Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization.

Successful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the green alga Chlamydomonas reinhardtii, Cu import is dependent on a transcription factor, Copper Response Regulator 1 (CRR1), responsible for activating genes in Cu-deficient cells. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1 and CTR2) and a related soluble protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation of CTR1, but not CTR2, recapitulates the poor growth of crr1 in Cu-deficient medium, consistent with a dominant role for CTR1 in high-affinity Cu(I) uptake. On the other hand, the overaccumulation of Cu(I) (20 times the quota) in zinc (Zn) deficiency depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation of CTR gene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation of CTR2 expression, again distinguishing CTR2 from CTR1 and consistent with the lower substrate affinity of CTR2. ONE SENTENCE SUMMARY: Regulation of Cu uptake and sequestration by members of the CTR family of proteins in Chlamydomonas.

Subscaling of a cytosolic RNA binding protein governs cell size homeostasis in the multiple fission alga Chlamydomonas.

Coordination of growth and division in eukaryotic cells is essential for populations of proliferating cells to maintain size homeostasis, but the underlying mechanisms that govern cell size have only been investigated in a few taxa. The green alga Chlamydomonas reinhardtii (Chlamydomonas) proliferates using a multiple fission cell cycle that involves a long G1 phase followed by a rapid series of successive S and M phases (S/M) that produces 2n daughter cells. Two control points show cell-size dependence: the Commitment control point in mid-G1 phase requires the attainment of a minimum size to enable at least one mitotic division during S/M, and the S/M control point where mother cell size governs cell division number (n), ensuring that daughter distributions are uniform. tny1 mutants pass Commitment at a smaller size than wild type and undergo extra divisions during S/M phase to produce small daughters, indicating that TNY1 functions to inhibit size-dependent cell cycle progression. TNY1 encodes a cytosolic hnRNP A-related RNA binding protein and is produced once per cell cycle during S/M phase where it is apportioned to daughter cells, and then remains at constant absolute abundance as cells grow, a property known as subscaling. Altering the dosage of TNY1 in heterozygous diploids or through mis-expression increased Commitment cell size and daughter cell size, indicating that TNY1 is a limiting factor for both size control points. Epistasis placed TNY1 function upstream of the retinoblastoma tumor suppressor complex (RBC) and one of its regulators, Cyclin-Dependent Kinase G1 (CDKG1). Moreover, CDKG1 protein and mRNA were found to over-accumulate in tny1 cells suggesting that CDKG1 may be a direct target of repression by TNY1. Our data expand the potential roles of subscaling proteins outside the nucleus and imply a control mechanism that ties TNY1 accumulation to pre-division mother cell size.

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii.

Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.

Outdoor cultivation and metabolomics exploration of Chlamydomonas engineered for bisabolene production.

Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineeredC. reinhardtii,establishing it as a one-cell two-wells biorefinery.

The Small Interactor of PKD2 protein promotes the assembly and ciliary entry of the Chlamydomonas PKD2-mastigoneme complexes.

In Chlamydomonas, the channel polycystin 2 (PKD2) is primarily present in the distal region of cilia, where it is attached to the axoneme and mastigonemes, extracellular polymers of MST1. In a smaller proximal ciliary region that lacks mastigonemes, PKD2 is more mobile. We show that the PKD2 regions are established early during ciliogenesis and increase proportionally in length as cilia elongate. In chimeric zygotes, tagged PKD2 rapidly entered the proximal region of PKD2-deficient cilia, whereas the assembly of the distal region was hindered, suggesting that axonemal binding of PKD2 requires de novo assembly of cilia. We identified the protein Small Interactor of PKD2 (SIP), a PKD2-related, single-pass transmembrane protein, as part of the PKD2-mastigoneme complex. In sip mutants, stability and proteolytic processing of PKD2 in the cell body were reduced and PKD2-mastigoneme complexes were absent from the cilia. Like the pkd2 and mst1 mutants, sip mutant cells swam with reduced velocity. Cilia of the pkd2 mutant beat with an increased frequency but were less efficient in moving the cells, suggesting a structural role for the PKD2-SIP-mastigoneme complex in increasing the effective surface of Chlamydomonas cilia.

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.

Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.

N-Terminal Processing and Modification of Ciliary Dyneins.

Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At least nineteen cytosolic factors are specifically needed to generate dynein holoenzymes and/or for their trafficking to the growing cilium. Many proteins are subject to N-terminal processing and acetylation, which can generate degrons subject to the AcN-end rule, alter N-terminal electrostatics, generate new binding interfaces, and affect subunit stoichiometry through targeted degradation. Here, we have used mass spectrometry of cilia samples and electrophoretically purified dynein heavy chains from Chlamydomonas to define their N-terminal processing; we also detail the N-terminal acetylase complexes present in this organism. We identify four classes of dynein heavy chain based on their processing pathways by two distinct acetylases, one of which is dependent on methionine aminopeptidase activity. In addition, we find that one component of both the outer dynein arm intermediate/light chain subcomplex and the docking complex is processed to yield an unmodified Pro residue, which may provide a setpoint to direct the cytosolic stoichiometry of other dynein complex subunits that contain N-terminal degrons. Thus, we identify and describe an additional level of processing and complexity in the pathways leading to axonemal dynein formation in cytoplasm.

An RNA thermometer in the chloroplast genome of Chlamydomonas facilitates temperature-controlled gene expression.

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.

Redox-mediated activation of ATG3 promotes ATG8 lipidation and autophagy progression in Chlamydomonas reinhardtii.

Autophagy is one of the main degradative pathways used by eukaryotic organisms to eliminate useless or damaged intracellular material to maintain cellular homeostasis under stress conditions. Mounting evidence indicates a strong interplay between the generation of reactive oxygen species and the activation of autophagy. Although a tight redox regulation of autophagy has been shown in several organisms, including microalgae, the molecular mechanisms underlying this control remain poorly understood. In this study, we have performed an in-depth in vitro and in vivo redox characterization of ATG3, an E2-activating enzyme involved in ATG8 lipidation and autophagosome formation, from 2 evolutionary distant unicellular model organisms: the green microalga Chlamydomonas (Chlamydomonas reinhardtii) and the budding yeast Saccharomyces cerevisiae. Our results indicated that ATG3 activity from both organisms is subjected to redox regulation since these proteins require reducing equivalents to transfer ATG8 to the phospholipid phosphatidylethanolamine. We established the catalytic Cys of ATG3 as a redox target in algal and yeast proteins and showed that the oxidoreductase thioredoxin efficiently reduces ATG3. Moreover, in vivo studies revealed that the redox state of ATG3 from Chlamydomonas undergoes profound changes under autophagy-activating stress conditions, such as the absence of photoprotective carotenoids, the inhibition of fatty acid synthesis, or high light irradiance. Thus, our results indicate that the redox-mediated activation of ATG3 regulates ATG8 lipidation under oxidative stress conditions in this model microalga.

IC2 participates in the cooperative activation of outer arm dynein densely attached to microtubules.

Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force. Studies using the green alga Chlamydomonas have revealed that ICs and LCs form a complex (IC/LC tower) at the base of the OAD tail and play a crucial role in anchoring OAD to specific sites on the microtubule. In this study, we isolated a novel slow-swimming Chlamydomonas mutant deficient in the IC2 protein. This mutation, E279K, is in the third of the seven WD repeat domains. No apparent abnormality was observed in electron microscope observations of axonemes or in SDS-PAGE analyses of dynein subunits. To explore the reason for the lowered motility in this mutant, in vitro microtubule sliding experiments were performed, which revealed that the motor activity of the mutant OAD was lowered. In particular, a large difference was observed between wild type (WT) and the mutant in the microtubule sliding velocity in microtubule bundles formed with the addition of OAD: ~35.3 µm/sec (WT) and ~4.3 µm/sec (mutant). From this and other results, we propose that IC2 in an OAD interacts with the ß HC of the adjacent OAD, and that an OAD-OAD interaction is important for efficient beating of cilia and flagella.Key words: cilia, axoneme, dynein heavy chain, cooperativity.

Photosystem II monomeric antenna CP26 plays a key role in nonphotochemical quenching in Chlamydomonas.

Thermal dissipation of excess excitation energy, called nonphotochemical quenching (NPQ), is 1 of the main photoprotective mechanisms in oxygenic photosynthetic organisms. Here, we investigated the function of the monomeric photosystem II (PSII) antenna protein CP26 in photoprotection and light harvesting in Chlamydomonas reinhardtii, a model organism for green algae. We used CRISPR/Cas9 genome editing and complementation to generate cp26 knockout mutants (named k6#) that did not negatively affect CP29 accumulation, which differed from previous cp26 mutants, allowing us to compare mutants specifically deprived of CP26, CP29, or both. The absence of CP26 partially affected PSII activity, causing reduced growth at low or medium light but not at high irradiances. However, the main phenotype observed in k6# mutants was a more than 70% reduction of NPQ compared to the wild type (Wt). This phenotype was fully rescued by genetic complementation and complemented strains accumulating different levels of CP26, demonstrating that ∼50% of CP26 content, compared to the Wt, was sufficient to restore the NPQ capacity. Our findings demonstrate a pivotal role for CP26 in NPQ induction, while CP29 is crucial for PSII activity. The genetic engineering of these 2 proteins could be a promising strategy to regulate the photosynthetic efficiency of microalgae under different light regimes.

Zn deficiency disrupts Cu and S homeostasis in Chlamydomonas resulting in over accumulation of Cu and Cysteine.

Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. We show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding "first responder" proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, γ-glutamylcysteine, and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ∼80-fold, corresponding to ∼2.8 × 109 molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.

The pyrenoid: the eukaryotic CO2-concentrating organelle.

The pyrenoid is a phase-separated organelle that enhances photosynthetic carbon assimilation in most eukaryotic algae and the land plant hornwort lineage. Pyrenoids mediate approximately one-third of global CO2 fixation, and engineering a pyrenoid into C3 crops is predicted to boost CO2 uptake and increase yields. Pyrenoids enhance the activity of the CO2-fixing enzyme Rubisco by supplying it with concentrated CO2. All pyrenoids have a dense matrix of Rubisco associated with photosynthetic thylakoid membranes that are thought to supply concentrated CO2. Many pyrenoids are also surrounded by polysaccharide structures that may slow CO2 leakage. Phylogenetic analysis and pyrenoid morphological diversity support a convergent evolutionary origin for pyrenoids. Most of the molecular understanding of pyrenoids comes from the model green alga Chlamydomonas (Chlamydomonas reinhardtii). The Chlamydomonas pyrenoid exhibits multiple liquid-like behaviors, including internal mixing, division by fission, and dissolution and condensation in response to environmental cues and during the cell cycle. Pyrenoid assembly and function are induced by CO2 availability and light, and although transcriptional regulators have been identified, posttranslational regulation remains to be characterized. Here, we summarize the current knowledge of pyrenoid function, structure, components, and dynamic regulation in Chlamydomonas and extrapolate to pyrenoids in other species.

Characterization of Chlamydomonas voltage-gated calcium channel and its interaction with photoreceptor support VGCC modulated photobehavioral response in the green alga.

Calcium (Ca2+) signaling plays a major role in regulating multiple processes in living cells. The photoreceptor potential in Chlamydomonas triggers the generation of all or no flagellar Ca2+ currents that cause membrane depolarization across the eyespot and flagella. Modulation in membrane potential causes changes in the flagellar waveform, and hence, alters the beating patterns of Chlamydomonas flagella. The rhodopsin-mediated eyespot membrane potential is generated by the photoreceptor Ca2+ current or P-current however, the flagellar Ca2+ currents are mediated by unidentified voltage-gated calcium (VGCC or CaV) and potassium channels (VGKC). The voltage-gated ion channel that associates with ChRs to generate Ca2+ influx across the flagella and its cellular distribution has not yet been identified. Here, we identified putative VGCCs from algae and predicted their novel properties through insilico analysis. We further present experimental evidence on Chlamydomonas reinhardtii VGCCs to predict their novel physiological roles. Our experimental evidences showed that CrVGCC4 localizes to the eyespot and flagella of Chlamydomonas and associates with channelrhodopsins (ChRs). Further in silico interactome analysis of CrVGCCs suggested that they putatively interact with photoreceptor proteins, calcium signaling, and intraflagellar transport components. Expression analysis indicated that these VGCCs and their putative interactors can be perturbed by light stimuli. Collectively, our data suggest that VGCCs in general, and VGCC4 in particular, might be involved in the regulation of the photobehavioral response of Chlamydomonas.

TurboID reveals the proxiomes of Chlamydomonas proteins involved in thylakoid biogenesis and stress response.

In Chlamydomonas (Chlamydomonas reinhardtii), the VESICLE-INDUCING PROTEIN IN PLASTIDS 1 and 2 (VIPP1 and VIPP2) play roles in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CHLOROPLAST GRPE HOMOLOG 1 (CGE1) and the stromal HEAT SHOCK PROTEIN 70B (HSP70B) as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in substantial biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B, and the CHLOROPLAST DNAJ HOMOLOG 2 (CDJ2). Proteins identified in the VIPP1/2 proxiomes can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport, including PROTON GRADIENT REGULATION 5-LIKE 1 (PGRL1). A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL). In reciprocal experiments, we confirmed VIPP1 in the proxiomes of VPL2 and PGRL1. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

Outer-arm dynein light chain LC1 is required for normal motor assembly kinetics, ciliary stability, and motility.

Light chain 1 (LC1) is a highly conserved leucine-rich repeat protein associated with the microtubule-binding domain of the Chlamydomonas outer-dynein arm γ heavy chain. LC1 mutations in humans and trypanosomes lead to motility defects, while its loss in oomycetes results in aciliate zoospores. Here we describe a Chlamydomonas LC1 null mutant (dlu1-1). This strain has reduced swimming velocity and beat frequency, can undergo waveform conversion, but often exhibits loss of hydrodynamic coupling between the cilia. Following deciliation, Chlamydomonas cells rapidly rebuild cytoplasmic stocks of axonemal dyneins. Loss of LC1 disrupts the kinetics of this cytoplasmic preassembly so that most outer-arm dynein heavy chains remain monomeric even after several hours. This suggests that association of LC1 with its heavy chain-binding site is a key step or checkpoint in the outer-arm dynein assembly process. Similarly to strains lacking the entire outer arm and inner arm I1/f, we found that loss of LC1 and I1/f in dlu1-1 ida1 double mutants resulted in cells unable to build cilia under normal conditions. Furthermore, dlu1-1 cells do not exhibit the usual ciliary extension in response to lithium treatment. Together, these observations suggest that LC1 plays an important role in the maintenance of axonemal stability.

Widening the landscape of transcriptional regulation of green algal photoprotection.

Availability of light and CO2, substrates of microalgae photosynthesis, is frequently far from optimal. Microalgae activate photoprotection under strong light, to prevent oxidative damage, and the CO2 Concentrating Mechanism (CCM) under low CO2, to raise intracellular CO2 levels. The two processes are interconnected; yet, the underlying transcriptional regulators remain largely unknown. Employing a large transcriptomic data compendium of Chlamydomonas reinhardtii's responses to different light and carbon supply, we reconstruct a consensus genome-scale gene regulatory network from complementary inference approaches and use it to elucidate transcriptional regulators of photoprotection. We show that the CCM regulator LCR1 also controls photoprotection, and that QER7, a Squamosa Binding Protein, suppresses photoprotection- and CCM-gene expression under the control of the blue light photoreceptor Phototropin. By demonstrating the existence of regulatory hubs that channel light- and CO2-mediated signals into a common response, our study provides an accessible resource to dissect gene expression regulation in this microalga.

Natural variation of nutrient homeostasis among laboratory and field strains of Chlamydomonas reinhardtii.

Natural variation among individuals and populations exists in all species, playing key roles in response to environmental stress and adaptation. Micro- and macronutrients have a wide range of functions in photosynthetic organisms, and mineral nutrition thus plays a sizable role in biomass production. To maintain nutrient concentrations inside the cell within physiological limits and prevent the detrimental effects of deficiency or excess, complex homeostatic networks have evolved in photosynthetic cells. The microalga Chlamydomonas reinhardtii (Chlamydomonas) is a unicellular eukaryotic model for studying such mechanisms. In this work, 24 Chlamydomonas strains, comprising field isolates and laboratory strains, were examined for intraspecific differences in nutrient homeostasis. Growth and mineral content were quantified in mixotrophy, as full nutrition control, and compared with autotrophy and nine deficiency conditions for macronutrients (-Ca, -Mg, -N, -P, and -S) and micronutrients (-Cu, -Fe, -Mn, and -Zn). Growth differences among strains were relatively limited. However, similar growth was accompanied by highly divergent mineral accumulation among strains. The expression of nutrient status marker genes and photosynthesis were scored in pairs of contrasting field strains, revealing distinct transcriptional regulation and nutrient requirements. Leveraging this natural variation should enable a better understanding of nutrient homeostasis in Chlamydomonas.

Palmelloid Formation and Cell Aggregation Are Essential Mechanisms for High Light Tolerance in a Natural Strain of Chlamydomonas reinhardtii.

Photosynthetic organisms, such as higher plants and algae, require light to survive. However, an excessive amount of light can be harmful due to the production of reactive oxygen species (ROS), which cause cell damage and, if it is not effectively regulated, cell death. The study of plants' responses to light can aid in the development of methods to improve plants' growth and productivity. Due to the multicellular nature of plants, there may be variations in the results based on plant age and tissue type. Chlamydomonas reinhardtii, a unicellular green alga, has also been used as a model organism to study photosynthesis and photoprotection. Nonetheless, the majority of the research has been conducted with strains that have been consistently utilized in laboratories and originated from the same source. Despite the availability of many field isolates of this species, very few studies have compared the light responses of field isolates. This study examined the responses of two field isolates of Chlamydomonas to high light stress. The light-tolerant strain, CC-4414, managed reactive oxygen species (ROS) slightly better than the sensitive strain, CC-2344, did. The proteomic data of cells subjected to high light revealed cellular modifications of the light-tolerant strain toward membrane proteins. The morphology of cells under light stress revealed that this strain utilized the formation of palmelloid structures and cell aggregation to shield cells from excessive light. As indicated by proteome data, morphological modifications occur simultaneously with the increase in protein degradation and autophagy. By protecting cells from stress, cells are able to continue to upregulate ROS management mechanisms and prevent cell death. This is the first report of palmelloid formation in Chlamydomonas under high light stress.