated on a chlorophyll basis in both wild-type and S9 cells grown in HL compared to LL, they are unchanged on a biomass basis. The de-epoxidation state is very low even in HL. Preliminary analyses suggest that other VHL R mutants behave similarly. Evidently, enhanced photoprotection via the xanthophyll cycle is not involved in survival of several different VHL R mutants under saturating light and CO 2 . Photosystem II properties and electron transfer rates in all VHL R mutants grown in 5% CO 2 under LL do not differ from those of their respective parental strains. These experiments also demonstrate that slow photosystem II electron transfer in strain A251L * due to altered D1 structure, which is present in L5 and L30 isolated from this strain, does not preclude selection of VHL R mutants. The ability of these mutants to survive under VHL in 5% CO 2 evidently involves processes other than photoprotection via the xanthophyll cycle and resistance to photodamage via alterations in D1 function.

Initiation of translation in Escherichia coli and related eubacteria involves well-defined interactions between a conserved Shine-Dalgarno (SD) sequence immediately upstream of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3' end of the 16S rRNA. SD-like sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in location, size, and base composition compared to those in E. coli, while anti-SD sequences in the respective 16S rRNAs remain highly conserved. We have examined the function of the SD-like sequences found in the leaders of four chloroplast genes of the green alga Chlamydomonas reinhardtii using replacement mutagenesis to eliminate complementarity with the anti-SD sequences and insertion of canonical SD sequences (GGAGG) at positions -9 to -5 relative to the initiation codon. Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diversity of chloroplast SD-like sequences were fused to aadA and uidA reporter genes encoding spectinomycin resistance and GUS activity respectively. Analysis of chloroplast transformants of C. reinhardtii and transformants of E. coli carrying the wild-type and mutant reporter constructs revealed that mutagenic replacement of the putative SD sequences had no effect on the expression of either the aadA or uidA reporter genes. Chloroplast transformants with the canonical SD sequence also showed no differences in reporter gene expression, whereas expression of the reporter genes was increased by 10 to 30% in the E. coli transformants. Collectively our results suggest that even though SD-dependent initiation predominates in E. coli, this bacterium also has the capacity to initiate translation by an SD-independent mechanism. In contrast, plant chloroplasts, and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation of most mRNAs.

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G→A change at 291 in the first putative exon, resulting in a Val→Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G→A change at the equivalent position (5751) within exon 10.

We elucidated the effects of substituting seven amino acids for Ala at residue 251 of the Chlamydomonas reinhardtii D1 protein on herbicide resistance and photoautotrophic growth. Ala251 has been suggested to play a key role in the structural integrity and function of the stromal loop between transmembrane helices IV and V of D1 and has previously been shown to affect resistance to “classical” PSII specific herbicides. Sensitive and rapid microtiter assays were employed to compare herbicide resistance and photoautotrophic growth in the various mutants. Substitution of Ala251 by Ile, Leu or Val conferred resistance to the PSII herbicides atrazine, bromacil and metribuzin but not to DCMU, and impaired photoautotrophic growth in high and low light. Compared to an otherwise isogenic wildtype strain, the lie and Val mutants exhibited nearly identical levels of herbicide resistance and reduced growth while the Leu mutant had even slower growth and higher levels of herbicide resistance. In contrast Cys, Pro, Ser and Gly mutants were phenotypically indistinguishable from wildtype in terms of herbicide sensitivity and photoautotrophic doubling times. Collectively the seven Ala251 mutations differed markedly from an Ala mutant (dr-1) at the well characterized Ser264 D1 residue in terms of herbicide resistance and photoautotrophic growth

The effects of introduced chloroplast gene mutations affecting D1 synthesis, turnover and function on photosynthesis, growth and competitive ability were examined in autotrophic cultures of Chlamydomonas reinhardtii (Chlorophyta) adapted to low or high irradiance. Few discernible effects were evident when the mutants were grown in low light (LL, 70 μmol m−2 s−1). The herbicide-resistant psbA mutation Ser264→ Ala (dr) slowed electron transfer and accelerated D1 degradation in cells grown under high light (HL, 600 μmol m−2 s−1). The maximum rate of light-and CO2-saturated photosynthesis, cell growth rate and competitive ability in the dr mutant were reduced compared to wild type under HL. However, the wild-type rate of D1 synthesis in dr was adequate to compensate for accelerated D1 degradation. 16S rRNA mutations conferring resistance to streptomycin and spectinomycin (spr/sr) that altered chloroplast ribosome structure and assembly were used to inhibit chloroplast protein synthesis. In spr/sr cells grown under HL, D1 synthesis was reduced by 40–60% compared to wild type and D1 degradation was accelerated, leading to a 4-fold reduction in D1 pool size. The reduced D1 levels were accompanied by an elevation of Fo and a decline in Fv/Fm, quantum yield and maximum rate of CO2-saturated photosynthesis. Chemostat experiments showed that the growth rate and competitive ability of spr/sr were reduced against both wild type and dr.

The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants. In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s). Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells. Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I. Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila. By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation.

We have constructed a dominant selectable marker for nuclear transformation of C. reinhardtii, composed of the coding sequence of the eubacterial aadA gene (conferring spectinomycin resistance) fused to the 5′ and 3′ untranslated regions of the endogenous RbcS2 gene. Spectinomycin-resistant transformants isolated by direct selection (1) contain the chimeric gene(s) stably integrated into the nuclear genome, (2) show cosegregation of the resistance phenotype with the introduced DNA, and (3) synthesize the expected mRNA and protein. Small linearized plasmids appeared to be inserted into the nuclear genome preferentially through their ends, with relatively few large deletions and/or rearrangements. Multiple copy transformants often integrated concatemers of transforming DNA. Our detailed analysis of the complex integration patterns of plasmid DNA in C. reinhardtii nuclear transformants should be useful for improving the technique of insertional mutagenesis. We also found that the spectinomycin-resistance phenotype was unstable in about half of the transformants. When maintained under nonselective conditions, neither the aadA mRNA nor the AadA protein were detected in these subclones. Moreover, since the integrated transforming DNA was not altered or lost, expression of the RbcS2::aadA::RbcS2 gene(s) appears to be repressed. Measurements of transcriptional activity, mRNA accumulation, and mRNA stability suggest that expression of this chimeric gene(s) may also be affected by rapid RNA degradation, presumably due to defects in mRNA processing and/or nuclear export. Thus, both gene silencing and transcript instability, rather than biased codon usage, may explain the difficulties encountered in the expression of foreign genes in the nuclear genome of Chlamydomonas.

In Cyanobacteria and Chlamydomonas reinhardtii, substitution of valine for alanine at position 251 of the photosystem II D1 protein in the loop between transmembrane helices IV and V confers resistance to herbicides that reduce photosystem II function and increases sensitivity to photoinhibition. Using site-directed mutagenesis and chloroplast transformation in Chlamydomonas we have examined further the role of residue 251 in relation to D1 structure, function, and photosynthetic performance. Of the 12 different amino acid substitutions for Ala251 introduced at this position, five (Arg, Asp, Gln, Glu, and His) resulted in a nonphotosynthetic phenotype. Transformants with the Arg251 substitution synthesize a normal sized 32-kDa D1 protein with greatly reduced stability. The Gln, Glu, His, and Asp transformants make a 33-34-kDa form of the D1 protein of varying stability as well as an immunologically related polypeptide of 24-25 kDa corresponding to the N-terminal portion of D1 that is unstable and appears to be an aborted D1 translation product. All mutant forms of the D1 protein are intrinsic to the thylakoids. In contrast to previous studies in Cyanobacteria showing that residues in the IV-V loop can be mutated or deleted without loss of photosynthetic competence, our results suggest that Ala251 has a key role in the structure and function of the IV-V loop region.

We have examined the effects of illumination, carbon source, and levels of chloroplast protein synthesis on trans-acting proteins that bind to the leaders of five representative chloroplast mRNAs. The accumulation of these five chloroplast mRNAs and the proteins they encode were measured in cells grown under identical conditions. Extracts from all cell types examined contain a minimum set of six chloroplast 5′-untranslated region (UTR)-binding proteins (81, 62, 56, 47, 38, and 15 kDa). Fractionation results suggest that multiple forms of the 81-, 62-, and 47-kDa proteins may exist. A 36-kDa protein was found in all cells except those deficient in chloroplast protein synthesis. Binding of the 81-, 47-, and 38-kDa proteins to the rps12 leader is effectively competed by the atpB or rbcL 5′-UTRs, indicating that the same proteins bind to all three leaders. In contrast, these three proteins do not bind to the nuclear-encoded α-1 tubulin leader, which bound novel proteins of 110, 70, and 43 kDa. Cis-acting sequences within the 5′-UTRs of two chloroplast mRNAs (rps7 and atpB) have been identified which are protected from digestion by RNase T1 by extracts enriched for the 81-, 47-, and 38-kDa proteins.

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.

The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51–59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg68 (CGT) to Len (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln53 to Len ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S 12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu68 mutations in rps4. Instead, six of 15 contained a Gln73 (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln73 to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.

A group of highly active N -phenylimide photobleaching herbicides have been synthesized. These N -phenylimide herbicides as well as diphenyl ether herbicides induce protoporphyrin IX accumulation and inhibit protoporphyrinogen oxidase activity at extremely low concentrations in higher plants. The binding of a 14C -labeled N -phenylimide herbicide S-23121 [N-[4-chloro- 2-fluoro-5-[(1-m ethyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide] to the solubilized plastid fractions of greening corn seedlings is competed by the diphenyl ether herbicide acifluorfen-ethyl, but not by diuron, an inhibitor of photosynthetic electron transport. These results indicate a similar mode of action for both N -phenylimide and diphenyl ether herbicides. In order to investigate the mechanism of photobleaching herbicides at the molecular level, a strain of Chlamydomonas reinhardtii RS-3 resistant to N -phenylimide S-23142 [N -(4-chloro- 2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide] was isolated by mutagenesis with N -m ethyl-N′-nitro-N -nitrosoguanidine. The 90% inhibition concentration of N -phenylimide S-23142 for growth of RS-3 was 100 times higher than that for wild type. Maximum accumulation of protoporphyrin IX was reached at 0.03 μᴍ of S-23142 for the wild type and 3 μᴍ for RS-3. RS-3 was resistant to oxadiazon, oxyfluorfen and acifluorfen-ethyl which had been shown to have the same mechanism of action as N -phenylimide herbicides, but not to paraquat, diuron or fluridone. Genetic analysis of RS-3 strain showed that the resistance results from a dominant mutation ( rs-3) in the nuclear genome. The magnesium protoporphyrin IX synthesizing activity from 5-am inolevulinic acid in chloroplast fragments isolated from RS-3 was less sensitive to S-23142 than that from wild type (CC-407). Protoporphyrinogen oxidase activity in Percoll™ -purified chloroplasts from RS-3 was also less sensitive to S-23142 than that from wild type. These results indicate that the resistance of RS-3 is specific for photobleaching herbicides, and that the mutation is related to protoporphyrinogen oxidase, the primary site of the photobleaching herbicide action.

The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 x 10(-7)) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 x 10(-7)). No colonies were seen on control plates (frequency < 0.96 x 10(-9)). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.

Intermolecular recombination of Chlamydomonas chloroplast genes has been analyzed in sexual crosses and following biolistic transformation. The pattern and position of specific exchange events within 15 kb of the 22-kb inverted repeat have been mapped with respect to known restriction fragment length polymorphism markers that distinguish the chloroplast genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii. Recombinant progeny were selected from two- and three-factor crosses involving point mutations conferring herbicide (dr) and antibiotic resistance (er and spr) in the psbA, 23S and 16S ribosomal RNA genes, respectively. Exchange events were not randomly distributed over the 15-kb region, but were found to occur preferentially in a 0.7-kb sequence spanning the 3' end of the psbA gene and were much less common in an adjacent region of ca. 2.0 kb. These findings are corroborated by data showing that the dr mutation is unlinked genetically (3% recombination/kb) to the er and spr rRNA mutations, which are themselves linked and show ca. 1% recombination/kb. This discrepancy is significant since the dr-er and er-spr intervals are about the same length (ca. 7 kb). During chloroplast transformation, the 0.7-kb recombination hotspot also functions as a preferential site for exchange events leading to the integration of donor psbA gene sequences. The 0.7-kb hotspot region contains four classes of 18-37-bp direct repeats also found in other intergenic regions, but no open reading frame. Using deletion constructs in a chloroplast transformation assay, the hotspot was localized to a 500-bp region that lacks most of these repeats, which suggests that the repeats themselves are not responsible for the increased recombination frequency. Within this region, a 400-bp sequence is highly conserved between the chloroplast genomes of C. reinhardtii and C. smithii and includes several structural motifs characteristic of recombination hotspots in other systems.

Synthesis of the photosystem II D1 protein as a precursor with a carboxyl-terminal extension occurs in almost all eukaryotic photosynthetic organisms examined so far, as well as in cyanobacteria. Processing of the D1 precursor has been recently postulated to play a regulatory role in the light-dependent migration of photosystem II units from the unstacked to the stacked thylakoids (Bowyer, J. M., Packer, J. C. L., McCormack, B. A., Whitelegge, J. P., Robinson, C., and Taylor, M. A. (1992) J. Biol. Chem. 267, 5424-5433). To test this hypothesis, site-directed mutagenesis and chloroplast transformation have been used to create a "preprocessed" mutant Chlamydomonas strain which synthesizes mature D1 protein directly. We have found that this strain is indistinguishable from wild type in terms of photosynthetic performance and cell doubling time under CO2- and light-saturated photoautotrophic growth conditions.

We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of non-photosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.

Summary Although the chloroplast genome of Chlamydomonas reinhardtii contains essentially the same complement of genes as chloroplast genomes of land plants, the arrangement of these genes within the circular DNA molecule is very different. Studies in several laboratories suggest that comparison of gene order, restriction fragment length polymorphisms, and sequences within chloroplast DNA may be valuable tools in phylogenetic analysis of Chlamydomonas species. We have found that six interfertile strains in the C. reinhardtii group differ by restriction fragment length polymorphisms throughout their chloroplast genomes. In two of these strains the chloroplast genomes are known to be colinear and the differences largely attributable to variations in the size and distribution of clusters of short dispersed repeat sequences occurring in intergenic regions. These repeats, which are marked by Aat II and Kpn I restriction sites, are present in the chloroplast genomes of all six interfertile strains of the C. reinhardtii group, but are absent from the chloroplast genomes of C. eugametos, C. moewusii, C. incerta and C. globosa. The latter two species, which are sensitive to the same vegetative cell autolysin as C. reinhardtii, do not appear to be interfertile with C. reinhardtii. Restriction fragment analysis of chloroplast DNA suggests that C. incerta and C. globosa are very similar to one another, but differ greatly from C. reinhardtii.

During the evolution of chloroplasts from cyanobacterial endosymbionts, many genes encoding components necessary for protein synthesis and photosynthesis have been transferred to the nucleus. Assembly of the machinery for both processes now relies on the concerted expression of genes in the nuclear and plastid genomes. Evidence accumulating in C. reinhardtii indicates that the expression and regulation of chloroplast genes probably differs in certain respects from the prokaryotic (E. coli) paradigm. Both mutational analysis of putative Shine-Dalgarno (SD) sequences, and creation of canonical SD sequences (-9 to -5) reveals that initiation is not mediated by SD-16S rRNA interactions for the majority of chloroplast-encoded mRNAs. Recent evidence in cyanobacteria, the most likely ancestors of the Chlamydomonas chloroplast, indicates that most genes lack SD sequences at the classical position. Interactions between cis-acting sequences in the 5′ untranslated regions (UTRs) and specific trans-acting nuclear gene products appear to mediate translation of chloroplast-encoded mRNAs for specific genes. Both UV crosslinking and gel mobility shift assays with several chloroplast leaders show that multiple proteins interact with these sequences. Some of these appear to be involved in gene-specific regulation, whereas others may be core proteins of a general ribonucleoprotein (RNP) complex. Inverted repeat sequences present in most chloroplast leaders predict higher order structures within the 5′ UTR of mRNAs that could serve as scaffolding for the formation of the RNP complex. One model predicts that the RNP complex directly enhances ribosome binding and translation initiation. A second model predicts that the RNP complex plays a role in making topological distinctions in the sites of synthesis of chloroplast mRNAs. A challenge in the coming years will be to determine which, if any, of these models are correct.

Certain mutations isolated in the 5′untranslated region (5′UTR) of the chloroplast rps7 gene in Chlamydomonas reduce expression of reporter genes. Second site suppressors in this 5′UTR sequence restore reporter expression. 5′UTR sequences with the original mutations fail to bind a 20-kD protein, one of five proteins that bind to leaders of several chloroplast genes. However, 5′UTRs from suppressed mutants restore binding to this protein but do not bind a 47-kD protein present on the wild type and the original mutant 5′UTRs. The 20-kD protein was shown to be the S7 protein of the chloroplast ribosomal small subunit encoded by rps7 , whereas the 47-kD protein was shown to be RB47, a poly(A) binding protein. Our data are consistent with the hypothesis that the S7 protein plays either a general or a specific regulatory role in translation initiation in the chloroplast.

Translational regulation plays a major role in controlling the expression of chloroplast-encoded genes (7, 13, 18, 26, 29). While the translation machinery of the chloroplast shares many functional characteristics with that of its prokaryotic relatives (14, 16), several differences are beginning to emerge with respect to the well-defined Escherichia coli model (2, 11, 27, 38). Interactions between evolutionarily conserved Shine-Dalgarno (SD) sequences (GGAGG) found in the vast majority of genes in E. coli 7 ± 2 nucleotides (nt) upstream of the initiator AUG codon and anti-SD sequences near the 3′ end of the 16S rRNA (CCUCC) are known to be essential for translational initiation in this bacterium. Although the anti-SD sequences in the chloroplast 16S rRNAs are highly conserved, SD-like sequences in the leaders of chloroplast mRNAs vary significantly in location, size, and nucleotide composition or are absent altogether (3, 8, 31). Neither elimination of the variable putative SD sequences in chloroplast leaders by deletion mutagenesis (1, 19, 22, 32) or replacement mutagenesis (8, 21) nor insertion of canonical SD sequences (8) has significant effects on chloroplast gene expression, indicating that translation initiation in the chloroplast occurs largely in an SD-independent manner. Surprisingly, the same chloroplast reporter constructs lacking SD sequences are also expressed efficiently in E. coli and addition of canonical SD sequences to the leaders of these constructs only modestly enhances translation of these mRNAs by the bacterial protein synthesizing system (8). In contrast to the majority of E. coli mRNAs, chloroplast mRNAs contain fairly long, AU-rich 5′ untranslated regions (5′UTRs) with little primary sequence conservation that appear to play a major role in the regulation of translation initiation (10, 13, 18). Analyses of representative chloroplast 5′UTRs, to determine likely secondary structure based on minimum energy models (42), predict that these leaders are highly folded, with several having large stem-loop structures directly upstream of the initiation codon (10, 18). Interactions between these structures and the translational apparatus of the chloroplast presumably allow for the regulation of translation initiation. We have carried out a random PCR mutagenesis on the sequence for the 5′UTR of the chloroplast rps7 gene from Chlamydomonas reinhardtii that specifies a protein of the small subunit of the chloroplast ribosome. The population of rps7 mutant leaders was then fused to the E. coli lacZ′ coding sequence in a pUC18 plasmid, and bacterial transformants that were unable to express β-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plates were identified as white colonies. Twelve of the 207 white colony mutants were found to have alterations in the coding sequence for the first 225 nt upstream of the initiation AUG codon of the rps7 5′UTR. Seven rps7 mutants that did not alter levels of lacZ′ mRNA accumulation affected expression of the aminoglycoside adenyltransferase (AAD) reporter protein to various degrees in both the C. reinhardtii chloroplast and in E. coli. The marked difference between the effect of a given mutant rps7 leader on translation of the lacZ′ and aadA reporter mRNAs in E. coli suggests that interactions between the coding sequences and the 5′UTR sequence play a role in the regulation of translation initiation. Three of the seven mutants altered the predicted structure of the second stem-loop upstream of the AUG initiation codon. Complementary nucleotide changes made in these mutants to reconstitute this predicted wild-type stem-loop structure restored spectinomycin-resistant growth and AAD enzyme activity. Additionally, site-directed mutants with base pair changes that strengthened or weakened the second predicted stem-loop of the rps7 5′UTR decreased growth and AAD enzyme activity in the C. reinhardtii chloroplast. These data strongly suggest that the secondary structure of this rps7 leader in vivo involves these predicted stem-loop structures that are essential for normal translational regulation. Changes in the predicted secondary and tertiary structures of the mutant 5′UTRs were also supported by differences in the RNA melting-reannealing profiles and RNase T1 gel shift protection patterns from those of wild-type and suppressed mutant strains. The combined genetic and physical evidence supports the hypothesis that the wild-type rps7 leader sequence resulting in the predicted folding pattern for the second stem-loop is essential for normal function.

The QB binding site of the D1 reaction center protein, located within a stromal loop between transmembrane helices IV and V formed by residues Ile219 to Leu272, is essential for photosynthetic electron transport through photosystem II (PSII). We have examined the function of the highly conserved Ala251 D1 residue in this domain in chloroplast transformants of Chlamydomonas reinhardtii and found that Arg, Asp, Gln, Glu, and His substitutions are nonphotosynthetic, whereas Cys, Ser, Pro, Gly, Ile, Val, and Leu substitutions show various alterations in D1 turnover, photosynthesis, and photoautotrophic growth. The latter mutations reduce the rate of QA to QB electron transfer, but this is not necessarily rate-limiting for photoautotrophic growth. The Cys mutant divides and evolves O2 at wild type rates, although it has slightly higher rates of D1 synthesis and turnover and reduced electron transfer between QA and QB. O2 evolution, D1 synthesis, and accumulation in the Ser, Pro, and Gly mutants in high light is reduced, but photoautotrophic growth rate is not affected. In contrast, the Ile, Val, and Leu mutants are impaired in photoautotrophic growth and photosynthesis in both low and high light and have elevated rates of D1 synthesis and degradation, but D1 accumulation is normal. While rates of synthesis/degradation of the D1 protein are not necessarily correlated with alterations in specific parameters of PSII function in these mutants, bulkiness of the substituted amino acids is highly correlated with the dissociation constant for QB in the seven mutants examined. These observations imply that the Ala251 residue plays a key role in D1 protein.

Hierarchical sampling from populations, incipient and recognized varieties within Phacelia dubia and P. maculata has revealed high levels of intraspecific polymorphism in chloroplast DNA. Much of the variation is partitioned between populations as evidenced by population-specific variants at fixation in all three populations of P. dubia var. interior and in both populations of P. maculata. Nine of 16 populations were polymorphic for cpDNA haplotypes. A total of 16 haplotypes was found in a sample of 106 individuals; the most common occurred in eight of the 16 populations and in 31 per cent of the individuals in the entire sample. A phylogenetic analysis revealed four basic plastome types. The two major groups of plastomes were separated by four independent base-pair mutations which suggests an ancient split in the evolution of plastid genomes. Representatives from each major plastome division were found in each of five populations spanning two allopatric varieties of P. dubia. The geographical distribution of haplotypes and lack of evidence for recent admixture argue against migration as a source of the polymorphism. It is more likely that the current taxonomic varieties are descendants of a polymorphic common ancestor.

Publisher Summary This chapter discusses genetics and transformation of mitochondria in the green alga Chlamydomonas . Molecular evidence strongly supports the endosymbiotic origin of mitochondria from alpha purple bacteria. Photosynthetic eukaryotes show the greatest diversity in mitochondrial genome size and structure, ranging from the 15-kb linear genome of the green alga Chlamydomonas reinhardtii to genomes of certain angiosperms that exceed one Mb. Although the mitochondrial genomes of numerous invertebrates and vertebrates and the liverwort Marchantia is completely sequenced, and the genes they encode are well characterized, the genetic systems in these organelles are poorly developed. Mitochondria of animal cells lacking DNA can be obtained by prolonged culture of cells in medium containing ethidium bromide, and pyruvate and uridine, which are essential for growth of the cell. Genetic dissection of mitochondrial structure and function in baker's yeast is possible because mitochondrial function is dispensable. Mitochondrial transformation in yeast is just beginning to be exploited to study expression of specific organelle genes, and much needs to be done to take advantage of this technique in Chlamydomonas.

Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival.

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostrepton-resistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.

Publisher Summary Chloroplast transformation provides the technology for dissecting the function of specific chloroplast regulatory sequences and probing structure or function relationships of individual chloroplast-encoded proteins by examining in vivo the consequences of in vitro mutations. This methodology also offers the opportunity for ascertaining the functions encoded by chloroplast open reading frame (ORF) sequences. Such investigations are already providing new approaches to the study of gene regulation and protein function, as the mutations created can be evaluated in vivo in their normal location in the chloroplast genome without complications resulting from position effects, which can arise in the case of nonhomologous recombination. In addition, successful transformation of higher plant chloroplasts brings with it the hope that these organelles may eventually be engineered for practical purposes, such as is currently being done with nuclear genes in important crop plants.

The green alga Chlamydomonas reinhardtii is an ideal model in which to study the cooperation of nuclear and chloroplast genes in chloroplast ribosome biogenesis. At least six of the 33 r-proteins identified in the large subunit and 14 of the 31 r-proteins of the small subunit appear to be made in the chloroplast of C. reinhardtii, with the remainder being synthesized in the cytoplasm (Schmidt et al, 1983). Antibodies to 16 chloroplast r-proteins from C. reinhardtii have been examined for cross reactivity with specific r-proteins from Anabaena, E. coli and the chloroplast of spinach (Randolph-Anderson et al, 1989; Schmidt et al, 1984). To date 11 chloroplast-encoded r-protein genes have been identified in C. reinhardtii (Harris, 1992). The completely sequenced chloroplast genomes of tobacco, rice and the liverwort Marchantia have revealed an additional 12 r-protein genes which have not yet been located in Chlamydomonas (Shimada and Sugiura, 1991). With a few exceptions, the same set of r-protein genes is found in all chloroplast genomes examined in green algae and land plants (Palmer, 1991). The exceptions include the location of the rpl5 gene in the chloroplast genome of Chlamydomonas and certain other algae, the rpl21 gene in the chloroplast genome of Marchantia, and the rpsl6 gene in the chloroplast genome of higher plants but not in Marchantia.

Of the more than 400 species of unicellular green algae in the genus Chlamydomonas (Ettl, 1976), only a few have been used for genetic investigations. The small cell size, rapid division time, and short sexual cycles of these green flagellates make them especially favorable organisms for genetic and molecular studies. The haploid heterothallic species C. reinhardtii has been the organism of choice for most molecular, cellular and developmental investigations (see Harris, 1989). Its nuclear genome of ca. 1-2 × 105 kb comprises 18 genetic linkage groups (Harris, 1989,1990), and physical maps are being developed for many of these (Ranum et al, 1988). Detailed molecular maps have also been established for the 196 kb chloroplast and 15.8 kb mitochondrial genomes of C. reinhardtii, and formal genetic studies are possible for both organelles (see Harris, 1989,1990). A fourth genome of 6–9 mb, localized in the basal body has recently been defined (Hall et al., 1989) and correlated with a unique quasi-Mendelian circular linkage group (Ramanis and Luck, 1986). Existence of closely related, interfertile isolates such as C. smithii mt + (CC-1373 = SAG 54.72 and UTEX 1062) and the Minnesota S-1 D-2 isolates (CC-2290 and CC-1952) that differ from C. reinhardtii by numerous restriction fragment length polymorphisms (RFLPs) in two or more genomes (Boynton et al, 1987; Gross et al., 1988; Hall et al., 1989; Ranum et al., 1988) makes this species group especially useful for molecular genetic studies.

The green alga Chlamydomonas reinhardtii is an ideal model organism in which to investigate the cooperation of nuclear and chloroplast genomes in chloroplast ribosome formation from both developmental and evolutionary perspectives. Mutations affecting a variety of ribosomal components have been isolated and mapped genetically (see Boynton et al., 1990a and Harris, 1989 for reviews), and both the chloroplast (Boynton et al., 1988, 1990b; Blowers et al., 1989) and nuclear genomes (Debuchy et al., 1989; Kindle et al., 1989; Mayfield and Kindle, 1990; Diener et al., 1990) can be manipulated using newly developed transformation methods. In this paper we review our recent work on 1) the genetics and molecular biology of mutations to antibiotic resistance affecting the rRNA genes; 2) identification of chloroplast and nuclear genes encoding chloroplast ribosomal proteins (r-proteins); 3) expression of chloroplast genes encoding r-proteins; and 4) the transformation of chloroplast rRNA genes. We also outline the direction our current research is taking to define more precisely the rRNA-r-protein interactions involved in chloroplast ribosome biogenesis and function, as well as the molecular mechanisms involved in regulating expression of chloroplast r-protein genes.

Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt + parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt - parent, arise at a low frequency. Mutations at the mt +-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt-parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt - parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically which the mat3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt - parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt + parent.

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.

We have characterized two independently isolated point mutants in Chlamydomonas reinhardtii, ac-u-a-1-15 and FUD 17, mapping to the chloroplast ac-u-a locus which corresponds to the atpE gene. Both mutants have a single A:T base pair deletion in a sequence of 6 A:T base pairs at nucleotide positions 102 to 107. This causes a frameshift, altering the coding sequence for the next 8 amino acids and creating a termination codon at amino acid position 44, 98 amino acids from the C-terminus of the protein. Assembly of the ATP synthase is impaired in the mutants; less than 5% of the wild-type level of alpha and beta subunits and no gamma or epsilon subunits are associated with thylakoid membranes of the mutants. The genes encoding the beta and epsilon subunits of the chloroplast ATP synthase from C. reinhardtii are not cotranscribed, in contrast to all other photosynthetic organisms examined to date. Four transcripts, of approximately 1.7, 2.9, 3.3 and 7.0 x 10(3) nucleotides (nt), are found for the atpE gene. S1 nuclease mapping of the 1.7 x 10(3) nt transcript shows that the atpE gene message is preceded by a leader of about 1250 nt. DNA sequence analysis of this region revealed a 159 bp open reading frame corresponding to the 3' half of the rps7 gene, encoding the S7 protein of the small subunit of the chloroplast ribosome. Only the 5' portion of this gene is located in the opposite unique sequence region of the C. reinhardtii chloroplast genome where the rps7 gene was previously mapped by heterologous hybridization.

In photosynthetic eukaryotes, genes encoding chloroplast proteins of multimeric complexes involved in photosynthesis and protein synthesis are partitioned between the cell’s nuclear and chloroplast genomes. The chloroplast genome’s relatively small size, high copy number and great evolutionary conservation have led to rapid progress in identifying and characterizing chloroplast genes. In fact, the entire chloroplast genomes of tobacco, liverwort and rice have been sequenced within the last three years (1–3). Somewhat less progress has been made in identifying the nuclear genes for the remainder of the chloroplast polypeptides, due to the larger number of genes and the much larger size and evolutionary diversity of the nuclear genomes themselves. However in many plant species, nuclear genes can be engineered in vitro, the constructs introduced by transformation, and their function studied in vivo.

Transfer of Chlamydomonas reinhardtii cells grown photoautotrophically in low light to higher light intensities has a dramatic transient effect on the differential expression of the two major chloroplast encoded photosynthetic proteins. Synthesis of the D1 protein of Photosystem II increases more than 10-fold during the first six hours in high light (HL), whereas synthesis of the large subunit (LSU) of Rubisco drops dramatically within 15 min and only gradually resumes at about 6 h. Synthesis of the chloroplast-encoded ATP synthase beta subunit, the nuclear-encoded Rubisco small subunit and the nuclear-encoded beta-tubulin is not noticeably affected. Up regulation of psbA mRNA translation accounts for a substantial fraction of the increased D1 synthesis, since accumulation of psbA mRNA increases 4.2- and 6.3-fold less than D1 synthesis at 6 and 18 h in HL. Down-regulation of LSU synthesis is not correlated with a reduction in the steady-state level of the rbcL transcript. Primer extension mapping of the 5' ends of the rbcL mRNAs reveals transcripts with start points located at -93 and -168 relative to the first translated ATG. Transfer of low light (LL)-grown cells to HL temporarily decreases the ratio of the -93 to -168 transcripts, but this ratio normalizes after 6 h in HL, coincident with the recovery in the synthesis of LSU. These several distinct effects of temporary light stress were correlated with a rapid, sustained increase in the reduction state of QA, a transient decline in photosynthetic efficiency, a less rapid drop in total chlorophyll content and a delay in cell division.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.

Treatment of Chlamydomonas reinhardtii with acriflavin induces, with nearly 100% efficiency, lethal minute colony mutants which are able to undergo only 8+9 mitotic divisions under photosynthetic conditions and almost no division in darkness. The minute mutation is inherited in a non-Mendelian biparental fashion and differs in this respect from the non-Mendelian uniparentally inherited mutations previously described in Chlamydomonas which are thought to reside in chloroplast DNA. The minute mutations may well be analogous to the respiratory deficient petite mutations which acriflavin induces in yeast and may arise from alterations in mitochondrial DNA.

Mitochondria and chloroplasts of eucaryotic cells contain populations of DNA molecules. In certain cases, e.g., the chloroplasts of Chlamydomonas reinhardtii and the mitochondria of Saccharomyces cerevisiae, organelles contributed by the two parents are known to fuse in the zygote, creating a single population of DNA molecules. In a cross, this population will include molecules of both parental genotypes. There is reason to suspect that organelle DNA molecules in this population are selected randomly for replication and recombination. This would result in random changes in the frequency of a particular allele or genotype within the organelle gene pool of a single zygote and also within its clone of progeny cells. A given gene frequency would increase in some zygote clones and decrease in others, analogous to random drift of gene frequencies in small Mendelian populations. To test this, we have examined the distribution of chloroplast gene frequencies among the zygote clones produced in each of a number of crosses of Chlamydomonas. These distributions are typically U or L shaped as predicted by the random drift hypothesis. They include uniparental zygote clones, in which a chloroplast allele from one parent has been fixed (frequency 100%) and the alternative allele from the other parent has been lost (frequency 0%). Among the remaining (biparental) zygote clones, there is a linear distribution of allele frequencies, showing a great increase in variance over the input frequencies. In these experiments both biparental and uniparental zygotes show a bias favoring chloroplast alleles from the mt+ (maternal) parent, and there is no statistically significant mode at the allele frequency of 0.5 corresponding to the equal input of alleles from the maternal and paternal (mt−) parents. The observed distributions support the hypothesis that both uniparental inheritance and the high variance of allele frequencies among zygote clones are due to random drift of allele frequencies, coupled with a directional force which favors fixation of the maternal allele. In addition, statistical analysis of the data shows a strong but incomplete tendency for linked chloroplast markers to be fixed or lost together in uniparental zygotes. Possible cellular and molecular mechanisms for these observations are discussed.

Among a collection of obligate photoautotrophic (dark-dier, dk) mutants isolated in Chlamydomonas reinhardtii, two have been found which are inherited in crosses to wild type in a non-Mendelian, biparental and apparently random fashion. F1 progeny include not only cells which show the dk and wildtype parental phenotypes but also many which possess intermediate phenotypes between wild type and dk. When F1 progeny with dk, intermediate or wild-type phenotype were backcrossed to wild type, the dk phenotype continued to be inherited in a biparental and random fashion. Upon selection, neither mutant formed stable clones producing only dk progeny, suggesting that the two mutants segregate dk and wild-type progeny somatically and that the homozygous dk condition may be lethal. The biparental transmission of these two non-Mendelian dk mutations resembles the transmission of acriflavin-induced minute mutations of Chlamydomonas and is distinct from the uniparentally inherited chloroplast mutations of this alga. Both the dk and minute mutations may alter mitochondrial DNA and thereby alter mitochondrial functions.

We have isolated nonphotosynthetic (acetate-requiring) mutants with physical alterations in chloroplast DNA following growth of haploid cells in the chloroplast specific mutagen 5-fluorodeoxyuridine (FdUrd) or treatment of FdUrd-grown diploid cells with X rays. About one-third of the nonphotosynthetic mutations resulting from FdUrd treatment alone show simple deletions. All eight of the mutants examined so far which were obtained with FdUrd plus X rays have deletions that are accompanied by rearrangements, including inversions or duplications. All the alterations extend into one of the two inverted repeat regions of the chloroplast genome which contain the ribosomal RNA cistrons. However, Southern hybridization experiments reveal that the rRNA cistrons are not deleted but instead are contained in new fragments. The relocated rRNA cistrons appear to be functional, since the mutants have normal levels of chloroplast ribosomes. In most cases the deletions and rearrangements are symmetrical and affect both inverted repeats in a similar fashion. An exception is the mutant ac-u-c-2–43, which lacks one inverted repeat region almost completely, including an entire set of rRNA genes. Three additional mutants, which fail to recombine with ac-u-c-2–43 to give photosynthetically competent cells, have smaller deletions in the same region of the genome. These physical mapping studies have allowed us to place the ac-u-c locus itself in a region of unique sequence DNA in a fragment, Ba10, which also includes the right-hand end of one inverted repeat.