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The 24-h cycle of light and darkness governs daily rhythms of complex behaviors across all domains of life. Intracellular photoreceptors sense specific wavelengths of light that can reset the internal circadian clock and/or elicit distinct phenotypic responses. In the surface ocean, microbial communities additionally modulate nonrhythmic changes in light quality and quantity as they are mixed to different depths. Here, we show that eukaryotic plankton in the North Pacific Subtropical Gyre transcribe genes encoding light-sensitive proteins that may serve as light-activated transcription factors, elicit light-driven electrical/chemical cascades, or initiate secondary messenger-signaling cascades. Overall, the protistan community relies on blue light-sensitive photoreceptors of the cryptochrome/photolyase family, and proteins containing the Light-Oxygen-Voltage (LOV) domain. The greatest diversification occurred within Haptophyta and photosynthetic stramenopiles where the LOV domain was combined with different DNA-binding domains and secondary signal-transduction motifs. Flagellated protists utilize green-light sensory rhodopsins and blue-light helmchromes, potentially underlying phototactic/photophobic and other behaviors toward specific wavelengths of light. Photoreceptors such as phytochromes appear to play minor roles in the North Pacific Subtropical Gyre. Transcript abundance of environmental light-sensitive protein-encoding genes that display diel patterns are found to primarily peak at dawn. The exceptions are the LOV-domain transcription factors with peaks in transcript abundances at different times and putative phototaxis photoreceptors transcribed throughout the day. Together, these data illustrate the diversity of light-sensitive proteins that may allow disparate groups of protists to respond to light and potentially synchronize patterns of growth, division, and mortality within the dynamic ocean environment., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits," namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating., Competing Interests: The authors declare no competing interest.

Photosynthetic organisms use nonphotochemical quenching (NPQ) mechanisms to dissipate excess absorbed light energy and protect themselves from photooxidation. In the model green alga Chlamydomonas reinhardtii , the capacity for rapidly reversible NPQ (qE) is induced by high light, blue light, and UV light via increased expression of LHCSR and PSBS genes that are necessary for qE. Here, we used a forward genetics approach to identify SPA1 and CUL4, components of a putative green algal E3 ubiquitin ligase complex, as critical factors in a signaling pathway that controls light-regulated expression of the LHCSR and PSBS genes in C. reinhardtii The spa1 and cul4 mutants accumulate increased levels of LHCSR1 and PSBS proteins in high light, and unlike the wild type, they express LHCSR1 and exhibit qE capacity even when grown in low light. The spa1-1 mutation resulted in constitutively high expression of LHCSR and PSBS RNAs in both low light and high light. The qE and gene expression phenotypes of spa1-1 are blocked by mutation of CrCO, a B-box Zn-finger transcription factor that is a homolog of CONSTANS, which controls flowering time in plants. CONSTANS-like cis -regulatory sequences were identified proximal to the qE genes, consistent with CrCO acting as a direct activator of qE gene expression. We conclude that SPA1 and CUL4 are components of a conserved E3 ubiquitin ligase that acts upstream of CrCO, whose regulatory function is wired differently in C. reinhardtii to control qE capacity via cis -regulatory CrCO-binding sites at key photoprotection genes., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The unicellular green alga Chlamydomonas reinhardtii displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that Chlamydomonas exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes PSBS , LHCSR1 , and LHCSR3 show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while LHCSR3 genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Cells have developed regulatory mechanisms that underlie flagellar assembly and maintenance, including the transcriptional regulation of flagellar genes, an initial step for making flagella. Although transcriptional regulation of flagellar gene expression is required for flagellar assembly in Chlamydomonas , no transcription factor that regulates the transcription of flagellar genes has been identified. We report that X chromosome-associated protein 5 (XAP5) acts as a transcription factor to regulate flagellar assembly in Chlamydomonas While XAP5 proteins are evolutionarily conserved across diverse organisms and play vital roles in diverse biological processes, nothing is known about the biochemical function of any member of this important protein family. Our data show that loss of XAP5 leads to defects in flagellar assembly. Posttranslational modifications of XAP5 track flagellar length during flagellar assembly, suggesting that cells possess a feedback system that modulates modifications to XAP5. Notably, XAP5 regulates flagellar gene expression via directly binding to a motif containing a CTGGGGTG-core. Furthermore, recruitment of RNA polymerase II (Pol II) machinery for transcriptional activation depends on the activities of XAP5. Our data demonstrate that, through recruitment of Pol II, XAP5 defines a class of transcription factors for transcriptional regulation of ciliary genes. This work provides insights into the biochemical function of the XAP5 family and the fundamental biology of the flagellar assembly, which enhance our understanding of the signaling and functions of flagella., Competing Interests: The authors declare no conflict of interest.

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.

Almost all living organisms need to obtain molybdenum from the external medium to achieve essential processes for life. Activity of important enzymes such as sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase, and nitrate reductase is strictly dependent on the presence of Mo in its active site. Cells take up Mo in the form of the oxianion molybdate, but the molecular nature of the transporters is still not well known in eukaryotes. MOT1 is the first molybdate transporter identified in plant-type eukaryotic organisms, but it is absent in animal genomes. Here we report a molybdate transporter different from the MOT1 family, encoded by the Chlamydomonas reinhardtii gene MoT2, that is also present in animals including humans. The knockdown of CrMoT2 transcription leads to the deficiency of molybdate uptake activity in Chlamydomonas. In addition, heterologous expression in Saccharomyces cerevisiae of MoT2 genes from Chlamydomonas and humans support the functionality of both proteins as molybdate transporters. Characterization of CrMOT2 and HsMOT2 activities showed an apparent Km of about 550 nM that, though higher than the Km reported for MOT1, still corresponds to high affinity systems. CrMoT2 transcription is activated when extracellular molybdate concentration is low but in contrast to MoT1 is not activated by nitrate. Analysis of protein databases revealed the presence of four motifs present in all the proteins with high similarity to MOT2, that label a previously undescribed family of proteins probably related to molybdate transport. Our results open the way toward the understanding of molybdate transport as part of molybdenum homeostasis and Moco biosynthesis in animals.

Changes in chromatin organization are emerging as key regulators in nearly every aspect of DNA-templated metabolism in eukaryotes. Histones undergo many, largely reversible, posttranslational modifications that affect chromatin structure. Some modifications, such as trimethylation of histone H3 on Lys 4 (H3K4me3), correlate with transcriptional activation, whereas others, such as methylation of histone H3 on Lys 27 (H3K27me), are associated with silent chromatin. Posttranslational histone modifications may also be involved in the inheritance of chromatin states. Histone phosphorylation has been implicated in a variety of cellular processes but, because of the dynamic nature of this modification, its potential role in long-term gene silencing has remained relatively unexplored. We report here that a Chlamydomonas reinhardtii mutant defective in a Ser/Thr protein kinase (MUT9p), which phosphorylates histones H3 and H2A, shows deficiencies in the heritable repression of transgenes and transposons. Moreover, based on chromatin immunoprecipitation analyses, phosphorylated H3T3 (H3T3ph) and monomethylated H3K4 (H3K4me1) are inversely correlated with di/trimethylated H3K4 and associate preferentially with silenced transcription units. Conversely, the loss of those marks in mutant strains correlates with the transcriptional reactivation of transgenes and transposons. Our results suggest that H3T3ph and H3K4me1 function as reinforcing epigenetic marks for the silencing of euchromatic loci in Chlamydomonas.

An inducible chloroplast gene expression system was developed in Chlamydomonas reinhardtii by taking advantage of the properties of the copper-sensitive cytochrome c(6) promoter and of the nucleus-encoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5' UTR. A construct containing the Nac2 coding sequence fused to the cytochrome c(6) promoter was introduced into the nac2-26 mutant strain deficient in Nac2. In this transformant, psbD is expressed in copper-depleted but not in copper-replete medium. Because psbD encodes the D2 reaction center polypeptide of photosystem II (PSII), the repression of psbD leads to the loss of PSII. We have tested this system for hydrogen production. Upon addition of copper to cells pregrown in copper-deficient medium, PSII levels declined to a level at which oxygen consumption by respiration exceeded oxygen evolution by PSII. The resulting anaerobic conditions led to the induction of hydrogenase activity. Because the Cyc6 promoter is also induced under anaerobic conditions, this system opens possibilities for sustained cycling hydrogen production. Moreover, this inducible gene expression system is applicable to any chloroplast gene by replacing its 5' UTR with the psbD 5' UTR in the same genetic background. To make these strains phototrophic, the 5' UTR of the psbD gene was replaced by the petA 5' UTR. As an example, we show that the reporter gene aadA driven by the psbD 5' UTR confers resistance to spectinomycin in the absence of copper and sensitivity in its presence in the culture medium.

MscS is a mechanosensitive channel that is ubiquitous among bacteria. Recent progress in the genome projects has revealed that homologs of MscS are also present in eukaryotes, but whether they operate as ion channels is unknown. In this study we cloned MSC1, a homolog of MscS in Chlamydomonas, and examined its function when expressed in Escherichia coli. Full-length MSC1 was not functional when expressed in E. coli cells. However, removal of the N-terminal signal sequence (DeltaN-MSC1) reversed this effect. DeltaN-MSC1 was found to open in response to membrane stretch and displayed a preference for anions over cations as permeable ions. DeltaN-MSC1 exhibited marked hysteretic behavior in response to ascending and descending stimuli. That is, channel gating occurred in response to significant stimuli but remained open until the stimulus was almost completely removed. Indirect immunofluorescence revealed that MSC1 is present as punctate spots in the cytoplasm and chloroplasts. Moreover, knockdown of MSC1 expression resulted in the abnormal localization of chlorophyll. These findings show that MSC1 is an intracellular mechanosensitive channel and is responsible for the organization of chloroplast in Chlamydomonas.

Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Deltand4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I.

The CRR1 (Copper Response Regulator) locus, required for both activating and repressing target genes of a copper- and hypoxia-sensing pathway in Chlamydomonas, encodes a 1,232-residue candidate transcription factor with a plant-specific DNA-binding domain named SBP, ankyrin repeats, and a C-terminal Cys-rich region, with similarity to a Drosophila metallothionein. The recombinant SBP domain of Crr1 shows zinc-dependent binding to functionally defined copper-response elements associated with the CYC6 and CPX1 promoters that contain a critical GTAC core sequence. Competition experiments indicate equivalent selectivity for copper-response elements from either promoter and 10-fold greater selectivity for the wild-type sequence vs. a sequence carrying a single mutation in the GTAC core. The SBP domain of Chlamydomonas Crr1 binds also to a related GTAC-containing sequence in the Arabidopsis AP1 promoter that is the binding site of a defining member of the SBP family of DNA-binding proteins. Chlamydomonas Crr1 is most similar to a subset of the Arabidopsis SBP domain proteins, which include SPL1, SPL7, and SPL12. The abundance of the CRR1 mRNA is only marginally copper-responsive, and although two mRNAs that differ with respect to splicing of the first intron are detected, there is no indication that the splicing event is regulated by metal nutrition or hypoxia. It is likely that the dramatic copper-responsive action of Crr1 occurs at the level of the polypeptide.

Biological speciation ultimately results in prezygotic isolation-the inability of incipient species to mate with one another-but little is understood about the selection pressures and genetic changes that generate this outcome. The genus Chlamydomonas comprises numerous species of unicellular green algae, including numerous geographic isolates of the species C. reinhardtii. This diverse collection has allowed us to analyze the evolution of two sex-related genes: the mid gene of C. reinhardtii, which determines whether a gamete is mating-type plus or minus, and the fus1 gene, which dictates a cell surface glycoprotein utilized by C. reinhardtii plus gametes to recognize minus gametes. Low stringency Southern analyses failed to detect any fus1 homologs in other Chlamydomonas species and detected only one mid homolog, documenting that both genes have diverged extensively during the evolution of the lineage. The one mid homolog was found in C. incerta, the species in culture that is most closely related to C. reinhardtii. Its mid gene carries numerous nonsynonymous and synonymous codon changes compared with the C. reinhardtii mid gene. In contrast, very high sequence conservation of both the mid and fus1 sequences is found in natural isolates of C. reinhardtii, indicating that the genes are not free to drift within a species but do diverge dramatically between species. Striking divergence of sex determination and mate recognition genes also has been encountered in a number of other eukaryotic phyla, suggesting that unique, and as yet unidentified, selection pressures act on these classes of genes during the speciation process.

Replacement of lysine-350 by methionine in the beta-tubulin gene of Chlamydomonas confers resistance to microtubule-depolymerizing drugs and increased sensitivity to the microtubule-stabilizing drug taxol. This mutation was created in cloned BTU1, one of two coexpressed beta-tublin genes of Tetrahymena thermophila. When introduced by electroporation, the mutated gene transformed Tetrahymena exclusively by gene replacement at the homologous locus. Taxol-sensitive transformants could be retransformed with a wild-type gene and selection for taxol resistance. Analyses of phenotypic assortment and of the mRNA in transformed cells suggest that complete replacement of the BTU1 gene in the polyploid macronucleus can be obtained. These studies demonstrate the utility of this marker for studying tublin gene function and show that electroporation allows facile gene replacement in Tetrahymena.

Using a cotransformation system to identify chloroplast transformants in Chlamydomonas reinhardtii, we converted histidine-195 of the photosystem II reaction center D1 protein to a tyrosine residue. The mutants were characterized by a reduced quantum efficiency for photosynthetic oxygen evolution, which varied in a pH-dependent manner, a reduced capacity to oxidize artificial donors to photosystem II, and P680+ reduction kinetics (microsecond) that were essentially similar to wild type. In addition, a dark-stable radical was detected by ESR in mutant photosystem II particles but not in wild-type particles. This radical was similar in g value and lineshape to chlorophyll or carotenoid cations but could have arisen from a tyrosine-195 cation. The ability of the photosystem II trap (P680+) to oxidize tyrosine residues suggests that the mutant tyrosine residue could be used as a redox-sensitive probe to investigate the environment around the photosystem II trap.

After conjugation between Chlamydomonas gametes of opposite mating type, a transient dikaryon is formed. The two nuclei fuse within 4-6 hr after mating. The young diploid zygote differentiates into dormant zygospore competent to complete meiosis, or more rarely (2-10% of cases) it undergoes mitosis to produce a stable diploid progeny. We here bring genetical, biochemical, and cytological evidence that among the mitotic zygotes, a large proportion of them undergo cytokinesis without fusion of the nuclei-a process that has been termed "cytoduction." By using appropriate genetic markers, haploid cytoductants that possess the nuclear genotype of one parent and the chloroplast marker of the other parent can easily be isolated. Genetical analysis and hybridization experiments moreover show that many haploid cytoductants transmit the chloroplast DNA molecules of both parents and that, as in diploids, these DNA copies occasionally recombine. This process of cytoduction extends the life cycle of Chlamydomonas and provides new tools for its genetic analysis.

Two copies of structurally related genes (CAH1 and CAH2) for carbonic anhydrase (EC 4.2.1.1) were found to be tandemly clustered on the Chlamydomonas reinhardtii genome. The previously isolated cDNA clones for carbonic anhydrase polypeptides were derived from the upstream gene, CAH1, which has 10 introns in its coding region. The downstream gene, CAH2, also has 10 introns, at positions identical to those of CAH1. Although amino acid sequences deduced from the two genes showed 91.8% identity, partial sequences of the authentic enzyme isolated from air-induced cells were identical only to those of the CAH1 product. Northern hybridization using gene-specific probes showed that the level of 2.0-kilobase CAH1 mRNA increased in response to a decrease in CO2 concentration in the presence of light. The CAH1 mRNA did not accumulate when CO2 was lowered in the dark. In contrast, the level of 2.0-kilobase CAH2 mRNA decreased in response to lowering of CO2 and increased upon transfer to the high-CO2 condition in light. The decrease of CAH2 mRNA under the low-CO2 condition was not observed in the dark. The fully induced mRNA level was much higher for CAH1 than for CAH2. These results indicate that CAH1 is a gene coding for the major periplasmic carbonic anhydrase whose level of transcript is rapidly induced under the low-CO2 condition in the presence of light, and that CAH2 may encode another periplasmic isozyme, which is made under the high-CO2 condition.

We have isolated telomeric DNA and telomere-associated sequences from Chlamydomonas reinhardtii. The terminal telomere sequences of the green alga Chlamydomonas are composed of (TTTTAGGG)n repeats that are similar, but not identical, to those of the higher plant Arabidopsis thaliana. We demonstrate that these repeats are telomeric by their preferential sensitivity to nuclease Bal-31 digestion, their similarity to A. thaliana telomeres, their orientation relative to the end of the chromosome, and the methods used for their isolation. Five independent telomere clones were isolated, and three of these clones include closely related telomere-associated sequences. One of these telomere-associated sequences hybridizes to a number of genomic fragments sensitive to digestion with the exonuclease Bal-31. Like telomere sequences from other organisms, the C. reinhardtii telomeres display a bias for guanine and thymine nucleotides on the 3'-end strand. However, the sequence of Chlamydomonas telomeres is more A + T-rich than any other known telomere sequence. We propose that the common feature of all known telomere is the frequent occurrence of tracts of three or more adjacent guanine residues.

The unicellular alga Chlamydomonas reinhardtii is surrounded by a cell wall composed entirely of hydroxyproline-rich glycoproteins (HRGPs). When the walls of vegetative cells are removed with the enzyme gamete autolysin (g-lysin), they regenerate a matrix within 3-4 hr. In vitro translation of mRNAs isolated from g-lysin-treated cells showed significant increases and decreases in abundance of several mRNAs encoding proline-rich polypeptides. Because the population of up-regulated mRNAs is likely to include species encoding cell wall components, expression of genes for two outer wall HRGPs (GP1 and GP2) was analyzed during wall regeneration by using cDNAs isolated from a C. reinhardtii lambda gt11 library. Transcripts encoding GP1 and GP2 were elevated severalfold within the first hour or regeneration, suggesting that upregulation of HRGP mRNAs is a primary response to cell wall removal by g-lysin. Cell wall regeneration in Chlamydomonas provides an accessible system to study HRGP gene expression during matrix development.

The biflagellate alga Chlamydomonas has been used extensively in the genetic and biochemical analysis of flagellar assembly and motility. We have restored motility to a paralyzed-flagella mutant of Chlamydomonas by transforming with the corresponding wild-type gene. A nitrate reductase-deficient paralyzed-flagella strain, nit1-305 pf-14, carrying mutations in the genes for nitrate reductase and radial spoke protein 3, was transformed with wild-type copies of both genes. Two-thirds of the cells that survived nitrate selection also regained motility, indicating that they had been transformed with both the nitrate reductase and radial spoke protein 3 genes. Transformants typically contained multiple copies of both genes, genetically linked to each other, but not linked to the original mutant loci. Complementation of paralyzed-flagella mutants by transformation is a powerful tool for investigating flagellar assembly and function.

cDNA clones for the periplasmic carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) of Chlamydomonas reinhardtii cells were isolated and characterized. The fact that the cloned cDNA fragments encoded a 377-amino acid polypeptide (41,626 Da) consisting of an NH2-terminal hydrophobic signal peptide of 20 amino acids, a large (35,603 Da) subunit polypeptide, and a small (4144 Da) subunit polypeptide indicates that the two subunits are cotranslated as a precursor polypeptide. The amino acid sequence of mature subunits deduced from the nucleotide sequence showed 20-22% homology with human CA isozymes (CAI, CAII, and CAIII). Three zinc-liganded histidine residues and those forming a hydrogen-bond network to zinc-bound solvent molecules were highly conserved in the plant and animal enzymes. Three possible asparagine-linked glycosylation sites were found in the large subunit. Northern blot analysis was carried out using the cDNA fragment as a probe. The level of 2.0-kilobase CA mRNA increased within 1 hr when CO2 concentration of the bubbling gas was changed from 5% to 0.04% (air level) in the presence of light. On the other hand, CA mRNA did not accumulate when CO2 concentration was lowered in the dark. Experiments using 3-(3,4-dichlorophenyl)-1,1-dimethylurea showed that photosynthesis is absolutely required for the accumulation of CA mRNA. These results indicate that CA biosynthesis is regulated by changes in environmental CO2 concentration as well as light at the level of mRNA abundance.

We have developed a stable nuclear transformation system for the unicellular green alga Chlamydomonas reinhardtii. Transformation was accomplished by introducing the cloned C. reinhardtii oxygen-evolving enhancer protein 1 (OEE1) gene into C. reinhardtii cells by bombardment with DNA-coated tungsten particles. The recipient strain was an OEE1-deficient, nonphotosynthetic, acetate-requiring mutant, which recovered photosynthetic competence after transformation, and was therefore able to grow in the absence of acetate. Analysis of several transformants indicates that transformation has proceeded via second-site integration of the cloned gene, leaving the endogenous mutant gene intact. In genetic crosses of transformants with wild type, both mutant and wild-type phenotypes were recovered, showing that the photosynthetic competence of transformants was due not to reversion of the original locus but rather to expression of the introduced gene. We suggest that the success of the present system is largely due to using a homologous C. reinhardtii gene, leading to stable maintenance and expression of the gene. Transformation with heterologous genes may be problematic because of poor expression due to an unusual codon bias in C. reinhardtii.

By using a method in which cell-wall-deficient Chlamydomonas reinhardtii cells were agitated in the presence of DNA, glass beads, and polyethylene glycol, nuclear transformation rates of approximately 10(3) transformants per micrograms of plasmid DNA were achieved. The nitrate reductase gene from wild-type Chlamydomonas was used to complement a mutation in the corresponding gene of a strain containing nit1-305. Transformants were selected by growth with nitrate as sole source of nitrogen. The transforming DNA integrated into the genome at a low-copy number in nit+ transformants. When cells carrying nit1-305 were agitated in the presence of two plasmids, one with the gene for nitrate reductase and the second with an unselected gene, the unselected gene was present in 10-50% of nit+ transformants. This high frequency of cotransformation will allow any cloned gene to be introduced into Chlamydomonas. Moreover, the overall efficiency of transformation should be high enough to permit isolation of genes from genomic libraries by complementation of stable nuclear mutants. The availability of efficient nuclear and chloroplast transformation in Chlamydomonas provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and nuclear-chloroplast genome interactions.

The accumulation pattern of a number of mRNAs during the cell cycle of Chlamydomonas was examined by two-dimensional gel analysis of in vitro translation products and by RNA blot hybridization analysis. Two-dimensional gel analysis revealed that 10-15% of the 300 most abundant translation products are differentially synthesized from RNA obtained at various cell cycle stages. RNAs that direct the synthesis of alpha- and beta-tubulins and that hybridize to cloned alpha- and beta-tubulin probes accumulate coordinately during the predivision period of the cell cycle, reaching peak levels before or during division. Other RNAs represented by selected cloned cDNA probes show a number of different cell cycle patterns of accumulation. The accumulation patterns of these RNAs are not directly influenced by ongoing illumination conditions, even though alternating light-dark illumination cycles are used to synchronize Chlamydomonas cells. The results suggest that there may be a complex program of gene expression correlated with cell cycle progression in Chlamydomonas.

The Chlamydomonas reinhardtii mutant FUD50 has a deletion in the atpB gene of the chloroplast ATP synthase [Woessner, J. P., Masson, A., Harris, E. H., Bennoun, P., Gillham, N. W., and Boynton, J. E. (1984) Plant Mol. Biol. 3, 177-190]. We have isolated a suppressed strain (FUD50su) that can grow under phototrophic conditions, although it still showed no synthesis of the beta subunit of coupling factor 1. Thylakoid membranes of the FUD50su strain were similar to those of the original FUD50 strain, in that they both lacked all the subunits making up the chloroplast ATP synthase complex. We show that photosynthesis in FUD50su is sensitive to inhibitors such as antimycin, specific for mitochondrial electron transport. This observation indicates that photosynthesis in the FUD50su strain is achieved through an unusual interaction between mitochondria and chloroplast. Exportation of light-induced reduced compounds from the chloroplast to the mitochondria elicits ATP formation in the latter, and ATP is subsequently imported to the chloroplast. The activation of such an ATP shuttle coupled to an NADPH shuttle would thus provide the reducing power and the free energy needed for carbon assimilation in a chloroplast that lacks chloroplast ATP synthase.

The phylogenetic relationships among the chlorophyte Chlamydomonas reinhardtii, the chrysophyte Ochromonas danica, and the oomycete Achyla bisexualis were explored by comparing the sequences of their small-subunit ribosomal RNA coding regions. Comparisons of similarity values or inspection of phylogenetic trees constructed by distance matrix methods reveal a very close relationship between oomycetes and chrysophytes. The separation of chrysophytes from chlorophytes is comparable to that of plants from animals, and both separations are far antedated by the divergence of a number of other protist groups.

We have used inhibitors of methylation to evaluate the proposal that the extent of methylation of chloroplast DNA ( cpDNA ) of the mating type-plus (mt+) parent occurring during gametogenesis in wild-type Chlamydomonas renhardtii is directly correlated with the uniparental transmission of chloroplast genes by this parent [ Sager , R., Grabowy , C. & Sano , H. (1981) Cell 24, 41-47]. As detected by high-pressure liquid chromatography, the methylation of cpDNA was at its lowest level in the vegetative stage; the mt+ cells had a deoxycytidine methylation index (the percentage of deoxycytidine methylated) of 0.5, while the mating type-minus (mt-) index was lower by at least a factor of 3. This basal level of cpDNA methylation increased more than 20-fold after gametogenesis to give a methylation index of 12.1 and 4.3 for mt+ and mt- gametes, respectively. Another striking increase was detected at the 7-hr-zygote stage, resulting in the methylation of nearly half of the total deoxycytidine residues. The extent of zygotic cpDNA methylation was shown to be dependent on the preexisting methylation level of both parental gametic cpDNAs . L-Ethionine and 5-azacytidine effectively inhibited cpDNA methylation during gametogenesis and ensuing zygotic development as shown by both Hpa II/Msp I digestion patterns and HPLC. The transmission of chloroplast genes was analyzed concomitantly with the inhibitor studies. The two inhibitors produced different patterns of inhibition of methylation in mt- and mt+ cells at a given developmental stage. Our overall results demonstrate that the extent of mating type-specific and gamete-specific methylation during gametogenesis is not correlated with the frequency of maternal transmission of chloroplast genes.

Mitochondrial DNA (mtDNA) has been isolated from a mitochondrial pellet of Chlamydomonas reinhardtii. The mtDNA has a buoyant density of 1.706 g/ml in CsCl, a melting temperature of 87.9 degrees in standard saline citrate, and a nucleoside composition of 47.5% deoxyguanidine plus deoxycytidine with no odd nucleosides. Thermal denaturation and renaturation studies have shown that (i) mtDNA contains no extensive intramolecular heterogeneity nor significant base bias between the complementary polynucleotide chains and (ii) mtDNA renatures as a single homogeneous class with a kinetic complexity of 9.78 X 10(6) daltons. Although rare (less than or equal to 1%), both open and supercoiled circular mtDNA molecules have been observed in the electron microscope. Contour lengths of linear and open and closed circular molecules are all within the range of 4.0-5.4 micron with a mean of 4.67 +/- 0.30 micron. This size is similar to that of animal mtDNA but approximately 1/8 that of the higher plant mtDNAs. The magnitude of mtDNA reiteration in C. reinhardtii is estimated to be of the same order as that of chloroplast DNA.

The motility mutant of Chlamydomonas reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its (35)S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spoke-heads. To determine whether any of the missing proteins represent the mutant gene product two experimental approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with (35)S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.

Three group I introns of bacteriophage T4 have been compared with respect to their sequence and structural properties. The introns include the td intervening sequence, as well as the two newly described introns in the nrdB and sunY genes of T4. The T4 introns are very closely related, containing phylogenetically conserved sequence elements that allow them to be folded into a core structure that is characteristic of eukaryotic group IA introns. Similarities extend outward to the exon sequences surrounding the three introns. All three introns contain open reading frames (ORFs). Although the intron ORFs are not homologous and occur at different positions, all three ORFs are looped-out of the structure models, with only the 3' ends of each of the ORFs extending into the secondary structure. This arrangement invites interesting speculations on the regulation of splicing by translation. The high degree of similarity between the T4 introns and the eukaryotic group I introns must reflect a common ancestry, resulting either from vertical acquisition of a primordial RNA element or from horizontal transfer.

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational components and Chlamydomonas reinhardtii chloroplast DNA. Labeled chloroplast DNA was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor Tu. The chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. The region homologous to the elongation factor genes was located on the physical map of the chloroplast genome by probing restriction fragments of chloroplast DNA with cloned fragments, labeled in vitro, carrying the E. coli elongation factor Tu genes.

The nitrate reductase structural gene of Chlamydomonas reinhardtii has been isolated from a genomic library by using a nitrate reductase cDNA probe from barley. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-1. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binding domains of tobacco and Arabidopsis thaliana nitrate reductases and human NADH cytochrome b5 reductase. The identity of the cloned gene as nitrate reductase was confirmed by its ability to complement a nit-1 mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, significantly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.

Physical differences between organelle genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii have been used to demonstrate that sexual zygotes transmit chloroplast and mitochondrial DNA from opposite mating types. Processes responsible can be separated functionally and genetically, although both are controlled by mating type. In vegetative diploids, chloroplast and mitochondrial genomes are transmitted biparentally, but a 1-kilobase insert present in the C. smithii mitochondrial genome spreads unidirectionally to all C. reinhardtii genomes in a manner reminiscent of the intron found in the mitochondrial 21S rRNA gene of omega + strains of yeast.

We have used heterologous hybridization and DNA sequence analysis to determine whether the 16-kilobase-pair (kbp) DNA from Chlamydomonas reinhardtii mitochondria is the functional equivalent of mtDNA in other eukaryotes. Restriction fragments corresponding to a continuous internal stretch spanning 75% of the 16-kbp DNA have been cloned and mapped, and regions hybridizing with probes specific for the cytochrome oxidase subunit I [CytOx I (acronym COI)] and apocytochrome b (Cyt b) genes of yeast and the mitochondrial 26S and 18S rRNA genes of wheat have been identified. Sequence analysis has verified the presence of CytOx I and the large and small subunit rRNA genes in the C. reinhardtii 16-kbp DNA. In the region of the 16-kbp DNA corresponding to exon 4 in the yeast CytOx I gene, the derived amino acid sequence is 61% and 63% identical with the CytOx I amino acid sequences of yeast and human mitochondria, respectively. Notably, tryptophan is specified by TGG rather than by TGA in this section of the C. reinhardtii CytOx I gene. A probe from the CytOx I region of the 16-kbp DNA hybridizes only with this 16-kbp DNA in Southern blots of total cellular DNA from C. reinhardtii but with a larger DNA species in the total cellular DNA of C. moewusii and C. eugametos--two species that lack a 16-kbp DNA. These observations provide evidence that C. reinhardtii 16-kbp DNA comprises at least part of the mitochondrial genome of this organism and that a homologous DNA exists in other species of Chlamydomonas.

Two beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii have been isolated on the basis of altered sensitivity to the growth-inhibitory effect of colchicine. The two mutations of colR4 and colR15 have been found to be tightly linked, mapping to a previously unmarked site in linkage group XII. The drug-resistance phenotypes of both mutations segregated in genetic crosses with the presence of distinct, acidic variant beta-tubulin isoforms found assembled into the microtubules of the flagella. Analysis of the in vitro translation products of total poly(A)+ RNA from the mutants provided evidence that the variant proteins are altered primary beta-tubulin gene products. Compared to wild type, strains carrying the mutations expressed an increased resistance to the inhibitory effects of colchicine in clonal growth, flagellar assembly, and germination of meiotic products, suggesting that the beta-tubulin altered in the mutants participates in multiple microtubule functions.

The uni1 mutant of Chlamydomonas reinhardtii lacks one of the paired flagella seen in wild-type cells. The missing flagellum is cis to the eye spot and at this site the basal body is incomplete, lacking a transition zone. Together with nine other loci affecting flagellar assembly and two loci affecting flagellar function uni1 defines an approximately equal to 100-centimorgan linkage group with a circular map. Measurements of gene-centromere distances on the uni linkage group are consistent with the ordering of loci determined by recombination analysis and place the centromere near pf29, a motility-defective mutant. In well-synchronized meiotic cycles, recombination frequencies between loci on the uni linkage group but not loci on other linkage groups show striking temperature sensitivity during a 1- to 2-hr period 5 days before meiosis.

mRNA coding for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from Chlamydomonas reinhardi has been isolated from small polyribosomes immunoadsorbed to column-bound anti-LS antibody. 32P-Labeled LS mRNA was used as a hybridization probe to detect LS genes. The probe hybridized to C. reinhardi chloroplast DNA and at hybridization saturation revealed that there are approximately 75 LS genes per chloroplast. When chloroplast DNA was digested with the restriction endonuclease EcoRI and the fragments were transferred to a nitrocellulose filter, the LS mRNA probe hybridized to a DNA fragment of molecular weight 3.2 X 10(6). This same fragment codes (in part) for 16S and 23S chloroplast rRNAs, which are also coded (in part) by fragments of molecular weights 9.0, 2.3, and 0.4 X 10(6). The restriction fragment containing the LS gene has been cloned in the Escherichia coli plasmid pMB9.

The genome of Arabidopsis thaliana (Linnaeus) Heynhold was shown to contain an alpha-tubulin gene family consisting of at least four genes and/or pseudogenes. The primary structure of a transcribed alpha-tubulin gene was determined. A comparison of the predicted amino acid sequence of the A. thaliana alpha-tubulin with the predicted amino acid sequences of alpha-tubulins of Chlamydomonas reinhardtii, Stylonychia lemnae, and Homo spaiens reveals a high degree of homology; 90%, 87%, and 83% identity, respectively. Thus, a plant alpha-tubulin exhibits a high degree of homology to the alpha-tubulins of protists and animals. The coding sequence of the A. thaliana alpha-tubulin gene is interrupted by four introns, which occur at positions different from those of the less numerous introns of C. reinhardtii and rat alpha-tubulin genes. S1 nuclease mapping data showed that transcription is initiated 99 +/- 1 base pairs upstream from the translation initiation codon. Both 5' and 3' noncoding gene-specific probes were used to examine the expression of the alpha-tubulin gene in leaves, roots, and flowers by hybridization to total RNA isolated from these tissues. The results showed that the alpha-tubulin gene was transcribed in all three tissues.

A cDNA library from Chlamydomonas reinhardtii, constructed in the phage expression vector lambda gt11, was probed with antiserum directed against the nuclear-encoded gamma subunit of the chloroplast H+-transporting ATP synthase [ATP phosphohydrolase (H+-transporting) or chloroplast coupling factors 0 and 1, EC 3.6.1.34] of C. reinhardtii. A cDNA was isolated and transcribed in vitro. The transcript was translated in vitro and immunoprecipitated with anti-gamma-subunit serum to yield a product that coelectrophoresed with the immunoprecipitated product from in vitro-translated polyadenylylated RNA. These proteins were larger than the mature gamma subunit, either immunoprecipitated as chloroplast coupling factor 1 or as the individual subunit. Thus, the gamma subunit is synthesized as a precursor of greater molecular weight in C. reinhardtii. Furthermore, the precursor protein encoded by the cDNA is imported into pea chloroplasts and processed to a lower molecular weight polypeptide that coelectrophoreses with mature C. reinhardtii gamma subunit. The largest cDNA isolated is about the same length as the corresponding mRNA (approximately equal to 1900 bases long) and probably contains the entire coding region. Southern blot analyses revealed restriction fragment length polymorphisms and that the gamma subunit is probably encoded by an intron-containing single-copy gene.

The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25 degrees C but fails to grow photosynthetically at 35 degrees C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25 degrees C, but less than 15% when grown at 35 degrees C. Pulse-labeling with 35S showed that the decrease in enzyme level at the restrictive temperature was not a result of reduced synthesis of enzyme subunits. The CO2/O2 specificity factor (VCKO/VOKC, where VC and VO are Vmax values for carboxylation and oxygenation and KC and KO are Km values for CO2 and O2) of the mutant enzyme was found to be significantly less than that of the wild-type enzyme (54 +/- 2 and 62 +/- 1, respectively), and this alteration was accompanied by increases in KO and KC and a decrease in VC/VO. DNA sequencing revealed a single missense mutation in the 68-4PP chloroplast large-subunit gene. This mutation causes leucine to be replaced by phenylalanine at position 290 in the large-subunit polypeptide sequence. These results (i) support previous studies that implicated this region of the large subunit as an important structural component of the enzyme's function and (ii) demonstrate that chloroplast genetic modification of the CO2/O2 specificity factor of a plant-type carboxylase/oxygenase is feasible.