The C-terminus of CFAP410 forms a tetrameric helical bundle that is essential for its localization to the basal body.

Cilia are antenna-like organelles protruding from the surface of many cell types in the human body. Defects in ciliary structure or function often lead to diseases that are collectively called ciliopathies. Cilia and flagella-associated protein 410 (CFAP410) localizes at the basal body of cilia/flagella and plays essential roles in ciliogenesis, neuronal development and DNA damage repair. It remains unknown how its specific basal body location is achieved. Multiple single amino acid mutations in CFAP410 have been identified in patients with various ciliopathies. One of the mutations, L224P, is located in the C-terminal domain (CTD) of human CFAP410 and causes severe spondylometaphyseal dysplasia, axial (SMDAX). However, the molecular mechanism for how the mutation causes the disorder remains unclear. Here, we report our structural studies on the CTD of CFAP410 from three distantly related organisms, Homo sapiens, Trypanosoma brucei and Chlamydomonas reinhardtii. The crystal structures reveal that the three proteins all adopt the same conformation as a tetrameric helical bundle. Our work further demonstrates that the tetrameric assembly of the CTD is essential for the correct localization of CFAP410 in T. brucei, as the L224P mutation that disassembles the tetramer disrupts its basal body localization. Taken together, our studies reveal that the basal body localization of CFAP410 is controlled by the CTD and provide a mechanistic explanation for how the mutation L224P in CFAP410 causes ciliopathies in humans.

Polyunsaturated triacylglycerol accumulation mainly attributes to turnover of de novo-synthesized membrane lipids in stress-induced starchless Chlamydomonas.

KEY MESSAGE: Assembly of PUFA-attached TAGs is intimately correlated to turnover of newly formed membrane lipids in starch-deficient Chlamydomonas exposed to high light and nitrogen stress under air-aerated mixotrophic conditions. Triacylglycerols (TAGs) rich in polyunsaturated fatty acids (PUFAs) in microalgae have attracted extensive attention due to its promising application in nutraceuticals and other high-value compounds. Previous studies revealed that PUFAs accumulated in TAG primarily derived from the dominant membrane lipids, monogalactosyldiacylglycerolipid, digalactosyldiacylglycerol and diacylglycerol-N,N,N-trimethylhomoserine (DGTS), in the model alga Chlamydomonas reinhardtii. However, their respective contribution to PUFA-attached TAG integration has not been clearly deciphered, particularly in starchless Chlamydomonas that hyper-accumulates TAG. In this study, the starchless C. reinhardtii BAFJ5 was mixotrophically cultivated in photobioreactors aerated with air (0.04% CO2), and we monitored the dynamic changes in growth, cellular carbon and nitrogen content, photosynthetic activity, biochemical compositions, and glycerolipid remodeling under high light and nitrogen starvation conditions. The results indicated that multiple PUFAs continually accumulated in total lipids and TAG, and the primary distributors of these PUFAs gradually shifted from membrane lipids to TAG in stress-induced BAFJ5. The stoichiometry analyses showed that the PUFA-attached TAG assembly attributed to turnover of not only the major glycerolipids, but also the phospholipids, phosphatidylethanolamine (PE) and phosphatidylglycerol. Specifically, the augmented C16:3n3 and C18:3n3 in TAG mainly originated from de novo-synthesized galactolipids, while the cumulative C18:3n6 and C18:4n3 in TAG were intimately correlated with conversion of the newly formed DGTS and PE. These findings emphasized significance of PUFA-attached TAG formation dependent on turnover of de novo assembled membrane lipids in starch-deficient Chlamydomonas, beneficial for enhanced production of value-added lipids in microalgae.

Co-utilization of microalgae and heterotrophic microorganisms improves wastewater treatment efficiency.

Wastewater treatment using the activated sludge method requires a large amount of electricity for aeration. Therefore, wastewater treatment using co-culture systems of microalgae and heterotrophic microorganisms, which do not require aeration, has attracted attention as an energy-saving alternative to the method. In this study, we investigated different combinations of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment. Three types of microalgae and five heterotrophic microorganisms were used in combination for wastewater treatment. The combination of Chlamydomonas reinhardtii NIES-2238 and Saccharomyces cerevisiae SH-4 showed the highest wastewater treatment efficiency. Using this combination for artificial wastewater treatment, the removal rates of total organic carbon, PO43-, and NH4+ reached 80%, 93%, and 63%, respectively, after 18 h of treatment. To the best of our knowledge, this is the first study to show that a combination of green algae and yeast improves the efficiency of wastewater treatment. Transcriptome analysis revealed that the combined wastewater treatment altered the expression of 1371 and 692 genes in C. reinhardtii and S. cerevisiae, respectively. One of the main reasons for the improved wastewater treatment performance of the combination of green algae and yeast was the increased expression of genes related to the uptake of phosphate and ammonium ions in the green algae. As both the green algae C. reinhardtii and the yeast S. cerevisiae are highly safe microorganisms, the establishment of their effective combination for wastewater treatment is highly significant. KEY POINTS: • Combination of various microalgae and heterotrophic microorganisms was tested • Combination of green algae and yeast showed the highest efficiency • This is the first report that this combination is effective for wastewater treatment.

A hybrid biosynthetic-catabolic pathway for norspermidine production.

The only known pathway for biosynthesis of the polyamine norspermidine starts from aspartate ß-semialdehyde to form the diamine 1,3-diaminopropane, which is then converted to norspermidine via a carboxynorspermidine intermediate. This pathway is found primarily in the Vibrionales order of the γ-Proteobacteria. However, norspermidine is also found in other species of bacteria and archaea, and in diverse single-celled eukaryotes, chlorophyte algae and plants that do not encode the known norspermidine biosynthetic pathway. We reasoned that products of polyamine catabolism could be an alternative route to norspermidine production. 1,3-diaminopropane is formed from terminal catabolism of spermine and spermidine, and norspermidine can be formed from catabolism of thermospermine. We found that the single-celled chlorophyte alga Chlamydomonas reinhardtii thermospermine synthase (CrACL5) did not aminopropylate exogenously-derived 1,3-diaminopropane efficiently when expressed in Escherichia coli. In contrast, it completely converted all E. coli native spermidine to thermospermine. Co-expression in E. coli of the polyamine oxidase 5 from lycophyte plant Selaginella lepidophylla (SelPAO5), together with the CrACL5 thermospermine synthase, converted almost all thermospermine to norspermidine. Although CrACL5 was efficient at aminopropylating norspermidine to form tetraamine norspermine, SelPAO5 oxidizes norspermine back to norspermidine, with the balance of flux being inclined fully to norspermine oxidation. The steady-state polyamine content of E. coli co-expressing thermospermine synthase CrACL5 and polyamine oxidase SelPAO5 was an almost total replacement of spermidine by norspermidine. We have recapitulated a potential hybrid biosynthetic-catabolic pathway for norspermidine production in E. coli, which could explain norspermidine accumulation in species that do not encode the known aspartate ß-semialdehyde-dependent pathway.

Processed microalgae: green gold for tissue regeneration and repair.

As novel biomedical materials, microalgae have garnered significant interest because of their ability to generate photosynthetic oxygen, their antioxidant activity, and their favorable biocompatibility. Many studies have concentrated on the hypoxia-alleviating effects of microalgae within tumor microenvironments. However, recent findings indicate that microalgae can significantly increase the regeneration of various tissues and organs. To augment microalgae's therapeutic efficacy and mitigate the limitations imposed by immune clearance, it is essential to process microalgae through various processing strategies. This review examines common microalgal species in biomedical applications, such as Chlorella, Chlamydomonas reinhardtii, diatoms, and Spirulina. This review outlines diverse processing methods, including microalgae extracts, microalgae‒nanodrug composite delivery systems, surface modifications, and living microalgae‒loaded hydrogels. It also discusses the latest developments in tissue repair using processed microalgae for skin, gastrointestinal, bone, cardiovascular, lung, nerve, and oral tissues. Furthermore, future directions are presented, and research gaps for processed microalgae are identified. Collectively, these insights may inform the innovation of processed microalgae for various uses and offer guidance for ongoing research in tissue repair.

Mitochondria dysfunction is one of the causes of diclofenac toxicity in the green alga Chlamydomonas reinhardtii.

Background: Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism. Methods: Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L-1, corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell's structure organization was analyzed using confocal microscopy and transmission electron microscopy. Results: The results indicate that short-term exposure to DCF leads to an increase in oxygen consumption rate, accompanied by low MMP and reduced mtROS production by the cells in the treated populations as compared to control ones. These observations suggest an uncoupling of oxidative phosphorylation due to the disruption of mitochondrial membranes, which is consistent with the malformations in mitochondrial structures observed in electron micrographs, such as elongation, irregular forms, and degraded cristae, potentially indicating mitochondrial swelling or hyper-fission. The assumption about non-specific DCF action is further supported by comparing mitochondrial parameters in DCF-treated cells to the same parameters in cells treated with selective respiratory inhibitors: no similarities were found between the experimental variants. Conclusions: The results obtained in this work suggest that DCF strongly affects cells that experience mild metabolic or developmental disorders, not revealed under control conditions, while more vital cells are affected only slightly, as it was already indicated in literature. In the cells suffering from DCF treatment, the drug influence on mitochondria functioning in a non-specific way, destroying the structure of mitochondrial membranes. This primary effect probably led to the mitochondrial inner membrane permeability transition and the uncoupling of oxidative phosphorylation. It can be assumed that mitochondrial dysfunction is an important factor in DCF phytotoxicity. Because studies of the effects of NSAIDs on the functioning of plant mitochondria are relatively scarce, the present work is an important contribution to the elucidation of the mechanism of NSAID toxicity toward non-target plant organisms.

Functional characterization of a novel Chlamydomonas reinhardtii hydrolase involved in biotransformation of chloramphenicol.

Microalgae-based biotechnology is one of the most promising alternatives to conventional methods for the removal of antibiotic contaminants from diverse water matrices. However, current knowledge regarding the biochemical mechanisms and catabolic enzymes involved in microalgal biodegradation of antibiotics is scant, which limits the development of enhancement strategies to increase their engineering feasibility. In this study, we investigated the removal dynamics of amphenicols (chloramphenicol, thiamphenicol, and florfenicol), which are widely used in aquaculture, by Chlamydomonas reinhardtii under different growth modes (autotrophy, heterotrophy, and mixotrophy). We found C. reinhardtii removed >92 % chloramphenicol (CLP) in mixotrophic conditions. Intriguingly, gamma-glutamyl hydrolase (GGH) in C. reinhardtii was most significantly upregulated according to the comparative proteomics, and we demonstrated that GGH can directly bind to CLP at the Pro77 site to induce acetylation of the hydroxyl group at C3 position, which generated CLP 3-acetate. This identified role of microalgal GGH is mechanistically distinct from that of animal counterparts. Our results provide a valuable enzyme toolbox for biocatalysis and reveal a new enzymatic function of microalgal GGH. As proof of concept, we also analyzed the occurrence of these three amphenicols and their degradation intermediate worldwide, which showed a frequent distribution of the investigated chemicals at a global scale. This study describes a novel catalytic enzyme to improve the engineering feasibility of microalgae-based biotechnologies. It also raises issues regarding the different microalgal enzymatic transformations of emerging contaminants because these enzymes might function differently from their counterparts in animals.

Engineering microalgae for robust glycolate biosynthesis: Targeted knockout of hydroxypyruvate reductase 1 combined with optimized culture conditions enhance glycolate production in Chlamydomonas reinhardtii.

Microalgae-based glycolate production through the photorespiratory pathway is considered an environmentally friendly approach. However, the potential for glycolate production is limited by photoautotrophic cultivation with low cell density and existing strains. In this study, a targeted knockout approach was used to disrupt the key photorespiration enzyme, Chlamydomonas reinhardtii hydroxypyruvate reductase 1 (CrHPR1), leading to a significant increase in glycolate production of 280.1 mg/L/OD750. The highest potency yield reached 2.1 g/L under optimized mixotrophic conditions, demonstrating the possibility of synchronizing cell growth with glycolate biosynthesis in microalgae. Furthermore, the hypothesis that the cell wall-deficient mutant facilitates glycolate excretion was proposed and validated by comparing the glycolate accumulation trends of various Chlamydomonas reinhardtii strains. This study will facilitate the development of microalgae-based biotechnology and shed lights on the continuous advancement of green biomanufacturing for industrial application.

Phosphorous accumulation associated with mitochondrial PHT3-mediated enhanced arsenate tolerance in Chlamydomonas reinhardtii.

Arsenate is a highly toxic element and excessive accumulation of arsenic in the aquatic environment easily triggers a problem threatening the ecological health. Phytoremediation has been widely explored as a method to alleviate As contamination. Here, the green algae, Chlamydomonas reinhardtii was investigated by profiling the accumulation of arsenate and phosphorus, which share the same uptake pathway, in response to arsenic stress. Both C. reinhardtii wild type C30 and the Crpht3 mutant were cultured under arsenic stress, and demonstrated a similar growth phenotype under limited phosphate supply. Sufficient phosphate obviously increased the uptake of polyphosphate and intercellular phosphate in the Crpht3 mutant, which increased the arsenic tolerance of the Crpht3 mutant under stress from 500 µmol L-1 As(V). Upregulation of the PHT3 gene in the Crpht3 mutant increased accumulation of phosphate in the cytoplasm under arsenate stress, which triggered a regulatory role for the differentially expressed genes that mediated improvement of the glutathione redox cycle, antioxidant activity and detoxification. While the wild type C30 showed weak arsenate tolerance because of little phosphate accumulation. These results suggest that the enhanced arsenic tolerance of the Crpht3 mutant is regulated by the PHT3 gene mediation. This study provides insight onto the responsive mechanisms of the PHT3 gene-mediated in alleviating arsenic toxicity in plants.

pyMS-Vis, an Open-Source Python Application for Visualizing and Investigating Deconvoluted Top-Down Mass Spectrometric Experiments: A Histone Proteoform Case Study.

We report the development of an open-source Python application that provides quantitative and qualitative information from deconvoluted liquid-chromatography top-down mass spectrometry (LC-TDMS) data sets. This simple-to-use program allows users to search masses-of-interest across multiple LC-TDMS runs and provides visualization of their ion intensities and elution characteristics while quantifying their abundances relative to one another. Focusing on proteoform-rich histone proteins from the green microalga Chlamydomonas reinhardtii, we were able to quantify proteoform abundances across different growth conditions and replicates in minutes instead of hours typically needed for manual spreadsheet-based analysis. This resulted in extending previously published qualitive observations on Chlamydomonas histone proteoforms into quantitative ones, leading to an exciting new discovery on alpha-amino termini processing exclusive to histone H2A family members. Lastly, the script was intentionally developed with readability and customizability in mind so that fellow mass spectrometrists can modify the code to suit their lab-specific needs.

Assigning roles in Chlamydomonas ribosome biogenesis: The conserved factor NIP7.

Ribosome biogenesis (RB) is a highly conserved process across eukaryotes that results in the assembly of functional ribosomal subunits. Studies in Saccharomyces cerevisiae and Homo sapiens have identified numerous RB factors (RBFs), including the NIP7 protein, which is involved in late-stage pre-60S ribosomal maturation. NIP7 expression has also been observed in Chlamydomonas reinhardtii, highlighting its evolutionary significance. This study aimed to characterize the function of the NIP7 protein from C. reinhardtii (CrNip7) through protein complementation assays and a paromomycin resistance test, assessing its ability to complement the role of NIP7 in yeast. Protein interaction studies were conducted via yeast two-hybrid assay to identify potential protein partners of CrNip7. Additionally, rRNA modeling analysis was performed using the predicted structure of CrNip7 to investigate its interaction with rRNA. The study revealed that CrNip7 can complement the role of NIP7 in yeast, implicating CrNip7 in the biogenesis of the 60S ribosomal subunit. Furthermore, two possible partner proteins of CrNip7, UNC-p and G-patch, were identified through yeast two-hybrid assay. The potential of these proteins to interact with CrNip7 was explored through in silico analyses. Furthermore, nucleic acid interaction was also evaluated, indicating the involvement of the N- and C-terminal domains of CrNIP7 in interacting with rRNA. Collectively, our findings provide valuable insights into the RBFs CrNip7, offering novel information for comparative studies on RB among eukaryotic model organisms, shedding light on its evolutionary conservation and functional role across species.

Channelrhodopsins with distinct chromophores and binding patterns.

Channelrhodopsins are popular optogenetic tools in neuroscience, but remain poorly understood mechanistically. Here we report the cryo-EM structures of channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii and H. catenoides kalium channelrhodopsin (KCR1). We show that ChR2 recruits an endogenous N-retinylidene-PE-like molecule to a previously unidentified lateral retinal binding pocket, exhibiting a reduced light response in HEK293 cells. In contrast, H. catenoides kalium channelrhodopsin (KCR1) binds an endogenous retinal in its canonical retinal binding pocket under identical condition. However, exogenous ATR reduces the photocurrent magnitude of wild type KCR1 and also inhibits its leaky mutant C110T. Our results uncover diverse retinal chromophores with distinct binding patterns for channelrhodopsins in mammalian cells, which may further inspire next generation optogenetics for complex tasks such as cell fate control.

Nitrogen starvation leads to TOR kinase-mediated downregulation of fatty acid synthesis in the algae Chlorella sorokiniana and Chlamydomonas reinhardtii.

BACKGROUND: When subject to stress conditions such as nutrient limitation microalgae accumulate triacylglycerol (TAG). Fatty acid, a substrate for TAG synthesis is derived from de novo synthesis or by membrane remodeling. The model industrial alga Chlorellasorokiniana accumulates TAG and other storage compounds under nitrogen (N)-limited growth. Molecular mechanisms underlying these processes are still to be elucidated. RESULT: Previously we used transcriptomics to explore the regulation of TAG synthesis in C. sorokiniana. Surprisingly, our analysis showed that the expression of several key genes encoding enzymes involved in plastidic fatty acid synthesis are significantly repressed. Metabolic labeling with radiolabeled acetate showed that de novo fatty acid synthesis is indeed downregulated under N-limitation. Likewise, inhibition of the Target of Rapamycin kinase (TOR), a key regulator of metabolism and growth, decreased fatty acid synthesis. We compared the changes in proteins and phosphoprotein abundance using a proteomics and phosphoproteomics approach in C. sorokiniana cells under N-limitation or TOR inhibition and found extensive overlap between the N-limited and TOR-inhibited conditions. We also identified changes in the phosphorylation status of TOR complex proteins, TOR-kinase, and RAPTOR, under N-limitation. This indicates that TOR signaling is altered in a nitrogen-dependent manner. We find that TOR-mediated metabolic remodeling of fatty acid synthesis under N-limitation is conserved in the chlorophyte algae Chlorella sorokiniana and Chlamydomonas reinhardtii. CONCLUSION: Our results indicate that under N-limitation there is significant metabolic remodeling, including fatty acid synthesis, mediated by TOR signaling. This process is conserved across chlorophyte algae. Using proteomic and phosphoproteomic analysis, we show that N-limitation affects TOR signaling and this in-turn affects the metabolic status of the cells. This study presents a link between N-limitation, TOR signaling and fatty acid synthesis in green-lineage.

Biomimetic Synchronization in Biciliated Robots.

Direct mechanical coupling is known to be critical for establishing synchronization among cilia. However, the actual role of the connections is still elusive-partly because controlled experiments in living samples are challenging. Here, we employ an artificial ciliary system to address this issue. Two cilia are formed by chains of self-propelling robots and anchored to a shared base so that they are purely mechanically coupled. The system mimics biological ciliary beating but allows fine control over the beating dynamics. With different schemes of mechanical coupling, artificial cilia exhibit rich motility patterns. Particularly, their synchronous beating display two distinct modes-analogous to those observed in C. reinhardtii, the biciliated model organism for studying synchronization. Close examination suggests that the system evolves towards the most dissipative mode. Using this guideline in both simulations and experiments, we are able to direct the system into a desired state by altering the modes' respective dissipation. Our results have significant implications in understanding the synchronization of cilia.

Extraction Optimization and Anti-Tumor Activity of Polysaccharides from Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii polysaccharides (CRPs) are bioactive compounds derived from C. reinhardtii, yet their potential in cancer therapy remains largely unexplored. This study optimized the ultrasound-assisted extraction conditions using response surface methodology and proceeded with the isolation and purification of these polysaccharides. The optimal extraction conditions were identified as a sodium hydroxide concentration of 1.5%, ultrasonic power of 200 W, a solid-to-liquid ratio of 1:25 g/mL, an ultrasonic treatment time of 10 min, and a water bath duration of 2.5 h, yielding an actual extraction rate of 5.71 ± 0.001%, which closely aligns with the predicted value of 5.639%. Infrared analysis revealed that CRP-1 and CRP-2 are α-pyranose structures containing furoic acid, while CRP-3 and CRP-4 are ß-pyranose structures containing furoic acid. Experimental results demonstrated that all four purified polysaccharides inhibited the proliferation of cervical (HeLa) hepatoma (HepG-2) and colon (HCT-116) cancer cells, with CRP-4 showing the most significant inhibitory effect on colon cancer and cervical cancer, achieving inhibition rates of 60.58 ± 0.88% and 40.44 ± 1.44%, respectively, and significantly reducing the migration of HeLa cells. DAPI staining confirmed that the four purified polysaccharides inhibit cell proliferation and migration by inducing apoptosis in HeLa cells. CRP-1 has the most significant inhibitory effect on the proliferation of liver cancer cells. This study not only elucidates the potential application of C. reinhardtii polysaccharides in cancer therapy but also provides a scientific basis for their further development and utilization.

Establishment of an RNA-based transient expression system in the green alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a unicellular green alga, is a prominent model for green biotechnology and for studying organelles' function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii, including the 5' UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120 h with a peak around 4 h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46-1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae.

The structure of Chlamydomonas LOV1 as revealed by time-resolved serial synchrotron crystallography.

The photo-reaction of the LOV1 domain of the Chlamydomonas reinhardtii phototropin is investigated by room-temperature time-resolved serial crystallography. A covalent adduct forms between the C4a atom of the central flavin-mononucleotide chromophore and a protein cysteine. The structure of the adduct is very similar to that of LOV2 determined 23 years ago from the maidenhair fern Phy3.

Singlet-Oxygen-Mediated Regulation of Photosynthesis-Specific Genes: A Role for Reactive Electrophiles in Signal Transduction.

During photosynthesis, reactive oxygen species (ROS) are formed, including hydrogen peroxide (H2O2) and singlet oxygen (1O2), which have putative roles in signalling, but their involvement in photosynthetic acclimation is unclear. Due to extreme reactivity and a short lifetime, 1O2 signalling occurs via its reaction products, such as oxidised poly-unsaturated fatty acids in thylakoid membranes. The resulting lipid peroxides decay to various aldehydes and reactive electrophile species (RES). Here, we investigated the role of ROS in the signal transduction of high light (HL), focusing on GreenCut2 genes unique to photosynthetic organisms. Using RNA seq. data, the transcriptional responses of Chlamydomonas reinhardtii to 2 h HL were compared with responses under low light to exogenous RES (acrolein; 4-hydroxynonenal), ß-cyclocitral, a ß-carotene oxidation product, as well as Rose Bengal, a 1O2-producing photosensitiser, and H2O2. HL induced significant (p < 0.05) up- and down-regulation of 108 and 23 GreenCut2 genes, respectively. Of all HL up-regulated genes, over half were also up-regulated by RES, including RBCS1 (ribulose bisphosphate carboxylase small subunit), NPQ-related PSBS1 and LHCSR1. Furthermore, 96% of the genes down-regulated by HL were also down-regulated by 1O2 or RES, including CAO1 (chlorophyllide-a oxygnease), MDH2 (NADP-malate dehydrogenase) and PGM4 (phosphoglycerate mutase) for glycolysis. In comparison, only 0-4% of HL-affected GreenCut2 genes were similarly affected by H2O2 or ß-cyclocitral. Overall, 1O2 plays a significant role in signalling during the initial acclimation of C. reinhardtii to HL by up-regulating photo-protection and carbon assimilation and down-regulating specific primary metabolic pathways. Our data support that this pathway involves RES.

Copper Toxicity in Acidic Phytoplankton: Impacts of Labile Cu Trafficking and Causes of Mitochondria Dysfunction.

Most studies on Cu toxicity relied on indirect physicochemical parameters to predict Cu toxicity resulting from adverse impacts. This study presents a systematic and intuitive picture of Cu toxicity induced by exogenous acidification in phytoplankton Chlamydomonas reinhardtii. We first showed that acidification reduced the algal resistance to environmental Cu stress with a decreased growth rate and increased Cu bioaccumulation. To further investigate this phenomenon, we employed specific fluorescent probes to visualize the intracellular labile Cu pools in different algal cells. Our findings indicated that acidification disrupted the intracellular labile Cu trafficking, leading to a significant increase in labile Cu(I) pools. At the molecular level, Cu toxicity resulted in the inhibition of the Cu(I) import system and activation of the Cu(I) export system in acidic algal cells, likely a response to the imbalance in intracellular labile Cu trafficking. Subcellular analysis revealed that Cu toxicity induced extensive mitochondrial dysfunction and impacted the biogenesis and assembly of the respiratory chain complex in acidic algal cells. Concurrently, we proposed that the activation of polyP synthesis could potentially regulate disrupted intracellular labile Cu trafficking. Our study offers an intuitive, multilevel perspective on the origins and impacts of Cu toxicity in living organisms, providing valuable insights on metal toxicity.

Genome-Wide Identification and Characterization of Ammonium Transporter (AMT) Genes in Chlamydomonas reinhardtii.

Ammonium transporters (AMTs) are vital plasma membrane proteins facilitating NH4+ uptake and transport, crucial for plant growth. The identification of favorable AMT genes is the main goal of improving ammonium-tolerant algas. However, there have been no reports on the systematic identification and expression analysis of Chlamydomonas reinhardtii (C. reinhardtii) AMT genes. This study comprehensively identified eight CrAMT genes, distributed across eight chromosomes, all containing more than 10 transmembrane structures. Phylogenetic analysis revealed that all CrAMTs belonged to the AMT1 subfamily. The conserved motifs and domains of CrAMTs were similar to those of the AMT1 members of OsAMTs and AtAMTs. Notably, the gene fragments of CrAMTs are longer and contain more introns compared to those of AtAMTs and OsAMTs. And the promoter regions of CrAMTs are enriched with cis-elements associated with plant hormones and light response. Under NH4+ treatment, CrAMT1;1 and CrAMT1;3 were significantly upregulated, while CrAMT1;2, CrAMT1;4, and CrAMT1;6 saw a notable decrease. CrAMT1;7 and CrAMT1;8 also experienced a decline, albeit less pronounced. Transgenic algas with overexpressed CrAMT1;7 did not show a significant difference in growth compared to CC-125, while transgenic algas with CrAMT1;7 knockdown exhibited growth inhibition. Transgenic algas with overexpressed or knocked-down CrAMT1;8 displayed reduced growth compared to CC-125, which also resulted in the suppression of other CrAMT genes. None of the transgenic algas showed better growth than CC-125 at high ammonium levels. In summary, our study has unveiled the potential role of CrAMT genes in high-ammonium environments and can serve as a foundational research platform for investigating ammonium-tolerant algal species.