Search Results (286)

Ulva prolifera is the main causative species of marine green tides and exhibits extreme tolerance to intertidal abiotic stress. However, the underlying molecular mechanisms remain largely unclear. In this study, we cloned and characterized an MYB-related transcription factor, UpMYB-PHL, from U. prolifera. Expression analysis showed that UpMYB-PHL is rapidly and significantly induced by high-salt stress. Furthermore, heterologous overexpression of UpMYB-PHL in the model microalga Chlamydomonas reinhardtii significantly improved its salt tolerance and biomass. By yeast one-hybrid and dual-luciferase assays, we demonstrated that UpMYB-PHL directly binds to and activates the promoter of the phosphate transporter gene UpPHT1, which is a typical target gene of phosphate response (PHR) transcription factor and participates in salt stress responses of plants. Interestingly, yeast two-hybrid assays revealed that UpMYB-PHL physically interacts with UpGAPDH, a core enzyme in energy metabolism. Taken together, our findings reveal a novel regulatory network in which UpMYB-PHL coordinates phosphorus transporter and energy metabolism in response to salt stress in U. prolifera. This study provides a vital molecular explanation for the rapid adaptation and massive growth of U. prolifera under severe intertidal salt stress. Full article

Chlorophyll (Chl) b is crucial for assembly of the light-harvesting antennae that are required for optimal photosynthetic activity in plants and green algae. Synthesis of its precursor, chlorophyllide (Chlide) b, is catalyzed by Chlide a oxygenase (CAO), which contains a stable tyrosyl radical. Studies with the model organism Chlamydomonas reinhardtii y-1 suggested that protochlorophyllide (Pchlide) a is a substrate for the enzyme in the dark when a ‘cofactor’ is present to form a heterodimer, which apparently decreases the redox potential of Pchlide a. Data described in the literature are consistent with reduction in the redox potential of Chlide a by dimerization, which produces a substrate that allows rapid synthesis and accumulation of Chl b during chloroplast development in oxygenic photosynthetic organisms. In this article, we provide an emerging perspective on CAO’s structure, its assumed radical-mediated catalytic mechanism, and its role in planta. Full article

Cadmium (Cd) is one of the most toxic heavy metals in the environment, and it severely represses photosynthesis, growth, development and nutrient uptake in photosynthetic organisms. Excessive cadmium (Cd) taken up by plants seriously threatens global food security and human health. Therefore, designing an eco-friendly and sustainable strategy that can reduce the accumulation of Cd in plants is a major challenge. Phosphorus (P), as an essential nutrient for plant growth, has been shown to play a pivotal role in mediating Cd-induced stress response. However, the molecular mechanisms underlying the crosstalk between phosphate signaling and Cd stress response remain largely uncharacterized, especially the role of the core phosphate homeostasis regulator Phosphate Starvation Response 1 (PSR1). Here, we used the model green microalga Chlamydomonas reinhardtii to investigate the physiological and transcriptomic responses to Cd stress in wild type (WT, CC-125) and PSR1 loss-of-function mutant (Crpsr1, CC-4267). Our results showed that the Crpsr1 mutant exhibited significantly enhanced Cd tolerance compared with WT under P-sufficient conditions, with a better growth phenotype and a significantly lower Cd accumulation. Transcriptome analysis revealed distinct gene expression profiles between WT and the Crpsr1 mutant in response to Cd treatment. Gene Ontology (GO) enrichment analysis showed that differentially expressed genes (DEGs) were mainly involved in primary metabolism, protein kinase activity, ion binding and transmembrane transport, which are critical processes for mitigating Cd stress. Notably, key genes associated with iron uptake and homeostasis were significantly upregulated in the Crpsr1 mutant under Cd stress, indicating a potential regulatory link between PSR1, iron homeostasis and Cd tolerance. Taken together, our findings establish a functional association between the central phosphate signaling regulator PSR1 and Cd stress response in green microalgae, and provide novel candidate genes and regulatory networks for developing engineered microalgae with enhanced Cd phytoremediation capacity. Full article

Background: Ultraviolet-C (UV-C) radiation is a high-energy physical mutagen capable of inducing DNA damage and oxidative stress, thereby generating genomic variability in photosynthetic organisms. However, its genome-wide effects in unicellular eukaryotic microalgae remain poorly characterized. This study developed a UV-C mutagenesis protocol in Chlamydomonas reinhardtii and evaluated its genomic and physiological impacts. Methods: Axenic cultures of Chlamydomonas reinhardtii (137c+) were exposed to UV-C (100–280 nm) for 12, 48, and 96 min. Viable colonies were analyzed by Random Amplification of Polymorphic DNA PCR (RAPD-PCR) to assess genetic variability, while chlorophyll content and the expression of stress-responsive genes were measured via spectrophotometry and Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR), respectively. Results: UV-C treatment induced extensive genomic polymorphism with heterogeneous clustering patterns independent of exposure time, consistent with stochastic mutagenesis. Several mutants exhibited reduced chlorophyll content, indicating impaired photosynthetic efficiency. In contrast, one genotype (pop18) maintained wild-type chlorophyll levels despite marked genetic divergence, coupled with upregulation of antioxidant, DNA repair, and stress-response genes. Conclusions: Overall, UV-C irradiation represents an effective approach to generate non-directional genomic variability in Chlamydomonas reinhardtii, with evidence that random mutagenesis can drive functional reorganization of stress-response pathways, supporting its application in microalgal strain improvement. Full article

Exosomes are revolutionizing skincare as natural messengers for cell communication, yet their transition into cosmetics is often limited by the ethical and regulatory hurdles of their animal or human sourcing. This study describes the development and validation of vegan exosome-like biomimetic vesicles (EBVs) generated from the microalgae Chlamydomonas reinhardtii that reproduce the structural and functional logic of mammalian exosomes. Their structural biomimetism was confirmed through physical, lipidomic, and proteomic characterizations, revealing bilamellar vesicles (average diameter ~160 nm) containing 109 membrane lipids and 1369 proteins. Their functional biomimetism was assessed via 3′ mRNA sequencing, which showed that the EBVs induced transcriptional responses in human fibroblasts functionally analogous to human-derived exosomes in matrix-remodeling and anti-aging pathways. In vitro, the EBVs showed a 166.7% higher dermal delivery bias than standard liposomes and accelerated wound healing. Ex vivo, 2% EBVs protected skin explants against UV-A stress, showing 92% protective efficacy for excessive melanin production upon oxidative stress. Furthermore, the EBVs supported hair follicle anagen markers and follicle stem cell metabolism, significantly upregulating SOX9 (p = 0.0022). A 56-day placebo-controlled clinical study confirmed significant improvements in wrinkle depth (−12.2%), elasticity (+4.9%), and radiance (+20.0%). These results position EBVs as a scalable, high-performance alternative for next-generation anti-aging cosmetic applications. Full article

Chlorophylls are the major light-harvesting pigments in photosynthetic organisms. Their biosynthesis requires the coordinated supply of metabolic intermediates from two independent upstream branches: the methylerythritol 4-phosphate (MEP)-derived terpenoid pathway, which supplies the phytyl side chain via geranylgeranyl diphosphate (GGPP), and the tetrapyrrole biosynthesis pathway (TBP), which provides the porphyrin ring. How flux through these two branches is coordinated remains poorly understood. In this study, we report the identification of a direct protein–protein interaction between δ-aminolevulinic acid dehydratase (ALAD), the second enzyme of the TBP, positioned immediately upstream of the first metabolic branch point, and the Type II small subunit of GGPP synthase (SSUII), a key regulator of terpenoid flux toward chlorophyll biosynthesis. ALAD was identified as a candidate SSUII-interacting protein by co-immunoprecipitation coupled with LC-MS analysis of rice leaf tissue, with a sequence coverage of 57.04%. The interactions between OsALAD1 and OsSSUII in rice, and between AtALAD1 and AtSSUII in Arabidopsis thaliana, were validated by yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts. BiFC imaging demonstrated that the interaction is localized to the chloroplast. Sequence analysis revealed that plant ALAD proteins are highly conserved, with 92% similarity between OsALAD1 and AtALAD1, and 76.9% similarity between OsALAD1 and the green alga Chlamydomonas reinhardtii CrALAD1, indicating cross-species conservation of the ALAD–SSUII interaction. In vitro enzyme activity assays showed that AtSSUII does not directly alter AtALAD1 catalytic activity, suggesting the interaction operates through post-translational rather than direct catalytic mechanisms. Overexpression of AtALAD1 caused severe chlorosis and seedling lethality, while AtSSUII overexpression produced no distinct phenotype; neither transgene altered the transcript level of the other. Together, our results reveal a conserved cross-pathway protein–protein interaction linking the terpenoid regulatory machinery to the early TBP, suggesting a molecular possibility for the coordinated regulation of chlorophyll biosynthesis. Full article

Bioplastics are increasingly expected to function not only as alternatives to fossil-derived plastics but also as components of circular plastic systems. However, currently bioplastics remain limited by cost, feedstock availability, achievable biomass content, and end-of-life compatibility. This review examines these limitations by organizing recent technological and policy trends in bioplastics, with particular attention to Japan’s social and industrial infrastructure. On this basis, we discuss a systems-level framework for circular plastics that integrates regionally underutilized non-edible biomass, decentralized production concepts, and the emerging possibility of cell-plastics based on unicellular green algae. We argue that the practical dissemination of biomass plastics requires not only material development but also compatibility with molding processes, recycling and biodegradation pathways, and regional collection and treatment systems. In this context, cell-plastics derived from Chlamydomonas reinhardtii are positioned as an emerging technological platform for direct biomass utilization and interfacial material design, although their large-scale implementation remains limited by current cultivation and manufacturing constraints. We propose that circular biomass-plastics systems in Japan should be developed as regionally adapted production frameworks with clearly defined end-of-life pathways, rather than as simple substitutes for petroleum-derived plastics. Full article

Cannabinoids are high-value bioactive compounds whose sustainable production remains challenging, prompting interest in biocatalytic and microbial platforms as alternatives to plant extraction. In this study, we investigated the heterologous expression and functionality of two key cannabinoid-related enzymes in the photosynthetic microalga Chlamydomonas reinhardtii: the aromatic prenyltransferase, NphB_G286S/Y288A from Streptomyces sp., and the plant-derived cannabidiolic acid synthase (CBDAS) from Cannabis sativa. Codon-optimized genes were introduced into the nuclear genome of C. reinhardtii using several construct configurations and promoters, and stable transformants were generated and characterized for genomic integration, transcript accumulation, protein production, enzymatic activity, and cannabinoid-related metabolite formation. While NphB protein accumulation was achieved under the PSAD promoter control, CBDAS was not detected at the protein level under any condition tested. In vitro enzymatic assays using soluble algal protein extracts from NphB-expressing lines confirmed catalytic activity, yielding cannabigerolic acid (CBGA), reaching up to 633 ± 58 µg L⁻¹. However, no CBGA production was detected in vivo, despite substrate supplementation. These results indicate that, although bacterial prenyltransferase can be functionally expressed in C. reinhardtii, efficient metabolic conversion in vivo is limited by cellular and biochemical constraints, including substrate availability, intracellular compartmentalization, and potential competition with endogenous pathways. In contrast, the absence of detectable CBDAS highlights the challenges associated with expressing complex plant oxidocyclases in this photosynthetic host. Overall, this work provides mechanistic insights into enzyme compatibility and metabolic bottlenecks in microalgal systems and outlines key considerations for the future development of photosynthetic platforms for cannabinoid biocatalysis. Full article

Low temperature critically influences cellular metabolism by impairing processes such as membrane fluidity, enzyme activity, and protein folding. However, the comprehensive genetic landscape and regulatory mechanisms governing cold acclimation remain poorly understood. Here, we performed high-throughput, pooled genetic screening in the model alga Chlamydomonas reinhardtii (C. reinhardtii) to identify genes essential for cold acclimation. Our screening revealed numerous candidate genes implicated not only in early cold response pathways but also in core cellular processes, including DNA dynamics, protein homeostasis, metabolic regulation, and substrate transport. Notably, we identified a member of the RUS (ROOT UVB SENSITIVE) family, encoding a conserved DUF647 domain protein, designated CrRUS1. CRISPR-generated rus1 mutant alleles in C. reinhardtii display a phenotype consistent with our screening: the mutants did not exhibit any visible growth defects, but show severe growth defects at low temperature. Interestingly, the cold-induced phenotypic changes in rus1 can be reversed by dark conditions, suggesting that CrRUS1 likely promotes cold acclimation in C. reinhardtii through a light-dependent pathway. Our work provides novel genetic resources and mechanistic insights into cold acclimation in C. reinhardtii, with potential translational relevance for enhancing cold tolerance in crop species. Full article

Norfloxacin has been widely found in water bodies and exhibits a strong toxic effect on aquatic organisms. To uncover its toxic mechanism on algae, the cell growth, reactive oxygen species (ROS) levels, physiological activities, mitochondrial membrane potential (MMP), caspase-3-like activity, cell morphology, TUNEL-positive nuclei and DNA ladders were determined in Chlamydomonas reinhardtii in exposure to norfloxacin. With raising norfloxacin concentration, the inhibitory and lethal effects on C. reinhardtii cells gradually enhanced, and the whole of the cells were dead under 50 μM for 24 h. During the cell death, respiratory and photosynthetic rate gradually reduced and disappeared after 24 h, while ROS quickly burst and maintained high levels during the 24 h. The MMP was markedly broken after 0.5 h, while caspase-3-like was activated, with the highest activity at the 2nd h. With prolonging the treatment time, the algal cells showed a gradual shrinking and wrinkling trend, while the numbers and fluorescence intensity of TUNEL-positive nuclei gradually increased. Meanwhile, the DNA was degraded by Ca²⁺-dependent endonucleases to show ladders after 6 h, and the degradation gradually enhanced during the death process. These characteristics demonstrate that norfloxacin can poison algae by triggering programmed cell death induced by the elevated ROS. Full article

The gelatinization and pasting behavior of starch play a critical role in governing its suitability for various food and non-food applications. Although Chlamydomonas reinhardtii is the most-studied microalga, its starch gelatinization and pasting properties have remained elusive. In this study, we applied nitrogen limitation to promote the starch accumulation of C. reinhardtii and recovered the starch using high-pressure homogenization. The multiscale structure and properties of C. reinhardtii starch (CRS) were comprehensively analyzed and compared with those of commonly used terrestrial plant starch. Results showed that CRS possesses a unique multiscale structure characterized by an exceptionally high degree of branching (18.6%) and a thinner crystalline lamellae (9.29 nm). While maintaining an A-type crystalline pattern, CRS granules exhibited higher crystallinity compared with other microalgal starches. CRS had an irregular red blood cell-like morphology with a small size (~1 μm diameter). Physicochemical analysis revealed that CRS has an intermediate gelatinization temperature and a pasting profile defined by low viscosity and remarkable shear resistance, suggesting high stability during hydrothermal processing. Significantly, cooked CRS demonstrated a lower hydrolysis rate and higher resistant starch content than several common terrestrial starches. It is attributed to its higher degree of branching and superior thermostability. This study extends the fundamental knowledge of CRS and provides a critical scientific basis for its application as a novel, sustainable ingredient with special gel properties in the future food industry. Full article

Nitrogen (N) and phosphorus (P) are essential macronutrients for plant growth and major pollutants driving aquatic eutrophication. Microalgae represent a sustainable biological platform for nutrient recovery and circular utilization from wastewater; however, the molecular mechanisms governing efficient urea assimilation and its coordination with phosphorus uptake remain inadequately characterized. This study investigated how overexpression of the high-affinity urea transporter gene DUR3 enhances nutrient scavenging capacity in the model green alga Chlamydomonas reinhardtii. The DUR3-overexpressing line exhibited concentration-dependent growth responses to urea, showing significant promotion at low-to-moderate levels but inhibition at high urea concentration or under pure-urea conditions, where DUR3-overexpressing (DUR3-OE) was more severely inhibited than the wild-type (WT). Notably, the DUR3-OE consistently increased chlorophyll content and photosynthetic efficiency (Fv/Fm) under ammonium, urea, and mixed-N regimes. Under low-urea conditions, the total P content of the DUR3-OE was 8.8% higher and total N content was 4.3% higher than in WT (p < 0.05). Except in pure-urea medium, the engineered strains exhibited significantly increased total P accumulation and superior P recovery efficiency from the culture medium. Transcriptomic analysis revealed that DUR3 overexpression reprograms a coordinated regulatory network associated with N/P metabolism, photosynthesis, and carbon transport pathways. RT-qPCR validation confirmed significant upregulation of PMA2 (plasma membrane H⁺-ATPase), phosphate transporters (PTB3, PTB7), the inorganic carbon transporter HLA3, and photosynthesis-related genes, which was associated with improved nutrient assimilation and photosynthetic performance. These findings establish DUR3 as a key genetic target for engineering microalgae with optimized N-P co-uptake capacity, providing a robust molecular framework for developing high-efficiency algal strains for wastewater bioremediation and nutrient circular economy applications. Full article

Hydroponic systems are gaining prominence in sustainable agriculture, yet their nutrient-rich effluents remain an underexplored source of microbial biodiversity with potential biotechnological interest. In this study, shotgun metagenomic sequencing was employed to profile, with a high taxonomic resolution, the photosynthetic microbial community in hydroponic effluent before and after a natural algal bloom, revealing pronounced shifts in microbial composition. Notably, relative abundance increased sixfold for Chlamydomonas reinhardtii and tenfold for Bigelowiella natans. Four dominant microalgal strains (PR1–PR4) were subsequently isolated and characterized through integrative morphological and molecular taxonomy, with phylogenetic analyses based on four genetic markers (18S rRNA, ITS, rbcL and tufA) confirming that each isolate represents a distinct lineage within Chlorophyceae families, including Chlorella sp., Chlamydomonas sp., and Scenedesmus sp. Growth kinetics under three temperature regimes, typical of Greek environmental conditions from spring to autumn (15 °C, 23 °C, 32 °C), demonstrated broad ecological plasticity and rapid biomass production, highlighting strains with strong adaptive resilience. Biochemical profiling of the isolates revealed significant inter-strain differences in primary and secondary metabolite content, including proteins (up to 43% DW), lipids (up to 31% DW), carbohydrates (up to 44% DW), photosynthetic pigments, phenolics, flavonoids, and antioxidant activity. The observed metabolic diversity of autochthonous microalgal strains from hydroponic environments, combined with their high growth rates, underscores their potential for applications in bioremediation, bioenergy, and the development of value-added products within a circular bioeconomy framework. Full article

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. Full article

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the first committed step in arginine biosynthesis in organisms that perform the cyclic pathway of ornithine synthesis. In cyanobacteria and most Archaeplastida, the activity of NAGK is controlled by the PII signal transduction protein. During evolution, representatives of the class Mamiellophyceae, Ostreococcus and Bathycoccus lost the gene encoding PII, while Micromonas retained this gene. Here, we perform coupled enzyme and pull-down assays and show that M. commoda NAGK is activated by N-acetyl-L-glutamate and inhibited by arginine but is not controlled by PII proteins. This loss may have been compensated for by the enzyme’s low sensitivity to arginine. In contrast, M. commoda PII relieved Chlamydomonas reinhardtii NAGK from feedback inhibition by arginine. These observations suggest that M. commoda NAGK possesses a unique feature: it has lost the ability to interact with PII protein. The findings are discussed in the context of the relationship between NAGK control and the PII role in Mamiellophyceae. Full article