Conservation and specialization of the Ycf2-FtsHi chloroplast protein import motor in green algae.

The protein import motor in chloroplasts plays a pivotal role in their biogenesis and homeostasis by driving the translocation of preproteins into chloroplasts. While the Ycf2-FtsHi complex serves as the import motor in land plants, its evolutionary conservation, specialization, and mechanisms across photosynthetic organisms are largely unexplored. Here, we isolated and determined the cryogenic electron microscopy (cryo-EM) structures of the native Ycf2-FtsHi complex from Chlamydomonas reinhardtii, uncovering a complex composed of up to 19 subunits, including multiple green-algae-specific components. The heterohexameric AAA+ ATPase motor module is tilted, potentially facilitating preprotein handover from the translocon at the inner chloroplast membrane (TIC) complex. Preprotein interacts with Ycf2-FtsHi and enhances its ATPase activity in vitro. Integrating Ycf2-FtsHi and translocon at the outer chloroplast membrane (TOC)-TIC supercomplex structures reveals insights into their physical and functional interplay during preprotein translocation. By comparing these findings with those from land plants, our study establishes a structural foundation for understanding the assembly, function, evolutionary conservation, and diversity of chloroplast protein import motors.

A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways.

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes.

The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-encoded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercomplex from Chlamydomonas, containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid ß-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110. A rigid intermembrane space (IMS) scaffold bridges two chloroplast membranes, and a large hydrophilic cleft on the IMS scaffold connects TOC and TIC, forming a pathway for preprotein translocation. Our study provides structural insights into the TOC-TIC supercomplex composition, assembly, and preprotein translocation mechanism, and lays a foundation to interpret the evolutionary conservation and diversity of this fundamental translocon machinery.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism.

Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.

The Eukaryotic CO2-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.

Approximately 30%-40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.

The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein.

Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.

Structure of a step II catalytically activated spliceosome from Chlamydomonas reinhardtii.

Pre-mRNA splicing, a fundamental step in eukaryotic gene expression, is executed by the spliceosomes. While there is extensive knowledge of the composition and structure of spliceosomes in yeasts and humans, the structural diversity of spliceosomes in non-canonical organisms remains unclear. Here, we present a cryo-EM structure of a step II catalytically activated spliceosome (C* complex) derived from the unicellular green alga Chlamydomonas reinhardtii at 2.6 Å resolution. This Chlamydomonas C* complex comprises 29 proteins and four RNA elements, creating a dynamic assembly that shares a similar overall architecture with yeast and human counterparts but also has unique features of its own. Distinctive structural characteristics include variations in protein compositions as well as some noteworthy RNA features. The splicing factor Prp17, with four fragments and a WD40 domain, is engaged in intricate interactions with multiple protein and RNA components. The structural elucidation of Chlamydomonas C* complex provides insights into the molecular mechanism of RNA splicing in plants and understanding splicing evolution in eukaryotes.

Methylation of ciliary dynein motors involves the essential cytosolic assembly factor DNAAF3/PF22.

Axonemal dynein motors drive ciliary motility and can consist of up to twenty distinct components with a combined mass of ~2 MDa. In mammals, failure of dyneins to assemble within the axonemal superstructure leads to primary ciliary dyskinesia. Syndromic phenotypes include infertility, rhinitis, severe bronchial conditions, and situs inversus. Nineteen specific cytosolic factors (Dynein Axonemal Assembly Factors; DNAAFs) are necessary for axonemal dynein assembly, although the detailed mechanisms involved remain very unclear. Here, we identify the essential assembly factor DNAAF3 as a structural ortholog of S-adenosylmethionine-dependent methyltransferases. We demonstrate that dynein heavy chains, especially those forming the ciliary outer arms, are methylated on key residues within various nucleotide-binding sites and on microtubule-binding domain helices directly involved in the transition to low binding affinity. These variable modifications, which are generally missing in a Chlamydomonas null mutant for the DNAAF3 ortholog PF22 (DAB1), likely impact on motor mechanochemistry fine-tuning the activities of individual dynein complexes.

A mutualistic bacterium rescues a green alga from an antagonist.

Photosynthetic protists, known as microalgae, are key contributors to primary production on Earth. Since early in evolution, they coexist with bacteria in nature, and their mode of interaction shapes ecosystems. We have recently shown that the bacterium Pseudomonas protegens acts algicidal on the microalga Chlamydomonas reinhardtii. It secretes a cyclic lipopeptide and a polyyne that deflagellate, blind, and lyse the algae [P. Aiyar et al., Nat. Commun. 8, 1756 (2017) and V. Hotter et al., Proc. Natl. Acad. Sci. U.S.A. 118, e2107695118 (2021)]. Here, we report about the bacterium Mycetocola lacteus, which establishes a mutualistic relationship with C. reinhardtii and acts as a helper. While M. lacteus enhances algal growth, it receives methionine as needed organic sulfur and the vitamins B1, B3, and B5 from the algae. In tripartite cultures with the alga and the antagonistic bacterium P. protegens, M. lacteus aids the algae in surviving the bacterial attack. By combining synthetic natural product chemistry with high-resolution mass spectrometry and an algal Ca2+ reporter line, we found that M. lacteus rescues the alga from the antagonistic bacterium by cleaving the ester bond of the cyclic lipopeptide involved. The resulting linearized seco acid does not trigger a cytosolic Ca2+ homeostasis imbalance that leads to algal deflagellation. Thus, the algae remain motile, can swim away from the antagonistic bacteria and survive the attack. All three involved genera cooccur in nature. Remarkably, related species of Pseudomonas and Mycetocola also act antagonistically against C. reinhardtii or as helper bacteria in tripartite cultures.

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts.

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology.

The chloroplast ATP synthase redox domain in Chlamydomonas reinhardtii eludes activity regulation for heterotrophic dark metabolism.

To maintain CO2 fixation in the Calvin-Benson-Bassham cycle, multistep regulation of the chloroplast ATP synthase (CF1Fo) is crucial to balance the ATP output of photosynthesis with protection of the apparatus. A well-studied mechanism is thiol modulation; a light/dark regulation through reversible cleavage of a disulfide in the CF1Fo γ-subunit. The disulfide hampers ATP synthesis and hydrolysis reactions in dark-adapted CF1Fo from land plants by increasing the required transmembrane electrochemical proton gradient ([Formula: see text]). Here, we show in Chlamydomonas reinhardtii that algal CF1Fo is differently regulated in vivo. A specific hairpin structure in the γ-subunit redox domain disconnects activity regulation from disulfide formation in the dark. Electrochromic shift measurements suggested that the hairpin kept wild-type CF1Fo active, whereas the enzyme was switched off in algal mutant cells expressing a plant-like hairpin structure. The hairpin segment swap resulted in an elevated [Formula: see text] threshold to activate plant-like CF1Fo, increased by ~1.4 photosystem (PS) I charge separations. The resulting dark-equilibrated [Formula: see text] dropped in the mutants by ~2.7 PSI charge separation equivalents. Photobioreactor experiments showed no phenotypes in autotrophic aerated mutant cultures. In contrast, chlorophyll fluorescence measurements under heterotrophic dark conditions point to an altered dark metabolism in cells with the plant-like CF1Fo as the result of bioenergetic deviations from wild-type. Our results suggest that the lifestyle of C. reinhardtii requires a specific CF1Fo dark regulation that partakes in metabolic coupling between the chloroplast and acetate-fueled mitochondria.

Dramatic changes in mitochondrial subcellular location and morphology accompany activation of the CO2 concentrating mechanism.

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for ~50% of global photosynthesis, compartmentalize their Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase (Rubisco) into a specialized structure known as the pyrenoid when the cells experience limiting CO2 conditions; this compartmentalization is a component of the CO2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO2 limitation, although a role for this reorganization in CCM function remains unclear. We used the green microalga Chlamydomonas reinhardtii to monitor changes in mitochondrial position and ultrastructure as cells transition between high CO2 and Low/Very Low CO2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral cell location and orient in parallel tubular arrays that extend along the cell's apico-basal axis. We show that these ultrastructural changes correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membranes, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, with the latter involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial respiration in VLC-acclimated cells reduces the affinity of the cells for Ci. Overall, our results suggest that mitochondrial repositioning functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

Quantitative proteomics reveals insights into the assembly of IFT trains and ciliary assembly.

Intraflagellar transport (IFT) is required for ciliary assembly. The IFT machinery comprises the IFT motors kinesin-2 and IFT dynein plus IFT-A and IFT-B complexes, which assemble into IFT trains in cilia. To gain mechanistic understanding of IFT and ciliary assembly, here, we performed an absolute quantification of IFT machinery in Chlamydomonas reinhardtii cilium. There are ∼756, ∼532, ∼276 and ∼350 molecules of IFT-B, IFT-A, IFT dynein and kinesin-2, respectively, per cilium. The amount of IFT-B is sufficient to sustain rapid ciliary growth in terms of tubulin delivery. The stoichiometric ratio of IFT-B:IFT-A:dynein is ∼3:2:1 whereas the IFT-B:IFT-A ratio in an IFT dynein mutant is 2:1, suggesting that there is a plastic interaction between IFT-A and IFT-B that can be influenced by IFT dynein. Considering diffusion of kinesin-2 during retrograde IFT, it is estimated that one kinesin-2 molecule drives eight molecules of IFT-B during anterograde IFT. These data provide new insights into the assembly of IFT trains and ciliary assembly.

Small RNA-mediated silencing of phototropin suppresses the induction of photoprotection in the green alga Chlamydomonas reinhardtii.

Small RNAs (sRNAs) form complexes with Argonaute proteins and bind to transcripts with complementary sequences to repress gene expression. sRNA-mediated regulation is conserved in a diverse range of eukaryotes and is involved in the control of various physiological functions. sRNAs are present in the unicellular green alga Chlamydomonas reinhardtii, and genetic analyses revealed that the core sRNA biogenesis and action mechanisms are conserved with those of multicellular organisms. However, the roles of sRNAs in this organism remain largely unknown. Here, we report that Chlamydomonas sRNAs contribute to the induction of photoprotection. In this alga, photoprotection is mediated by LIGHT HARVESTING COMPLEX STRESS-RELATED 3 (LHCSR3), whose expression is induced by light signals through the blue-light receptor phototropin (PHOT). We demonstrate here that sRNA-defective mutants showed increased PHOT abundance leading to greater LHCSR3 expression. Disruption of the precursor for two sRNAs predicted to bind to the PHOT transcript also increased PHOT accumulation and LHCSR3 expression. The induction of LHCSR3 in the mutants was enhanced by light containing blue wavelengths, but not by red light, indicating that the sRNAs regulate the degree of photoprotection via regulation of PHOT expression. Our results suggest that sRNAs are involved not only in the regulation of photoprotection but also in biological phenomena regulated by PHOT signaling.

A conserved RWP-RK transcription factor VSR1 controls gametic differentiation in volvocine algae.

Volvocine green algae are a model for understanding the evolution of mating types and sexes. They are facultatively sexual, with gametic differentiation occurring in response to nitrogen starvation (-N) in most genera and to sex inducer hormone in Volvox. The conserved RWP-RK family transcription factor (TF) MID is encoded by the minus mating-type locus or male sex-determining region of heterothallic volvocine species and dominantly determines minus or male gametic differentiation. However, the factor(s) responsible for establishing the default plus or female differentiation programs have remained elusive. We performed a phylo-transcriptomic screen for autosomal RWP-RK TFs induced during gametogenesis in unicellular isogamous Chlamydomonas reinhardtii (Chlamydomonas) and in multicellular oogamous Volvox carteri (Volvox) and identified a single conserved ortho-group we named Volvocine Sex Regulator 1 (VSR1). Chlamydomonas vsr1 mutants of either mating type failed to mate and could not induce expression of key mating-type-specific genes. Similarly, Volvox vsr1 mutants in either sex could initiate sexual embryogenesis, but the presumptive eggs or androgonidia (sperm packet precursors) were infertile and unable to express key sex-specific genes. Yeast two-hybrid assays identified a conserved domain in VSR1 capable of self-interaction or interaction with the conserved N terminal domain of MID. In vivo coimmunoprecipitation experiments demonstrated association of VSR1 and MID in both Chlamydomonas and Volvox. These data support a new model for volvocine sexual differentiation where VSR1 homodimers activate expression of plus/female gamete-specific-genes, but when MID is present, MID-VSR1 heterodimers are preferentially formed and activate minus/male gamete-specific-genes.

Pumping iron: A multi-omics analysis of two extremophilic algae reveals iron economy management.

Marine algae are responsible for half of the world's primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hypersaline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multiomics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a unique family of siderophore-iron-uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, which is closely related but identifiably distinct from the chlorophyll binding protein, LHCA3. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms.

Two specific domains of the γ subunit of chloroplast FoF1 provide redox regulation of the ATP synthesis through conformational changes.

Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the ß-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the ß-hairpin and the redox loop domains specific to CFoCF1.

The thylakoid phosphatase TEF8 is involved in state transition and high light stress resistance in Chlamydomonas.

The sophisticated regulation of state transition is required to maintain optimal photosynthetic performance under fluctuating light condition, through balancing the absorbed light energy between photosystem II and photosystem I. This exquisite process incorporates phosphorylation and dephosphorylation of light-harvesting complexes and PSII core subunits, accomplished by thylakoid membrane-localized kinases and phosphatases that have not been fully identified. In this study, one Chlamydomonas high light response gene, THYLAKOID ENRICHED FRACTION 8 (TEF8), was characterized. The Chlamydomonas tef8 mutant showed high light sensitivity and defective state transition. The enzymatic activity assays showed that TEF8 is a bona fide phosphatase localized in thylakoid membranes. Biochemical assays, including BN-PAGE, pull-down, and phosphopeptide mass spectrometry, proved that TEF8 associates with photosystem II and is involved in the dephosphorylation of D2 and CP29 subunits during state 2 to state 1 transition. Taken together, our results identified TEF8 as a thylakoid phosphatase with multiple dephosphorylation targets on photosystem II, and provide new insight into the regulatory mechanism of state transition and high light resistance in Chlamydomonas.

Utilizing high-resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas.

Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq yields genome-wide insight into translational dynamics and post-transcriptional control of gene expression. Here, we provide details on the Ribo-seq method and the subsequent analysis with the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas) for generating high-resolution data covering more than 10 000 different transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5' and 3' sections of transcripts and characteristics of eukaryotic translation termination, which are fundamentally distinct for chloroplast translation. In chloroplasts, a heterogeneous RPF size distribution along the coding sequence indicates specific regulatory phases during protein synthesis. For example, local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly uncovering an uncharacterized regulatory step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light-harvesting complex proteins, LHCs. By providing high-quality datasets for all subcellular genomes and attaching our data to the Chlamydomonas reference genome, we aim to make ribosome profiles easily accessible for the broad research community. The data can be browsed without advanced bioinformatic background knowledge for translation output levels of specific genes and their splice variants and for monitoring genome annotation.