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Riboregulators such as riboswitches and RNA thermometers provide simple, protein-independent tools to control gene expression at the post-transcriptional level. In bacteria, RNA thermometers regulate protein synthesis in response to temperature shifts. Thermometers outside of the bacterial world are rare, and in organellar genomes, no RNA thermometers have been identified to date. Here we report the discovery of an RNA thermometer in a chloroplast gene of the unicellular green alga Chlamydomonas reinhardtii. The thermometer, residing in the 5' untranslated region of the psaA messenger RNA forms a hairpin-type secondary structure that masks the Shine-Dalgarno sequence at 25°C. At 40°C, melting of the secondary structure increases accessibility of the Shine-Dalgarno sequence to initiating ribosomes, thus enhancing protein synthesis. By targeted nucleotide substitutions and transfer of the thermometer into Escherichia coli, we show that the secondary structure is necessary and sufficient to confer the thermometer properties. We also demonstrate that the thermometer provides a valuable tool for inducible transgene expression from the Chlamydomonas plastid genome, in that a simple temperature shift of the algal culture can greatly increase recombinant protein yields., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest regarding this study., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Small RNAs (sRNAs) associate with ARGONAUTE (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including 3 AGOs and 3 DICER-like proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long (>26 nt) sRNAs that associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these >26 nt sRNAs are derived from moderately repetitive genomic clusters and their biogenesis is DICER-independent. Interestingly, the sequences generating these >26-nt sRNAs have been conserved and amplified in several Chlamydomonas species. Moreover, expression of these longer sRNAs increases substantially under nitrogen or sulfur deprivation, concurrently with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences from which >26-nt sRNAs are produced might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, certain sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks., Competing Interests: Conflict of interest statement. The authors declare that they have no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Five versions of the Chlamydomonas reinhardtii reference genome have been produced over the last two decades. Here we present version 6, bringing significant advances in assembly quality and structural annotations. PacBio-based chromosome-level assemblies for two laboratory strains, CC-503 and CC-4532, provide resources for the plus and minus mating-type alleles. We corrected major misassemblies in previous versions and validated our assemblies via linkage analyses. Contiguity increased over ten-fold and >80% of filled gaps are within genes. We used Iso-Seq and deep RNA-seq datasets to improve structural annotations, and updated gene symbols and textual annotation of functionally characterized genes via extensive manual curation. We discovered that the cell wall-less classical reference strain CC-503 exhibits genomic instability potentially caused by deletion of the helicase RECQ3, with major structural mutations identified that affect >100 genes. We therefore present the CC-4532 assembly as the primary reference, although this strain also carries unique structural mutations and is experiencing rapid proliferation of a Gypsy retrotransposon. We expect all laboratory strains to harbor gene-disrupting mutations, which should be considered when interpreting and comparing experimental results. Collectively, the resources presented here herald a new era of Chlamydomonas genomics and will provide the foundation for continued research in this important reference organism., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

In the green alga Chlamydomonas reinhardtii, regulation of the cell cycle in response to external cues is critical for survival in a changing environment. The loss of the nuclear COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS 7 (CHT7) protein affects the expression of many genes especially in response to nitrogen availability. Cells lacking CHT7 exhibit abnormal cell morphology following nitrogen deprivation and fail to resume normal cell division after N resupply. To investigate the function of CHT7 in the regulation of cell cycle-related pathways, cells were synchronized, and RNA-seq analysis was performed during various stages of the cell cycle. In the cht7 mutant following nitrogen deprivation, the cells were not dividing, but a subset of cell cycle genes involved in DNA replication and mitosis were found to be derepressed, suggesting that the CHT7 protein plays a role in cell cycle regulation that is opposite to that of the mitotic cyclin-dependent kinases. Furthermore, genes for cell wall synthesis and remodeling were found to be abnormally induced in nondividing cht7 cells; this misregulation may deplete cellular resources and thus contribute to cell death following nitrogen deprivation. Lastly, 43 minimally characterized kinases were found to be highly misregulated in cht7. Further analysis suggested that some of these CHT7-regulated kinases may be related to the MAP3K and Aurora-like kinases, while others are unique. Together, these results suggest a role of CHT7 in transcriptional regulation of the cell cycle and reveal several pathways and genes whose expression appears to be subject to a CHT7-mediated regulatory network., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Proteases can regulate myriad biochemical pathways by digesting or processing target proteins. While up to 3% of eukaryotic genes encode proteases, only a tiny fraction of proteases are mechanistically understood. Furthermore, most of the current knowledge about proteases is derived from studies of a few model organisms, including Arabidopsis thaliana in the case of plants. Proteases in other plant model systems are largely unexplored territory, limiting our mechanistic comprehension of post-translational regulation in plants and hampering integrated understanding of how proteolysis evolved. We argue that the unicellular green alga Chlamydomonas reinhardtii has a number of technical and biological advantages for systematic studies of proteases, including reduced complexity of many protease families and ease of cell phenotyping. With this end in view, we share a genome-wide inventory of proteolytic enzymes in Chlamydomonas, compare the protease degradomes of Chlamydomonas and Arabidopsis, and consider the phylogenetic relatedness of Chlamydomonas proteases to major taxonomic groups. Finally, we summarize the current knowledge of the biochemical regulation and physiological roles of proteases in this algal model. We anticipate that our survey will promote and streamline future research on Chlamydomonas proteases, generating new insights into proteolytic mechanisms and the evolution of digestive and limited proteolysis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

De novo mutations are central for evolution, since they provide the raw material for natural selection by regenerating genetic variation. However, studying de novo mutations is challenging and is generally restricted to model species, so we have a limited understanding of the evolution of the mutation rate and spectrum between closely related species. Here, we present a mutation accumulation (MA) experiment to study de novo mutation in the unicellular green alga Chlamydomonas incerta and perform comparative analyses with its closest known relative, Chlamydomonas reinhardtii. Using whole-genome sequencing data, we estimate that the median single nucleotide mutation (SNM) rate in C. incerta is μ = 7.6 × 10-10, and is highly variable between MA lines, ranging from μ = 0.35 × 10-10 to μ = 131.7 × 10-10. The SNM rate is strongly positively correlated with the mutation rate for insertions and deletions between lines (r > 0.97). We infer that the genomic factors associated with variation in the mutation rate are similar to those in C. reinhardtii, allowing for cross-prediction between species. Among these genomic factors, sequence context and complexity are more important than GC content. With the exception of a remarkably high C→T bias, the SNM spectrum differs markedly between the two Chlamydomonas species. Our results suggest that similar genomic and biological characteristics may result in a similar mutation rate in the two species, whereas the SNM spectrum has more freedom to diverge., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent nonhomologous end-joining (cNHEJ) and DNA polymerase theta (POLQ)-mediated end joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair (HDR)/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect HDR of the inactivated paromomycin-resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate HDR/gene inactivation. Our data suggest that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced HDR and random integration of transgenes, whereas KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in C. reinhardtii and can be used for future development of algal biotechnology., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Despite its role as a reference organism in the plant sciences, the green alga Chlamydomonas reinhardtii entirely lacks genomic resources from closely related species. We present highly contiguous and well-annotated genome assemblies for three unicellular C. reinhardtii relatives: Chlamydomonas incerta, Chlamydomonas schloesseri, and the more distantly related Edaphochlamys debaryana. The three Chlamydomonas genomes are highly syntenous with similar gene contents, although the 129.2 Mb C. incerta and 130.2 Mb C. schloesseri assemblies are more repeat-rich than the 111.1 Mb C. reinhardtii genome. We identify the major centromeric repeat in C. reinhardtii as a LINE transposable element homologous to Zepp (the centromeric repeat in Coccomyxa subellipsoidea) and infer that centromere locations and structure are likely conserved in C. incerta and C. schloesseri. We report extensive rearrangements, but limited gene turnover, between the minus mating type loci of these Chlamydomonas species. We produce an eight-species core-Reinhardtinia whole-genome alignment, which we use to identify several hundred false positive and missing genes in the C. reinhardtii annotation and >260,000 evolutionarily conserved elements in the C. reinhardtii genome. In summary, these resources will enable comparative genomics analyses for C. reinhardtii, significantly extending the analytical toolkit for this emerging model system., (� The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS7 (CHT7) in Chlamydomonas ( Chlamydomonas reinhardtii ) was previously shown to affect the transcription of a subset of genes during nitrogen (N)-replete growth and following N refeeding. Here, we show that an extensive derepression of genes involved in DNA metabolism and cell cycle-related processes, as well as downregulation of genes encoding oxidoreductases and nutrient transporters, occurs in the cht7 mutant during N deprivation. Cellular mutant phenotypes are consistent with the observed transcriptome misregulation, as cht7 cells fail to properly arrest growth, nuclear replication, and cell division following N deprivation. Reduction in cht7 colony formation following N refeeding is explained by its compromised viability during N deprivation and by the occurrence of abortive divisions during N refeeding. Surprisingly, the largely unstructured C-terminal half of CHT7 with predicted protein binding domains, but not the canonical CXC DNA binding domain, is essential for the ability of CHT7 to form stable complexes and reverse the cellular phenotypes and transcription levels in the cht7 mutant. Hence, although lacking the presumed DNA binding domain, CHT7 modulates the expression of cell cycle genes in response to N availability, which is essential for establishing an effective quiescent state and the coordinated resumption of growth following N refeeding., (© 2020 American Society of Plant Biologists. All rights reserved.)

A major finding in organelle biology over the past decade is that land plant mitochondrial genomes, which are the largest among eukaryotes, can have a "Jekyll and Hyde" mutational pattern: low for synonymous sites, high for intergenic ones. This has led to the theory that double-strand breaks (DSBs) in the intergenic DNA of plant mitogenomes are repaired by inaccurate mechanisms, such as break-induced replication, which can result in large insertions and, thus, could explain why these genomes are so prone to expansion. But how universal is this theory? Can it apply to other giant organelle DNAs, such as the massive plastid DNAs (ptDNAs) of chlamydomonadalean green algae? Indeed, it can. Analysis of the expanded plastomes from two distinct isolates of the unicellular chlamydomonadalean Chlorosarcinopsis eremi uncovered exceptionally low rates of synonymous substitution in the coding regions but high substitution rates, including frequent indels, in the noncoding ptDNA, mirroring the trend from land plant mitogenomes. Remarkably, nearly all of the substitutions and indels identified in the noncoding ptDNA of C. eremi occur adjacent to or within short inverted palindromic repeats, suggesting that these elements are mutational hotspots. Building upon earlier studies, I propose that these palindromic repeats are predisposed to DSBs and that error-prone repair of these breaks is contributing to genomic expansion. Short palindromic repeats are a common theme among bloated plastomes, including the largest one on record, meaning that these data could have wide-reaching implications for our understanding of ptDNA expansion., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas ( Chlamydomonas reinhardtii ) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas., (© 2020 American Society of Plant Biologists. All rights reserved.)

Microalgae such as Chlamydomonas reinhardtii accumulate triacylglycerol (TAG), which is a potential source of biofuels, under stress conditions such as nitrogen deprivation, whereas Chlamydomonas debaryana NIES-2212 has previously been identified and characterized as one of the rare species of Chlamydomonas, which massively accumulates TAG in the stationary phase without external stress. As the high density of the cells in the stationary phase was supposed to act as a trigger for the accumulation of TAG in C. debaryana, in this study, C. debaryana was encapsulated in a Ca2+-alginate gel for the culture with high cell density. We discovered that the growth of the encapsulated cells resulted in the formation of spherical palmelloid colonies with high cell density, where daughter cells with truncated flagella remained wrapped within the mother cell walls. Interestingly, gel encapsulation markedly promoted proliferation of C. debaryana cells, and the encapsulated cells reached the stationary phase earlier than that of the free-living cells. Gel encapsulation also enhanced TAG accumulation. Gene expression analysis revealed that two genes of acyltransferases, DGAT1 and DGTT3, were upregulated in the stationary phase of free-living C. debaryana. In addition, the gene expression of these acyltransferases increased earlier in the encapsulated cells than that in the free-living cells. The enhanced production of TAG by alginate gel encapsulation was not found in C. reinhardtii which is known to use a different repertoire of acyltransferases in lipid accumulation., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Intracellular processes can be localized for efficiency or regulation. For example, localized mRNA translation by chloroplastic ribosomes occurs in the biogenesis of PSII, one of the two photosystems of the photosynthetic electron transport chain in the chloroplasts of plants and algae. The biogenesis of PSI and PSII requires the synthesis and assembly of their constituent polypeptide subunits, pigments, and cofactors. Although these biosynthetic pathways are well characterized, less is known about when and where they occur in developing chloroplasts. Here, we used fluorescence microscopy in the unicellular alga Chlamydomonas reinhardtii to reveal spatiotemporal organization in photosystem biogenesis. We focused on translation by chloroplastic ribosomes and chlorophyll biosynthesis in two developmental contexts of active photosystem biogenesis: (1) growth of the mature chloroplast and (2) greening of a nonphotosynthetic chloroplast. The results reveal that a translation zone is the primary location of the biogenesis of PSI and PSII. This discretely localized region within the chloroplast contrasts with the distributions of photosystems throughout this organelle and, therefore, is likely a hub where anabolic pathways converge for photosystem biogenesis.plantcell;31/12/3057/FX1F1fx1., (© 2019 American Society of Plant Biologists. All rights reserved.)

The unicellular alga Chlamydomonas reinhardtii is a classical reference organism for studying photosynthesis, chloroplast biology, cell cycle control, and cilia structure and function. It is also an emerging model for studying sensory cilia, the production of high-value bioproducts, and in situ structural determination. Much of the early appeal of Chlamydomonas was rooted in its promise as a genetic system, but like other classic model organisms, this rise to prominence predated the discovery of the structure of DNA, whole-genome sequences, and molecular techniques for gene manipulation. The haploid genome of C. reinhardtii facilitates genetic analyses and offers many of the advantages of microbial systems applied to a photosynthetic organism. C. reinhardtii has contributed to our understanding of chloroplast-based photosynthesis and cilia biology. Despite pervasive transgene silencing, technological advances have allowed researchers to address outstanding lines of inquiry in algal research. The most thoroughly studied unicellular alga, C. reinhardtii , is the current standard for algal research, and although genome editing is still far from efficient and routine, it nevertheless serves as a template for other algae. We present a historical retrospective of the rise of C. reinhardtii to illuminate its past and present. We also present resources for current and future scientists who may wish to expand their studies to the realm of microalgae., (© 2019 American Society of Plant Biologists. All rights reserved.)

The unicellular alga Chlamydomonas ( Chlamydomonas reinhardtii ) exhibits oriented movement responses (phototaxis) to light over more than three log units of intensity. Phototaxis thus depends on the cell's ability to adjust the sensitivity of its photoreceptors to ambient light conditions. In Chlamydomonas , the photoreceptors for phototaxis are the channelrhodopsins (ChR)1 and ChR2; these light-gated cation channels are located in the plasma membrane. Although ChRs are widely used in optogenetic studies, little is known about ChR signaling in algae. We characterized the in vivo phosphorylation of ChR1. Its reversible phosphorylation occurred within seconds as a graded response to changes in the light intensity and ionic composition of the medium and depended on an elevated cytosolic Ca 2+ concentration. Changes in the phototactic sign were accompanied by alterations in the phosphorylation status of ChR1. Furthermore, compared with the wild type, a permanently negative phototactic mutant required higher light intensities to evoke ChR1 phosphorylation. C-terminal truncation of ChR1 disturbed its reversible phosphorylation, whereas it was normal in ChR2-knockout and eyespot-assembly mutants. The identification of phosphosites in regions important for ChR1 function points to their potential regulatory role(s). We propose that multiple ChR1 phosphorylation, regulated via a Ca 2+ -based feedback loop, is an important component in the adaptation of phototactic sensitivity in Chlamydomonas ., (© 2019 American Society of Plant Biologists. All rights reserved.)

Autophagy is a recycling system for amino acids and carbon- and nitrogen (N)-containing compounds. To date, the functional importance of autophagy in microalgae in nutrient-deficient conditions has not been evaluated by using autophagy-defective mutants. Here, we provide evidence which supports the following notions by characterizing an insertional mutant of the autophagy-related gene ATG8, encoding a ubiquitin-like protein necessary for the formation of the autophagosome in the green alga, Chlamydomonas reinhardtii. First, ATG8 is required for maintenance of cell survival and Chl content in N-, sulfur- and phosphate-deficient conditions. Secondly, ATG8 supports the degradation of triacylglycerol and lipid droplets after the resupply of N to cells cultured in N-limiting conditions. Thirdly, ATG8 is also necessary for accumulation of starch in phosphate-deficient conditions. Additionally, autophagy is not essential for maternal inheritance of the organelle genomes in gametogenesis.

The fast-growing biflagellated single-celled chlorophyte Chlamydomonas reinhardtii is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from Staphylococcus aureus and Streptococcus pyogenes , and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP1/2 , COP3 (encoding channelrhodopsin 1 [ChR1]), COP4 (encoding ChR2), COP5 , PHOT , UVR8 , VGCC , MAT3 , and aCRY and created the chr1 chr2 and uvr8 phot double mutants. Characterization of the chr1 , chr2 , and mat3 mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae., (© 2017 American Society of Plant Biologists. All rights reserved.)

Epigenetic modifications, such as DNA methylation or histone modifications, can be transmitted between cellular or organismal generations. However, there are no experiments measuring their role in adaptation, so here we use experimental evolution to investigate how epigenetic variation can contribute to adaptation. We manipulated DNA methylation and histone acetylation in the unicellular green alga Chlamydomonas reinhardtii both genetically and chemically to change the amount of epigenetic variation generated or transmitted in adapting populations in three different environments (salt stress, phosphate starvation, and high CO2) for two hundred asexual generations. We find that reducing the amount of epigenetic variation available to populations can reduce adaptation in environments where it otherwise happens. From genomic and epigenomic sequences from a subset of the populations, we see changes in methylation patterns between the evolved populations over-represented in some functional categories of genes, which is consistent with some of these differences being adaptive. Based on whole genome sequencing of evolved clones, the majority of DNA methylation changes do not appear to be linked to cis-acting genetic mutations. Our results show that transgenerational epigenetic effects play a role in adaptive evolution, and suggest that the relationship between changes in methylation patterns and differences in evolutionary outcomes, at least for quantitative traits such as cell division rates, is complex., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In many contexts, the problem arises of determining which of many candidate mutations is the most likely to be causative for some phenotype. It is desirable to have a way to evaluate this probability that relies as little as possible on previous knowledge, to avoid bias against discovering new genes or functions. We have isolated mutants with blocked cell cycle progression in Chlamydomonas and determined mutant genome sequences. Due to the intensity of UV mutagenesis required for efficient mutant collection, the mutants contain multiple mutations altering coding sequence. To provide a quantitative estimate of probability that each individual mutation in a given mutant is the causative one, we developed a Bayesian approach. The approach employs four independent indicators: sequence conservation of the mutated coding sequence with Arabidopsis ; severity of the mutation relative to Chlamydomonas wild-type based on Blosum62 scores; meiotic mapping information for location of the causative mutation relative to known molecular markers; and, for a subset of mutants, the transcriptional profile of the candidate wild-type genes through the mitotic cell cycle. These indicators are statistically independent, and so can be combined quantitatively into a single probability calculation. We validate this calculation: recently isolated mutations that were not in the training set for developing the indicators, with high calculated probability of causality, are confirmed in every case by additional genetic data to indeed be causative. Analysis of "best reciprocal BLAST" (BRB) relationships among Chlamydomonas and other eukaryotes indicate that the temperature sensitive-lethal (Ts-lethal) mutants that our procedure recovers are highly enriched for fundamental cell-essential functions conserved broadly across plants and other eukaryotes, accounting for the high information content of sequence alignment to Arabidopsis ., (Copyright © 2017 Cross et al.)

With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2 Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone., (Copyright © 2016 Hilton et al.)

The Chlamydomonas genome has been sequenced, assembled, and annotated to produce a rich resource for genetics and molecular biology in this well-studied model organism. However, the current reference genome contains ∼1000 blocks of unknown sequence ('N-islands'), which are frequently placed in introns of annotated gene models. We developed a strategy to search for previously unknown exons hidden within such blocks, and determine the sequence, and exon/intron boundaries, of such exons. These methods are based on assembly and alignment of short cDNA and genomic DNA reads, completely independent of prior reference assembly or annotation. Our evidence indicates that a substantial proportion of the annotated intronic N-islands contain hidden exons. For most of these, our algorithm recovers full exonic sequence with associated splice junctions and exon-adjacent intronic sequence. These new exons represent de novo sequence generally present nowhere in the assembled genome, and the added sequence improves evolutionary conservation of the predicted encoded peptides., (Copyright © 2016 Tulin, Cross.)

The Chlamydomonas genome has been sequenced, assembled, and annotated to produce a rich resource for genetics and molecular biology in this well-studied model organism. The annotated genome is very rich in open reading frames upstream of the annotated coding sequence ('uORFs'): almost three quarters of the assigned transcripts have at least one uORF, and frequently more than one. This is problematic with respect to the standard 'scanning' model for eukaryotic translation initiation. These uORFs can be grouped into three classes: class 1, initiating in-frame with the coding sequence (CDS) (thus providing a potential in-frame N-terminal extension); class 2, initiating in the 5' untranslated sequences (5UT) and terminating out-of-frame in the CDS; and class 3, initiating and terminating within the 5UT. Multiple bioinformatics criteria (including analysis of Kozak consensus sequence agreement and BLASTP comparisons to the closely related Volvox genome, and statistical comparison to cds and to random sequence controls) indicate that of ∼4000 class 1 uORFs, approximately half are likely in vivo translation initiation sites. The proposed resulting N-terminal extensions in many cases will sharply alter the predicted biochemical properties of the encoded proteins. These results suggest significant modifications in ∼2000 of the ∼20,000 transcript models with respect to translation initiation and encoded peptides. In contrast, class 2 uORFs may be subject to purifying selection, and the existent ones (surviving selection) are likely inefficiently translated. Class 3 uORFs are found in more than half of transcripts, frequently multiple times per transcript; however, they are remarkably similar to random sequence expectations with respect to size, number, and composition, and therefore may in most cases be selectively neutral., (Copyright © 2016 Cross.)

By enabling a tight control of cell excitation, optogenetics is a powerful approach to study the function of neurons and neural circuits. With its transparent body, a fully mapped nervous system, easily quantifiable behaviors and many available genetic tools, Caenorhabditis elegans is an extremely well-suited model to decipher the functioning logic of the nervous system with optogenetics. Our goal was to establish an efficient dual color optogenetic system for the independent excitation of different neurons in C. elegans. We combined two recently discovered channelrhodopsins: the red-light sensitive Chrimson from Chlamydomonas noctigama and the blue-light sensitive CoChR from Chloromonas oogama. Codon-optimized versions of Chrimson and CoChR were designed for C. elegans and expressed in different mechanosensory neurons. Freely moving animals produced robust behavioral responses to light stimuli of specific wavelengths. Since CoChR was five times more sensitive to blue light than the commonly used ChR2, we were able to use low blue light intensities producing no cross-activation of Chrimson. Thanks to these optogenetics tools, we revealed asymmetric cross-habituation effects between the gentle and harsh touch sensory motor pathways. Collectively, our results establish the Chrimson/CoChR pair as a potent tool for bimodal neural excitation in C. elegans and equip this genetic model organism for the next generation of in vivo optogenetic analyses., (Copyright © 2015 by the Genetics Society of America.)

We characterized two spontaneous and dominant nuclear mutations in the unicellular alga Chlamydomonas reinhardtii, ncc1 and ncc2 (for nuclear control of chloroplast gene expression), which affect two octotricopeptide repeat (OPR) proteins encoded in a cluster of paralogous genes on chromosome 15. Both mutations cause a single amino acid substitution in one OPR repeat. As a result, the mutated NCC1 and NCC2 proteins now recognize new targets that we identified in the coding sequences of the chloroplast atpA and petA genes, respectively. Interaction of the mutated proteins with these targets leads to transcript degradation; however, in contrast to the ncc1 mutation, the ncc2 mutation requires on-going translation to promote the decay of the petA mRNA. Thus, these mutants reveal a mechanism by which nuclear factors act on chloroplast mRNAs in Chlamydomonas. They illustrate how diversifying selection can allow cells to adapt the nuclear control of organelle gene expression to environmental changes. We discuss these data in the wider context of the evolution of regulation by helical repeat proteins., (© 2015 American Society of Plant Biologists. All rights reserved.)

Reverse transcription of mRNA is thought to be an important first step in a model that explains certain evolutionary changes within genes, such as the loss of introns or RNA editing sites. In this model, reverse transcription of mRNA produces cDNA molecules that replace part of the parental gene by homologous recombination. In vivo evidence of reverse transcription of physiologically relevant mRNAs is generally lacking, however, except in genetically engineered cells. Here, we provide in vivo evidence for reverse transcription of the chloroplast psbA mRNA in two naturally occurring species of Chlamydomonas (raudensis and subcaudata) that is based on the presence of spliced cDNAs in both organisms. The psbA cDNAs, which lack the group II intron of the genomic gene, are nearly full length, and the majority of them--though not all--are in the form of RNA-cDNA hybrids. Moreover, the presence in these species of psbA cDNAs is correlated with the loss of an early group I intron from the same psbA gene. The group II intron that interrupts psbA in C. raudensis and C. subcaudata potentially encodes a protein with a reverse transcriptase domain, and the C. raudensis protein was shown to have reverse transcriptase activity in vitro. These results provide strong evidence for reverse transcription of a physiologically important mRNA (psbA) in two species of Chlamydomonas that have also lost an intron from the same gene, possibly through recombination with the cDNA.

Microbial experimental evolution uses a few well-characterized model systems to answer fundamental questions about how evolution works. This special section highlights novel model systems for experimental evolution, with a focus on marine model systems that can be used to understand evolutionary responses to global change in the oceans., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

The molecular bases for the evolution of male-female sexual dimorphism are possible to study in volvocine algae because they encompass the entire range of reproductive morphologies from isogamy to oogamy. In 1978, Charlesworth suggested the model of a gamete size gene becoming linked to the sex-determining or mating type locus (MT) as a mechanism for the evolution of anisogamy. Here, we carried out the first comprehensive study of a candidate MT-linked oogamy gene, MAT3/RB, across the volvocine lineage. We found that evolution of anisogamy/oogamy predates the extremely high male-female divergence of MAT3 that characterizes the Volvox carteri lineage. These data demonstrate very little sex-linked sequence divergence of MAT3 between the two sexes in other volvocine groups, though linkage between MAT3 and the mating locus appears to be conserved. These data implicate genetic determinants other than or in addition to MAT3 in the evolution of anisogamy in volvocine algae.

The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.

Mutations at the APM1 and APM2 loci in the green alga Chlamydomonas reinhardtii confer resistance to phosphorothioamidate and dinitroaniline herbicides. Genetic interactions between apm1 and apm2 mutations suggest an interaction between the gene products. We identified the APM1 and APM2 genes using a map-based cloning strategy. Genomic DNA fragments containing only the DNJ1 gene encoding a type I Hsp40 protein rescue apm1 mutant phenotypes, conferring sensitivity to the herbicides and rescuing a temperature-sensitive growth defect. Lesions at five apm1 alleles include missense mutations and nucleotide insertions and deletions that result in altered proteins or very low levels of gene expression. The HSP70A gene, encoding a cytosolic Hsp70 protein known to interact with Hsp40 proteins, maps near the APM2 locus. Missense mutations found in three apm2 alleles predict altered Hsp70 proteins. Genomic fragments containing the HSP70A gene rescue apm2 mutant phenotypes. The results suggest that a client of the Hsp70-Hsp40 chaperone complex may function to increase microtubule dynamics in Chlamydomonas cells. Failure of the chaperone system to recognize or fold the client protein(s) results in increased microtubule stability and resistance to the microtubule-destabilizing effect of the herbicides. The lack of redundancy of genes encoding cytosolic Hsp70 and Hsp40 type I proteins in Chlamydomonas makes it a uniquely valuable system for genetic analysis of the function of the Hsp70 chaperone complex.

Molybdenum (Mo) is a very scarce element whose function is fundamental in living beings within the active site of Mo-oxidoreductases, playing key roles in the metabolism of N, S, purines, hormone biosynthesis, transformation of drugs and xenobiotics, etc. In eukaryotes, each step from Mo acquisition until its incorporation into a biologically active molybdenum cofactor (Moco) together with the assembly of this Moco in Mo-enzymes is almost understood. The deficiency in function of a particular molybdoenzyme can be critical for the survival of the organism dependent on the pathway involved. However, incapacity in forming a functional Moco has a pleiotropic effect in the different processes involving this cofactor. A detailed overview of Mo metabolism: (a) specific transporters for molybdate, (b) the universal biosynthesis pathway for Moco from GTP, (c) Moco-carrier and Moco-binding proteins for Moco transfer and (d) Mo-enzymes, is analyzed in light of recent findings and three systems are compared, the unicellular microalga Chlamydomonas, the plant Arabidopsis and humans.

Extending the carotenoid pathway to astaxanthin in plants is of scientific and industrial interest. However, expression of a microbial β-carotene ketolase (BKT) that catalyses the formation of ketocarotenoids in transgenic plants typically results in low levels of astaxanthin. The low efficiency of BKTs in ketolating zeaxanthin to astaxanthin is proposed to be the major limitation for astaxanthin accumulation in engineered plants. To verify this hypothesis, several algal BKTs were functionally characterized using an Escherichia coli system and three BKTs were identified, with high (up to 85%), moderate (∼38%), and low (∼1%) conversion rate from zeaxanthin to astaxanthin from Chlamydomonas reinhardtii (CrBKT), Chlorella zofingiensis (CzBKT), and Haematococcus pluvialis (HpBKT3), respectively. Transgenic Arabidopsis thaliana expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g(-1) dry weight with a 1.8-fold increase in total carotenoids. In contrast, the expression of CzBKT resulted in much lower astaxanthin content (0.24 mg g(-1) dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in A. thaliana. The none-native astaxanthin was found mostly in a free form integrated into the light-harvesting complexes of photosystem II in young leaves but in esterified forms in senescent leaves. The alteration of carotenoids did not affect chlorophyll content, plant growth, or development significantly. The astaxanthin-producing plants were more tolerant to high light as shown by reduced lipid peroxidation. This study advances a decisive step towards the utilization of plants for the production of high-value astaxanthin.

In this work, we query the Chlamydomonas reinhardtii copper regulon at a whole-genome level. Our RNA-Seq data simulation and analysis pipeline validated a 2-fold cutoff and 10 RPKM (reads per kilobase of mappable length per million mapped reads) (~1 mRNA per cell) to reveal 63 CRR1 targets plus another 86 copper-responsive genes. Proteomic and immunoblot analyses captured 25% of the corresponding proteins, whose abundance was also dependent on copper nutrition, validating transcriptional regulation as a major control mechanism for copper signaling in Chlamydomonas. The impact of copper deficiency on the expression of several O₂-dependent enzymes included steps in lipid modification pathways. Quantitative lipid profiles indicated increased polyunsaturation of fatty acids on thylakoid membrane digalactosyldiglycerides, indicating a global impact of copper deficiency on the photosynthetic apparatus. Discovery of a putative plastid copper chaperone and a membrane protease in the thylakoid suggest a mechanism for blocking copper utilization in the chloroplast. We also found an example of copper sparing in the N assimilation pathway: the replacement of copper amine oxidase by a flavin-dependent backup enzyme. Forty percent of the targets are previously uncharacterized proteins, indicating considerable potential for new discovery in the biology of copper.

We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit-RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle-regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.

We investigate how different rates of environmental change affect adaptive outcomes and dynamics by selecting Chlamydomonas populations for over 200 generations in environments where the rate of change varies. We find that slower rates of environmental change result in end populations that grow faster and pay a lower cost of adaptation than populations that adapt to a sudden change of the same magnitude. We detected partial selective sweeps in adapting populations by monitoring changes in marker frequency in each population. Although populations adapting to a sudden environmental change showed evidence of mutations of large effect segregating early on, populations adapting to slow rates of change showed patterns that were consistent with mutations of relatively small effect occurring at less predictable times. This work suggests that rates of environmental change may fundamentally alter adaptive dynamics and outcomes of adaptation by changing the size and timing of fitness increases. We suggest that using mutations of smaller effect during adaptation may result in lower levels of pleiotropy and historical constraints, which could in turn result in higher fitness by the end of the experiment.

The Chlamydomonas reinhardtii PSR1 gene is required for proper acclimation of the cells to phosphorus (P) deficiency. P-starved psr1 mutants show signs of secondary sulfur (S) starvation, exemplified by the synthesis of extracellular arylsulfatase and the accumulation of transcripts encoding proteins involved in S scavenging and assimilation. Epistasis analysis reveals that induction of the S-starvation responses in P-limited psr1 cells requires the regulatory protein kinase SNRK2.1, but bypasses the membrane-targeted activator, SAC1. The inhibitory kinase SNRK2.2 is necessary for repression of S-starvation responses during both nutrient-replete growth and P limitation; arylsulfatase activity and S deficiency-responsive genes are partially induced in the P-deficient snrk2.2 mutants and become fully activated in the P-deficient psr1snrk2.2 double mutant. During P starvation, the sac1snrk2.2 double mutants or the psr1sac1snrk2.2 triple mutants exhibit reduced arylsulfatase activity compared to snrk2.2 or psr1snrk2.2, respectively, but the sac1 mutation has little effect on the abundance of S deficiency-responsive transcripts in these strains, suggesting a post-transcriptional role for SAC1 in elicitation of S-starvation responses. Interestingly, P-starved psr1snrk2.2 cells bleach and die more rapidly than wild-type or psr1 strains, suggesting that activation of S-starvation responses during P deprivation is deleterious to the cell. From these results we infer that (i) P-deficient growth causes some internal S limitation, but the S-deficiency responses are normally inhibited during acclimation to P deprivation; (ii) the S-deficiency responses are not completely suppressed in P-deficient psr1 cells and consequently these cells synthesize some arylsulfatase and exhibit elevated levels of transcripts for S-deprivation genes; and (iii) this increased expression is controlled by regulators that modulate transcription of S-responsive genes during S-deprivation conditions. Overall, the work strongly suggests integration of the different circuits that control nutrient-deprivation responses in Chlamydomonas.

We recovered microorganisms from five ice core samples from three glaciers (Puruogangri, Malan, and Dunde) located in the Tibetan Plateau in China and analyzed their small subunit rRNA gene sequences. Most of the bacterial sequences were unknown previously; the most closely related known sequences were from bacteria of the Proteobacteria, Bacteroidetes, and Actinobacteria phyla. Chlorophyta, Streptophyta, Ciliophora, and fungal groups were represented among the 18S rRNA gene sequences that we obtained. The most abundantly represented glacial bacteria were Bacteroidetes, and Chlamydomonas was the predominant eukaryote. Comparative analysis showed that the Bacteroidetes sequences obtained from this study were highly similar to one another but most were only distantly related to previously characterized Bacteroidetes (<92% identity). We propose that our Bacteroidetes sequences represent two novel subgroups: one at the family level and one at the genus level. The unique ice environment and the high abundance of Bacteroidetes, combined with the coexistence of a high abundance of psychrophilic Chlamydomonas, strongly suggests that there is a viable ecosystem on the surface of Tibetan glaciers. Comparisons of microbial community structures in the five ice samples showed distinct differences, likely due to environmental differences in the locations in which the samples were obtained.

Plastids are descended from a cyanobacterial symbiosis which occurred over 1.2 billion years ago. During the course of endosymbiosis, most genes were lost from the cyanobacterium's genome and many were relocated to the host nucleus through endosymbiotic gene transfer (EGT). The issue of how many genes were acquired through EGT in different plant lineages is unresolved. Here, we report the genome-wide frequency of gene acquisitions from cyanobacteria in 4 photosynthetic eukaryotes--Arabidopsis, rice, Chlamydomonas, and the red alga Cyanidioschyzon--by comparison of the 83,138 proteins encoded in their genomes with 851,607 proteins encoded in 9 sequenced cyanobacterial genomes, 215 other reference prokaryotic genomes, and 13 reference eukaryotic genomes. The analyses entail 11,569 phylogenies inferred with both maximum likelihood and Neighbor-Joining approaches. Because each phylogenetic result is dependent not only upon the reconstruction method but also upon the site patterns in the underlying alignment, we investigated how the reliability of site pattern generation via alignment affects our results: if the site patterns in an alignment differ depending upon the order in which amino acids are introduced into multiple sequence alignment--N- to C-terminal versus C- to N-terminal--then the phylogenetic result is likely to be artifactual. Excluding unreliable alignments by this means, we obtain a conservative estimate, wherein about 14% of the proteins examined in each plant genome indicate a cyanobacterial origin for the corresponding nuclear gene, with higher proportions (17-25%) observed among the more reliable alignments. The identification of cyanobacterial genes in plant genomes affords access to an important question: from which type of cyanobacterium did the ancestor of plastids arise? Among the 9 cyanobacterial genomes sampled, Nostoc sp. PCC7120 and Anabaena variabilis ATCC29143 were found to harbor collections of genes which are-in terms of presence/absence and sequence similarity-more like those possessed by the plastid ancestor than those of the other 7 cyanobacterial genomes sampled here. This suggests that the ancestor of plastids might have been an organism more similar to filamentous, heterocyst-forming (nitrogen-fixing) representatives of section IV recognized in Stanier's cyanobacterial classification. Members of section IV are very common partners in contemporary symbiotic associations involving endosymbiotic cyanobacteria, which generally provide nitrogen to their host, consistent with suggestions that fixed nitrogen supplied by the endosymbiont might have played an important role during the origin of plastids.

The retinoblastoma (RB) protein is a eukaryotic tumor suppressor and negative cell-cycle regulator. Chlamydomonas reinhardtii cells that lack the RB homolog MAT3 show loss of size checkpoint control and deregulated cell-cycle progression leading to the production of tiny cells. We carried out an insertional mutagenesis screen to isolate bypass suppressors of mat3 (smt mutants) that reverted the mat3 cell-size defect. Previously we reported that the loci encoding Chlamydomonas homologs of E2F and DP were frequently disrupted in this screen, indicating that the architecture of the canonical RB pathway is conserved in Chlamydomonas with MAT3/RB acting as a negative regulator upstream of E2F/DP. Here, we describe four novel smt mutants that moderately suppressed the cell-size checkpoint and cell-cycle phenotypes of mat3. As single mutants, three of the smt strains displayed no obvious phenotypes, and one had a slightly small phenotype. Strikingly, several smt double-mutant combinations synergized to cause enhanced suppression of mat3 and even to cause a large-cell phenotype that is comparable to that caused by loss of DP1. Molecular characterization of one smt mutant revealed that suppression is due to a defect in a gene encoding a putative small ubiquitin-like modifier (SUMO) peptidase. Our results reveal a complex genetic network that lies downstream of MAT3/RB and implicate protein sumoylation as an important step for cell-cycle progression in cells that are missing MAT3/RB.

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.

The transition from unicellular to differentiated multicellular organisms constitutes an increase in the level complexity, because previously existing individuals are combined to form a new, higher-level individual. The volvocine algae represent a unique opportunity to study this transition because they diverged relatively recently from unicellular relatives and because extant species display a range of intermediate grades between unicellular and multicellular, with functional specialization of cells. Following the approach Darwin used to understand "organs of extreme perfection" such as the vertebrate eye, this jump in complexity can be reduced to a series of small steps that cumulatively describe a gradual transition between the two levels. We use phylogenetic reconstructions of ancestral character states to trace the evolution of steps involved in this transition in volvocine algae. The history of these characters includes several well-supported instances of multiple origins and reversals. The inferred changes can be understood as components of cooperation-conflict-conflict mediation cycles as predicted by multilevel selection theory. One such cycle may have taken place early in volvocine evolution, leading to the highly integrated colonies seen in extant volvocine algae. A second cycle, in which the defection of somatic cells must be prevented, may still be in progress.

Positive signaling by nitrate in its assimilation pathway has been studied in Chlamydomonas reinhardtii. Among >34,000 lines generated by plasmid insertion, 10 mutants were unable to activate nitrate reductase (NIA1) gene expression and had a Nit(-) (no growth in nitrate) phenotype. Each of these 10 lines was mutated in the nitrate assimilation-specific regulatory gene NIT2. The complete NIT2 cDNA sequence was obtained, and its deduced amino acid sequence revealed GAF, Gln-rich, Leu zipper, and RWP-RK domains typical of transcription factors and transcriptional coactivators associated with signaling pathways. The predicted Nit2 protein sequence is structurally related to the Nin (for nodule inception) proteins from plants but not to NirA/Nit4/Yna proteins from fungi and yeast. NIT2 expression is negatively regulated by ammonium and is optimal in N-free medium with no need for the presence of nitrate. However, intracellular nitrate is required to allow Nit2 to activate the NIA1 promoter activity. Nit2 protein was expressed in Escherichia coli and shown to bind to specific sequences at the NIA1 gene promoter. Our data indicate that NIT2 is a central regulatory gene required for nitrate signaling on the Chlamydomonas NIA1 gene promoter and that intracellular nitrate is needed for NIT2 function and to modulate NIA1 transcript levels.