Discovery and molecular mechanism of a novel antihypertensive peptide from Chlamydomonas reinhardtii based on molecular docking, molecular dynamics simulation, in vitro, and in vivo analysis.

The physiological role of chloroplast carbonic anhydrases (CAs) has long been debated, particularly in the context of photosynthesis. While early hypotheses proposed that CAs enhance CO2 assimilation by rapidly accessing the HCO3- pool, direct evidence has been lacking. In this study, we examined Arabidopsis (Arabidopsis thaliana) mutants lacking both chloroplast-localized βCA1 (AT3G01500) and βCA5 (AT4G33580) to assess their impact on plant growth and photosynthetic performance. Our results show that plants deficient in chloroplast CA activity are unable to grow under ambient CO2 conditions (400 μL L⁻1) but can complete their life cycle under elevated CO2 levels (≥12,000 μL L⁻1). However, CO2 assimilation rates and ΦII measurements in CA-deficient plants were comparable to those in wild-type plants under 0.04% (400 μL L⁻1), 0.4% (4,000 μL L⁻1), and 4% CO2 (40,000 μL L⁻1) concentrations, indicating that chloroplast CAs are not essential for photosynthetic CO2 fixation. Instead, our findings suggest that chloroplast CA activity is critical for supporting other metabolic pathways, namely amino acid, nucleic acid, and fatty acid biosynthesis. Expression of the Chlamydomonas (Chlamydomonas reinhardtii) bicarbonate transporter LCIA in chloroplast CA mutants partially rescued the growth phenotype under near-ambient CO2 conditions. These LCIA-complemented lines showed no difference in photosynthesis, further supporting the role of CAs in non-photosynthetic reactions. This work provides direct evidence that while chloroplast CAs are dispensable for photosynthesis, they are essential for plant growth and development under ambient CO2 due to their role in increasing the bicarbonate concentration for specific anaplerotic pathways.

Autophagy-like induction by the overexpression of the chloroplast protease subunit CrClpP5 increases nickel tolerance in Chlamydomonas reinhardtii.

Green algae, such as Chlamydomonas reinhardtii, are promising candidates for various industrial applications. For microorganisms, cell size relates to metabolic activity and cellular composition, among others, and is thus a highly relevant property for bioprocess operations. Besides time-consuming microscopy measurements, cell sizes can be estimated indirectly for example with flow cytometry based on light scattering, resulting in a measured size distribution. However, calibration for absolute cell sizes is obstructed by the optical properties of the involved particles, resulting in less accurate size distributions, which in turn can hinder applications such as model development or process monitoring. In this study, a novel approach is proposed to improve the estimation accuracy of the cell size distribution by utilizing a physical model on light scattering around a sphere. Benchmarked with microscopy image analysis, the model shows substantial improvement. Using the corrected size distribution, the cell division rate is inferred with an extended Gaussian process regression used upon a population balance model. The resulting model is able to accurately describe the observed size distribution with the estimated division kinetics. The approach is tested using data obtained from the cultivation of C. reinhardtii as a model organism. The results provide mechanistic insight into C. reinhardtii cell division and cell size heterogeneity.

The green alga Chlamydomonas reinhardtii is an interesting organism for production of heterologous isoprenoid natural products through metabolic engineering as it can grow photosynthetically and represents a plant-like cell environment for synthase expression. Here, we investigated expression of the patchoulol synthase from Pogostemon cablin (Benth) (PcPS) to probe the production of sesquiterpenoids from different sub-cellular compartments of the algal cell. We observed that knock-down of squalene synthase (SQS) in the cytoplasm did not affect sesquiterpene production titers in the plastid, suggesting no backward movement of FPP from cytoplasmic pools. Fusion of PcPS with a farnesyl pyrophosphate synthase (FPPS) did not enhance patchoulol production in the cytoplasm but did in the plastid, as also reported elsewhere. Secondary transformations with cytoplasmic and chloroplast-localized PcPS constructs increased synthase titers and improved patchoulol yields from both compartments. In multi-parallel photobioreactor experiments with patchoulol extraction through two-phase solvent contact, different daily patchoulol production behaviors were observed based on subcellular localization of the PcPS and carbon source. Those with plastid localization of PcPS-FPPS exhibited continued production into later stages of cultivation, while cytoplasm-localized PcPS with SQS k.d. had the highest production rates in mid-logarithmic phase with low productivity in later growth stages. Cultivations in high-density photobioreactors with nutrient-enriched medium generated 15 mg patchoulol/L culture from CO2, the highest yield reported from the alga to date. The results indicate that the algal cell has flexible pools of isoprenoid precursors in different subcellular compartments from which the biotechnologist can design an expression strategy tailored to carbon-feeding regimes.

Edible hydrogel coating films encapsulating α-lipoic acid and Chlamydomonas reinhardtii: A novel approach for blueberry preservation.

Friends or foes? Allelopathic effects within a 3-microalgal consortium.

Ferredoxins (FDXs) are ubiquitous proteins that bind iron-sulfur (Fe-S) clusters and usually catalyse electron transfer reactions. In eukaryotic photosynthetic organisms, a relatively high number of [2Fe-2S] cluster-containing FDXs is present in plastids and mitochondria. These are mostly redox-active FDXs, except one mitochondrial FDX that no longer binds an Fe-S cluster and is a component of the respiratory complex I. We have performed a phylogenomic study to describe the content and distribution of FDXs in different phylogenetic groups of the Archaeplastida clade, including the two models Arabidopsis thaliana and Chlamydomonas reinhardtii. Important differences exist since the number of FDXs ranges from four to 19. From the sequence characteristics and phylogenetic analyses, they cluster in 10 clades: eight containing plastidial FDXs and two mitochondrial FDXs. Six clades are present in most organisms, while four clades comprising plastidial FDXs (FDX5, FDX7, FDX8, and FDX9) are present in a small subset of organisms, mostly algae and lower Embryophytes; the FDX5 and FDX9 clades are even only present in Chlorophyceae. The expression patterns of these two FDXs in Chlamydomonas combined with the physiological and biochemical studies performed with FDX5 suggest specific roles of FDX5 in anoxia and of FDX9 in the dark. Structural analyses provide additional support to the functional divergence among plastidial FDXs. Overall, these analyses revealed the existence of an important diversity within the FDX family and allowed refining the FDX classification in Archaeplastida. It also provides clues for future physiological analyses to decipher the functions of the uncharacterised FDXs.

Branch-point engineering of carotenoid pathways reshapes ketocarotenoid profiles and photosynthetic performance in Chlamydomonas reinhardtii

Microalgal lipid droplets (LDs) are a promising and potentially more sustainable alternative to seed oils. This study aimed to investigate the extraction and stability of microalgal LDs. LDs from cell wall-deficient Chlamydomonas reinhardtii were extracted in water using pulsed electric fields, achieving 81 ± 1 wt% lipid recovery. The extracts were evaluated for stability and stabilisation mechanisms under various processing and environmental conditions. LDs remained stable during pasteurisation and homogenisation, whereas sterilisation and freeze–thaw cycles induced coalescence. Stability testing across pH and ionic strengths indicated electrostatic repulsion as the main stabilisation mechanism, with possible steric contributions. Compared with seed-derived LDs, C. reinhardtii LDs displayed a broader pH stability range but weaker steric stabilisation. Odour changes, likely initiated during cell disruption, highlight the need to improve oxidative stability during extraction. Despite this, the study showcases microalgal LDs as a viable platform for additive-free emulsions, offering reduced land and fertiliser requirements relative to terrestrial crops. • Aqueous lipid droplet (LD) extraction from C. reinhardtii yielded 81 % of the lipids. • LDs were stable for ≥30 days and unaffected by pasteurisation. • LDs were mainly electrostatically stabilised and had an isoelectric point at pH 4.0 • Sterilisation and freeze-thawing led to LD aggregation and coalescence. • Aldehydes in LD extracts may stem from enzymatic oxidation during cell disruption.

Rising atmospheric CO₂ underscores the need for carbon mitigation strategies. Microalgae have emerged as a promising nature-based solution due to their exceptional photosynthetic efficiency, rapid biomass production, and adaptability to diverse environmental conditions. It satisfies the mandate of different sustainable development goals viz. SDG-02, 06, 07, 09, 12 and 13. This study investigates the carbon sequestration potential of various microalgae along with different microalgal cultivation systems, factors affecting cultivation, genetic interventions and solution-based applications. It was investigated that carbon sequestration potential of various microalgal strains varied in between 0.06 and 2.57g L⁻¹ d⁻¹, where Haematococcus pluvialis exhibited the highest rate (2.57g L⁻¹ d⁻¹). Among the different types of microalgae cultivation systems, the biofilm reactors and hybrid reactor designs were evaluated for higher efficiency. Environmental and operational factors influencing carbon fixation capacity are critically compared. The study explores genetic interventions for enhanced microalgal productivity both quantitatively and qualitatively. Chlamydomonas reinhardtii showed an increase in carbon fixation (35%) and biomass production (25%) with genetic intervention. Alongside, microalgae have diverse industrial applications. In bioenergy production, species viz. Desmodesmus spp. and Nannochloropsis oceanica had broader applications, while Spirulina platensis and Chlorella variabilis were recommended for ensuring nutritional security. Pharmaceutical applications of Dunaliella bardawil and Chlamydomonas reinhardtii were also observed in anti-inflammatory drugs. Additionally, microalgae like Anabaena spp. and Nostoc spp. are utilised as biofertilizers; Dunaliella bardawil and Haematococcus lacustris for industrial bioproducts and Scenedesmus spp. and Chlorella spp. for bioremediation of wastewater treatment.

Soil salinization is becoming a more serious environmental issue. Excessive salt will hinder the growth and development of plants, reduce crop yields, limit global agricultural production, and increasingly threaten the sustainability of global food supplies. Salt sensing refers to the process in which external Na+ stimulates plants. GIPC binds Na+ to activate Ca2+ channels, leading to Ca2+ influx; this cytosolic Ca²+ signal is subsequently transmitted to the SOS signaling pathway, triggering intracellular Na+ efflux and vacuolar storage. The salt tolerance of plants is a trait that emerged gradually through evolutionary adaptation, rather than an inherent property of the first land plants. Although terrestrial plants appeared about 450–470 million years ago, the origin and evolution of plant salt sensing remain unclear. In this study, two potential salt-sensitive genes, PGSIP7 and PGSIP8, in glycosyltransferase family 8 were identified through bioinformatics analysis. Further salt stress treatment found that the PGSIP7 gene responded to salt stress, which showed limited growth and development and decreased Na+ transport capacity in the SOS pathway, while the PGSIP8 gene was insensitive to salt stress. In addition, the origin and evolution of the MOCA1 gene were preliminarily explored by cloning homologous genes of the Arabidopsis salt-sensing gene MOCA1 from lower to higher plants to obtain transgenic plants. It was found that the MOCA1 gene originated from the single-celled plant Chlamydomonas reinhardtii, and its homologous gene is Cre10g422450v5. The MOCA1 gene has weak salt-sensing ability in lower plants, but with evolution, its ability to sense Na+ gradually increases in higher plants. Although the MOCA1 gene has different salt sensitivity in different plants, its overexpression can improve salt tolerance. Our results have laid a certain experimental framework for subsequent investigations concerning the evolution of salt-sensing genes in plants and the identification of new salt-sensitive genes. They also provide research ideas for enhancing crops’ resistance against salt.

IntroductionThe chloroplast, a living relic of an ancient endosymbiotic interaction between a microalga and a microbe and the principal subcellular organelle responsible for biological CO2 assimilation, is emerging as a key target for research to enhance photosynthetic efficiency beyond its current limitations. Given that accurate protein localization is a prerequisite for the in-depth scientific investigation and practical application of the membrane-compartmentalized photosynthetic organelle, numerous computational prediction tools have been proposed, yet their accuracy remains unsatisfactory.MethodsTo address the limitation, we herein present Chlamy_ChloroPred, a newly developed deep learning-based framework composed of multi-layered artificial neural networks, carefully designed to perform binary classification of chloroplast proteins in the model photosynthetic microorganism, Chlamydomonas reinhardtii. The model captures locality-aware features of determinant amino acid residues in the chloroplast transit peptide (cTP), generally located within the ~50-amino-acid N-terminal region of mature chloroplast proteins, through the integration of ProtBERT-BFD embeddings, stacked bidirectional long short-term memory (BiLSTM) networks, and an attentive pooling layer.Results and discussionOur model achieved an accuracy of 0.8462 for the C. reinhardtii proteome, outperforming widely used localization predictors, including TargetP 1.1 (0.4970), TargetP 2.0 (0.7396), and PredAlgo (0.7738) under a binary classification scheme. Comparative analyses further demonstrated that Chlamy_ChloroPred exhibits competitive performance relative to the current state-of-the-art model, PB-Chlamy (0.8521), under identical evaluation conditions. Notably, despite being trained solely on the algal proteome, Chlamy_ChloroPred showed substantial cross-species versatility when applied to the proteome of the terrestrial plant, Arabidopsis thaliana, achieving an accuracy of 0.7316 – representing a 12.6% improvement over TargetP 2.0, a predictor with previously demonstrated cross-proteome versatility. This likely stems from the model’s robust ability to capture conserved features of chloroplast proteins across proteomes from diverse photosynthetic lineages.ConclusionWe developed a deep learning–based framework, Chlamy_ChloroPred, that integrates carefully designed neural layers with low computational complexity, achieving high predictive accuracy and interpretability. We believe that Chlamy_ChloroPred represents a compelling alternative to existing predictors, especially when accurate inference of chloroplast proteins is required.

Chlamydomonas reinhardtii is a model green microalga that has great industrial potential as a sustainable bio-factory for recombinant protein and high-value chemical production. Efficient genome editing tools are required to redesign this organism for synthetic biology applications. CRISPR-Cas editing technologies have already been adapted for gene knockout, transgene knock-in, and precise gene editing in C. reinhardtii. However, the efficacy of CRISPR/Cas-mediated gene knockout (KO) is low, which hampers pathway engineering and functional genomic studies. Here we report that co-delivery of CRISPR-Cas gene editing reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHO) increases gene knockout efficacy up to 100-fold in C. reinhardtii. This phenomenon, referred to as non-homologous oligonucleotide enhancement (NOE), is heavily affected by the length, structure, and chemical modifications of dsNHO, and is largely mediated by the DNA double-stranded break sensor KU70/80 (KU) heterodimer in a Cas nuclease-, locus-, and strain-independent manner. Our data suggest that dsNHOs disrupt the cell's double-stranded break (DSB) sensing pathways, consequently shifting the balance of DNA repair from canonical non-homologous end joining (c-NHEJ) towards the more error-prone, microhomology-mediated end joining (MMEJ), which could be harnessed as a strategy for improving gene KO efficiency in Chlamydomonas and beyond.

The C2a projection of the central pair of flagella in Chlamydomonas reinhardtii harbours the A-kinase anchoring protein FAP65, FAP174, FAP147 and FAP70. FAP174, an RII-like protein with its N-terminal dimerization and docking domain, binds to the amphipathic helices of FAP65. Cryo-EM data do not reveal the entire sequences for FAP174 and FAP147. Hence, the interacting domains within this scaffold remain elusive. This study has identified the interacting domains of FAP174 with FAP147. The FAP147 protein and its MYCBPAP domain (129-639 a.a.) bind to the C-terminus of FAP174 (47-92 a.a.). In silico docking analyses using CABS-Dock to delineate the interaction identified several MYCBPAP-derived peptides, such as p3 (310-339), p4 (319-348), p9 (547-576), p13 (528-557) and p15 (350-379), to form stable interacting complexes with RMSD < 3 Å 2-3 times, and are potentially amphipathic. To gain atomistic details of the interaction, molecular dynamics (MD) simulations of the FAP147 MYCBPAP domain in complex with the FAP174 C-terminus were performed. It revealed stable interfacial contacts, a subset of which overlap with residues within the p15 peptide region of the MYCBPAP domain, while identifying G48, S49, P52, Y55, L79, Q80 and V83 as key interacting residues within the C-terminus of FAP174. Individual alanine substitution of the FAP174 residues, followed by overlay assay with FAP147, retained the interaction, indicating that the interaction does not depend solely on discrete amino acids but on broader interface interactions.

Mitochondrial biogenesis requires coordinated expression from both nuclear and mitochondrial genomes. To understand the consequences of mitochondrial genome loss, we generated a mitochondrial DNA-depleted line (crm-) in Chlamydomonas reinhardtii via long-term ethidium bromide treatment. We then examined how mtDNA disruption affects mitochondrial ultrastructure, chloroplast function, and the mitochondrial transcription termination factor (mTERF) gene family. Our results reveal that mitochondrial dysfunction is associated with severe organelle remodeling, including mitochondrial elongation, matrix condensation, and cristae collapse. Consequently, mitochondria reduce the electron sink capacity which appears to over-reduce the chloroplast electron transport chain, correlating with causing damage to photosystem II (PSII), as indicated by higher plastoquinone PQ redox state and PSII excitation pressure and lower non-photochemical quantum yield [Y(NPQ)]. Furthermore, we identified and characterized eight nuclear-encoded mTERF genes in C. reinhardtii (CrmTERFs). Phylogenetic analysis grouped them into three clades with potential functional conservation. Collinearity analysis suggested potential evolutionary relationships between mTERF genes in Chlamydomonas and Marchantia polymorpha. Gene ontology annotation linked CrmTERFs to transcription termination and RNA biosynthesis regulation. Additionally, in silico prediction identified twelve putative miRNAs targeting seven of the eight CrmTERFs, with CrmTERF3 as the only exception, providing candidates for future experimental validation. This study provides the first comprehensive analysis of the nuclear encoded mTERF gene family in Chlamydomonas and demonstrates that mtDNA loss is correlated with mTERF genes expression, as well as mitochondrial structure and chloroplast photoprotective impairments. These findings suggest a potential role for CrmTERFs in mitochondrial retrograde signaling and organellar crosstalk, though functional validation is required to establish causality.

Heterogeneity is an intrinsic characteristic of living organisms, yet the single-cell heterogeneity in metal uptake and toxicity remains poorly understood. Here, we investigated the single-cell heterogeneity of labile Cu(I) metabolism in the model microalga Chlamydomonas reinhardtii using an image-enabled flow cytometer platform. Algal cells exposed to chronic Cu stress exhibited distinct labile Cu(I) bioaccumulation patterns, forming two subpopulations: "LCu(I) cells" and "HCu(I) cells," differentiated by intracellular labile Cu(I) content. These results provide direct evidence of heterogeneous Cu(I) distribution at the single-cell level in microalgae. Higher Cu stress induced a shift from LCu(I) cells to HCu(I) cells, suggesting differential cellular sensitivity to Cu stress and varying labile Cu(I) accumulation. At the molecular level, multiomics analyses identified Ctr3p as a potential key regulator of labile Cu(I) homeostasis in algal cells. Confocal imaging revealed abnormal aggregation of glutathione (GSH) in granules within HCu(I) cells. Complementary GSEA results indicated an aberrant GSH compartmentalization in HCu(I) cells that might contribute to Cu(I) hyperaccumulation. At the physiological level, hyperaccumulated Cu(I) in HCu(I) cells likely caused cytotoxicity and photosynthesis inhibition. This study highlights the complexity and variability of labile Cu metabolism at the single-cell level, emphasizing the importance of accounting for subpopulation-specific responses in metal toxicity assessments, rather than relying solely on bulk-level analyses.

Chloroplast ribosomes (chlororibosomes) synthesize the core protein components of the photosynthetic apparatus, yet their structural diversity outside flowering plants remains largely unexplored. Here, we combine in situ cryo-electron tomography (cryo-ET) with single-particle cryo-electron microscopy (cryo-EM) to determine the structure of the chlororibosome from the unicellular green alga Chlamydomonas reinhardtii . Subtomogram averaging of chlororibosomes in their native environment, resolved to ∼5 Å resolution and in distinct translational states, reveals particles both free in the stroma and loosely tethered to thylakoid membranes. These in situ reconstructions uncover an additional "arm" domain on the small subunit. High-resolution single-particle reconstruction of isolated chlororibosomes to ∼2.5 Å, in states bound either to the inhibitory translation factor pY or to a nascent chain-linked P-site tRNA, reveals that this domain is built primarily from extensive chloroplast-encoded insertions and extensions of conserved small subunit proteins, supported by chlororibosome-specific ribosomal proteins. The arm domain is located around the mRNA entry and exit channels, suggesting a role in stabilizing the mRNA trajectory through the small subunit and organizing chloroplast polysomes. Together, these data reveal unexpected structural variation of algal chlororibosomes and suggest that chloroplast translation has diversified substantially even among relatively closely related photosynthetic lineages.

This study characterizes a translin-like protein from Chlamydomonas reinhardtii, a unicellular alga. The efficient binding of the Crtranslin protein to both single-stranded DNA and RNA aligns it with the nucleic acid-binding properties of the translin protein family, known for its roles in DNA repair, RNA metabolism, and mRNA transport. We report for the first time the presence of a translin-like protein that forms octameric rings, is more closely related to rice translin, and is localized to an organelle not yet known to harbor such a family of proteins, viz., in the basal body and flagella of C. reinhardtii. This study lays the groundwork for future investigations into the molecular functions of Crtranslin and its potential regulatory roles in flagellar dynamics. Impact statement This is the first report of the presence of a nucleic acid-binding protein, Translin, in the basal body and cilia.

Abstract Algae are used intensively in bioremediation studies due to their cell structure organization, doubling their weight in one day, ease of application in biotechnological processes, low cost, content of many useful substances, ability to remove some harmful substances from the environment, and resistance to environmental factors. In this work, Spirogyra sp . was isolated from freshwater sources in Ankara, and the cultivated algal biomass was entrapped in magnetic alginate beads (MA@ALG). The MA@ALG beads were used for the biosorption and degradation of three different endocrine-disrupting chemicals (EDCs), namely Bisphenol A (BPA), Diclofenac (DCF), and Congo red (CR), using free algal biomass as a control system. The biosorption capacities of the free algal biomass for BPA, CR and DCF were 89.4, 163.1, and 68.2 mg/g, respectively. Whereas the biosorption capacities of MA@ALG for BPA, DCF, and CR were 73.6, 104.4, and 49.5 mg/g, respectively. The presence of laccase-like activity of the Spirogyra sp . in the cell-free medium was assayed after contact with the EDCs. The degradation efficiencies of BPA, CR, and DCF were 79.7, 88.2, and 81.7% by the free algal biomass, and 68.9, 73.3, and 73.4% for MA@ALG, respectively, in a batch reactor for 7 days. Biosorption of BPA, DCF, and CR on the free algal biomass and MA@ALG is described by the Langmuir isotherm model and the pseudo-second order kinetic model. The toxicities of the tested compounds and degradation products were assessed with Daphnia magna , Chlamydomonas reinhardtii , and Triticum aestivum L . The degradation products of BPA, DCF, and CR had no remarkable toxic effect on the test organisms used compared to the parent compounds.