The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis and micronutrient homeostasis. Ten years since its genome project was initiated, an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the “omics” era. Housed at Phytozome, the Joint Genome Institute’s (JGI) plant genomics portal, the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of RNA-Seq data. Here, we present the past, present and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes.

With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment.

SUMMARY Reactive oxygen species (ROS) are produced by and have the potential to be damaging to all aerobic organisms. In photosynthetic organisms, they are an unavoidable byproduct of electron transfer in both the chloroplast and mitochondrion. We employ the reference unicellular green alga, Chlamydomonas reinhardtii, to identify the effect of H2O2 on gene expression by monitoring the transcriptome changes in a timecourse experiment. Comparison of transcriptomes from cells sampled immediately prior to addition of H2O2, and 0.5 and 1 h subsequently revealed 1278 differentially abundant transcripts. Of those transcripts that increase in abundance, many encode proteins involved in ROS detoxification, protein degradation and stress-responses, whereas among those that decrease are transcripts encoding proteins involved in photosynthesis and central carbon metabolism. In addition to these transcriptomic adjustments, we observe that H2O2 addition is followed by an accumulation and oxidation of the total intracellular glutathione pool, and a decrease in photosynthetic O2 output. Additionally, we analyze our transcriptomes in the context of transcript abundance changes in response to singlet O2 (O2*), and relate our H2O2-induced transcripts to a diurnal transcriptome, where we demonstrate enrichments of H2O2-induced transcripts early in the light phase, late in the light phase and 2 h prior to light. On this basis several genes that are highlighted in this work may be involved in previously undiscovered stress remediation pathways or acclimation responses.

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.

Autophagy is an intracellular self-degradation pathway by which eukaryotic cells recycle their own material in response to specific stress conditions. Exposure to high concentrations of metals causes cell damage, although the effect of metal stress on autophagy has not been explored in photosynthetic organisms. In this study, we investigated the effect of metal excess on autophagy in the model unicellular green alga Chlamydomonas reinhardtii. We show in cells treated with nickel an upregulation of ATG8 that is independent of CRR1, a global regulator of copper signaling in Chlamydomonas. A similar effect on ATG8 was observed with copper and cobalt but not with cadmium or mercury ions. Transcriptome sequencing data revealed an increase in the abundance of the protein degradation machinery, including that responsible for autophagy, and a substantial overlap of that increased abundance with the hydrogen peroxide response in cells treated with nickel ions. Thus, our results indicate that metal stress triggers autophagy in Chlamydomonas and suggest that excess nickel may cause oxidative damage, which in turn activates degradative pathways, including autophagy, to clear impaired components and recover cellular homeostasis. Eukaryotic cells are able to degrade and recycle their own material when they are exposed to nutrient starvation or other adverse conditions through a catabolic pathway known as macroautophagy or autophagy. This process is characterized by the formation of double-membrane vesicles termed autophagosomes that engulf and deliver cytosolic components to the vacuole/lysosome for degradation (1-4). The primary function of autophagy is to recycle cytoplasmic material as well as to clear damaged organelles or toxic cellular components generated during stress in order to maintain cellular homeostasis. In higher eukaryotes, autophagy has also been implicated in cell differentiation, development and cell death, and several human pathologies, such as cancer and neurodegenerative diseases (5, 6).Autophagy is mediated by highly conserved autophagy-related (ATG) genes, which have been described in organisms ranging from yeasts to mammals. Some ATG proteins are required for the formation of the autophagosome and constitute the core autophagy machinery (4,7,8). This group of proteins includes the ATG8 and ATG12 ubiquitin-like systems required for vesicle expansion. The ATG8 protein has been widely used to monitor autophagy in many systems (9) because, unlike other ATG proteins, this protein firmly binds to the autophagosome membrane through a covalent bond to phosphatidylethanolamine (PE). Most of the core ATG proteins are conserved in land plants (10-12) and in evolutionarily distant algae, including freshwater species, such as the model green alga Chlamydomonas reinhardtii (herein referred to as Chlamydomonas) (13) and marine species (14). Our current knowledge about autophagy in algae is still limited compared to our knowledge about autophagy in other eukaryotes, but recent studies, mainly performed in Chlamydomon...