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The eyespot apparatus is an organelle that forms carotenoid-rich globules in diverse flagellated microalgae and functions in phototaxis. The euglenophytes have structurally and functionally distinct eyespot apparatuses from chlorophytes. β-Carotene is the most abundant pigment detected in chlorophytes’ eyespots, while xanthophylls such as zeaxanthin and diadinoxanthin have been suggested to function in euglenophytes’ eyespots. Here, we investigated the association between carotenoid composition and eyespot formation via pathway-scale mutagenesis using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing in the euglenophyte Euglena gracilis. Lycopene cyclase (lcy) mutants exhibited sole lycopene accumulation, defective red eyespots, and phototactic insensitivity. Conversely, β-carotene hydroxylase (cytochrome P450 97h1, cyp97h1) mutants accumulated β-carotene and its hydroxylated products β-cryptoxanthin and zeaxanthin and formed phototactic eyespot apparatuses, while cyp97h1 cyp97f2 double mutants were deficient in β-carotene hydroxylation and mostly lacked functional eyespots. Thus, zeaxanthin is required for the stable formation of functional eyespots in E. gracilis, highlighting evolutionary differences between euglenophytes and chlorophytes in the metabolic regulation of photoreactive organelle formation.

Carotenoids are photosynthetic pigments and hydrophobic antioxidants that are necessary for the survival of photosynthetic organisms, including the microalga Euglena gracilis. In the present study, we identified an uncharacterized gene encoding the E. gracilis β-carotene synthetic enzyme lycopene cyclase (EgLCY) and discovered a relationship between EgLCY-mediated carotenoid synthesis and the reactive oxygen species (ROS) scavenging system ascorbate-glutathione cycle. The EgLCY cDNA sequence was obtained via homology searching E. gracilis transcriptome data. An enzyme assay using Escherichia coli demonstrated that EgLCY converts lycopene to β-carotene. E. gracilis treated with EgLCY double-stranded RNA (dsRNA) produced colorless cells with hypertrophic appearance, inhibited growth, and marked decrease in carotenoid and chlorophyll content, suggesting that EgLCY is essential for the synthesis of β-carotene and downstream carotenoids, which are abundant and physiologically functional. In EgLCY dsRNA-treated cells, the ascorbate-glutathione cycle, composed of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR), was unusually modulated; APX and GR activities significantly decreased, whereas DHAR and MDAR activities increased. Ascorbate content was significantly increased and glutathione content significantly decreased in EgLCY dsRNA-treated cells and was correlated with their recycling enzyme activities. Fluorescent imaging demonstrated that EgLCY dsRNA-treated cells accumulated higher levels of H2O2 compared to wild-type cells. Taken together, this study revealed that EgLCY-mediated synthesis of β-carotene and downstream carotenoid species upregulates APX activity and increases glutathione pool size for H2O2 scavenging. Our study suggests a possible relationship between carotenoid synthesis and the ascorbate-glutathione cycle for ROS scavenging in E. gracilis.

Carotenoids are photosynthetic pigments and hydrophobic antioxidants that are necessary for the survival of photosynthetic organisms, including the microalga Euglena gracilis. In the present study, we identified an uncharacterized gene encoding the E. gracilis β-carotene synthetic enzyme lycopene cyclase (EgLCY) and discovered a relationship between EgLCY-mediated carotenoid synthesis and the reactive oxygen species (ROS) scavenging system ascorbate-glutathione cycle. The EgLCY cDNA sequence was obtained via homology searching E. gracilis transcriptome data. An enzyme assay using Escherichia coli demonstrated that EgLCY converts lycopene to β-carotene. E. gracilis treated with EgLCY double-stranded RNA (dsRNA) produced colorless cells with hypertrophic appearance, inhibited growth, and marked decrease in carotenoid and chlorophyll content, suggesting that EgLCY is essential for the synthesis of β-carotene and downstream carotenoids, which are abundant and physiologically functional. In EgLCY dsRNA-treated cells, the ascorbate-glutathione cycle, composed of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR), was unusually modulated; APX and GR activities significantly decreased, whereas DHAR and MDAR activities increased. Ascorbate content was significantly increased and glutathione content significantly decreased in EgLCY dsRNA-treated cells and was correlated with their recycling enzyme activities. Fluorescent imaging demonstrated that EgLCY dsRNA-treated cells accumulated higher levels of H2O2 compared to wild-type cells. Taken together, this study revealed that EgLCY-mediated synthesis of β-carotene and downstream carotenoid species upregulates APX activity and increases glutathione pool size for H2O2 scavenging. Our study suggests a possible relationship between carotenoid synthesis and the ascorbate-glutathione cycle for ROS scavenging in E. gracilis.

Sequence-specific DNA-binding transcription factors (TFs) are key components of gene regulatory networks. Advances in high-throughput sequencing have facilitated the rapid acquisition of whole genome assembly and TF repertoires in microalgal species. In this review, we summarize recent advances in gene discovery and functional analyses, especially for transcription factors in microalgal species. Specifically, we provide examples of the genome-scale identification of transcription factors in genome-sequenced microalgal species and showcase their application in the discovery of regulators involved in various cellular functions. Herein, we highlight TF-based genetic engineering as a promising framework for designing microalgal strains for microalgal-based bioproduction.

Some carotenoids are found in the Euglena gracilis, including β-carotene, diadinoxanthin, diatoxanthins, and neoxanthin as the major species; however, the molecular mechanism underlying carotenoid biosynthesis in E. gracilis is not well understood. To clarify the pathway and regulation of carotenoid biosynthesis in this alga, we functionally characterized the cytochrome P450 (CYP)-type carotene hydroxylase gene EgCYP97H1. Heterologous in vivo enzyme assay in E. coli indicated that EgCYP97H1 hydroxylated β-carotene to β-cryptoxanthin. E. gracilis cells suppressing EgCYP97H1 resulted in marked growth inhibition and reductions in total carotenoid and chlorophyll contents. Analysis of carotenoid composition revealed that suppression of EgCYP97H1 resulted in higher level of β-carotene, suggesting that EgCYP97H1 is physiologically essential for carotenoid biosynthesis and thus normal cell growth. To our knowledge, this is the first time EgCYP97H1 has been suggested to be β-carotene monohydroxylase, but not β-carotene dihydroxylase. Moreover, during light adaptation of dark-grown E. gracilis, transcript levels of the carotenoid biosynthetic genes (EgCYP97H1, geranylgeranyl pyrophosphate synthase EgcrtE, and phytoene synthase EgcrtB) remained virtually unchanged. In contrast, carotenoid accumulation in E. gracilis grown under the same conditions was inhibited by treatment with a translational inhibitor but not a transcriptional inhibitor, indicating that photo-responsive carotenoid biosynthesis is regulated post-transcriptionally in this alga.

Eukaryotic microalgae have been classified into several biological divisions and have evolutionarily acquired diverse morphologies, metabolisms, and life cycles. They are naturally exposed to environmental stresses that cause oxidative damage due to reactive oxygen species accumulation. To cope with environmental stresses, microalgae contain various antioxidants, including carotenoids, ascorbate (AsA), and glutathione (GSH). Carotenoids are hydrophobic pigments required for light harvesting, photoprotection, and phototaxis. AsA constitutes the AsA-GSH cycle together with GSH and is responsible for photooxidative stress defense. GSH contributes not only to ROS scavenging, but also to heavy metal detoxification and thiol-based redox regulation. The evolutionary diversity of microalgae influences the composition and biosynthetic pathways of these antioxidants. For example, α-carotene and its derivatives are specific to Chlorophyta, whereas diadinoxanthin and fucoxanthin are found in Heterokontophyta, Haptophyta, and Dinophyta. It has been suggested that AsA is biosynthesized via the plant pathway in Chlorophyta and Rhodophyta and via the Euglena pathway in Euglenophyta, Heterokontophyta, and Haptophyta. The GSH biosynthetic pathway is conserved in all biological kingdoms; however, Euglenophyta are able to synthesize an additional thiol antioxidant, trypanothione, using GSH as the substrate. In the present study, we reviewed and discussed the diversity of microalgal antioxidants, including recent findings.

Cell division of unicellular microalgae is a fascinating process of proliferation, at which whole organelles are regenerated and distributed to two new lives. We performed dynamic live cell imaging of Euglena gracilis using optical microscopy to elucidate the mechanisms involved in the regulation of the eyespot and flagellum during cell division and distribution of the organelles into the two daughter cells. Single cells of the wild type (WT) and colorless SM-ZK cells were confined in a microfluidic device, and the appearance of the eyespot (stigma) and emergent flagellum was tracked in sequential video-recorded images obtained by automatic cell tracking and focusing. We examined 12 SM-ZK and 10 WT cells and deduced that the eyespot diminished in size and disappeared at an early stage of cell division and remained undetected for 26–97 min (62 min on average, 22 min in deviation). Subsequently, two small eyespots appeared and were distributed into the two daughter cells. Additionally, the emergent flagellum gradually shortened to zero-length, and two flagella emerged from the anterior ends of the daughter cells. Our observation revealed that the eyespot and flagellum of E. gracilis are degraded once in the cell division, and the carotenoids in the eyespot are also decomposed. Subsequently, the two eyespots/flagella are regenerated for distribution into daughter cells. As a logical conclusion, the two daughter cells generated from a single cell division possess the equivalent organelles and each E. gracilis cell has eternal or non-finite life span. The two newly regenerated eyespot and flagellum grow at different rates and mature at different timings in the two daughter cells, resulting in diverse cell characteristics in E. gracilis.

Euglena gracilis exhibits photomovements in response to various light stimuli, such as phototactic and photophobic responses. Our recent study revealed that carotenoids in the eyespot apparatus are required for triggering phototaxis in this alga. However, the role of chloroplasts in eyespot formation is not understood. Here, we isolated carotenoid-less (cl) strains of E. gracilis from cells silenced gene expression of phytoene synthase (EgcrtB). Unlike WT, the culture colors of cl1, cl3, and the non-photosynthetic mutant SM-ZK were orange, while that of cl4 was white. Electron microscope observations showed that SM-ZK, cl1, and cl3 had no developed chloroplast and formed a normal eyespot apparatus, similar to that of WT, but this was not the case for cl4. Carotenoids detected in WT were diadinoxanthin, neoxanthin, and β-carotene. However, the most abundant species of SM-ZK, cl1, and cl3 was zeaxanthin, and there was no diadinoxanthin or neoxanthin. Photomovement analysis showed that SM-ZK, cl1, and cl3 exhibited negative phototactic and photophobic responses, similar to those of WT, whereas cl4 lacked negative phototaxis. Taken together, the formation of the eyespot apparatus required for phototaxis is independent of chloroplast development in E. gracilis, suggesting that this property is different from other photosynthetic flagellates.

The adaptation to a strong light is one of the essential characteristics of green algae, yet lacking relatively the information about the photophobic responses of Eukaryotic microalgae. We investigated the photophobic step-up responses of Euglena gracilis over a time course of several hours with alternated repetition of blue-light pulse illumination and spatially patterned blue-light illumination. Four distinctive photophobic motions in response to strong blue light were identified in a trace image analysis, namely on-site rotation, running and tumbling, continuous circular swimming, and unaffected straightforward swimming. The cells cultured in autotrophic conditions under weak light showed mainly the on-site rotation response at the beginning of blue-light illumination, but they acquired more blue-light tolerant responses of running and tumbling, circular swimming, or straightforward swimming. The efficiency of escaping from a blue-light illuminated area improved markedly with the development of these photophobic motions. Time constant of 3.0 h was deduced for the evolution of photophobic responses of E. gracilis. The nutrient-rich metabolic status of the cells resulting from photosynthesis during the experiments, i.e., the accumulation of photosynthesized nutrient products in balance between formation and consumption, was the main factor responsible for the development of photophobic responses. The reduction-oxidation status in and around E. gracilis cells did not affect their photophobic responses significantly, unlike the case of photophobic responses and phototaxis of Chlamydomonas reinhardtii cells. This study shows that the evolution of photophobic motion type of E. gracilis is dominated mainly by the nutrient metabolic status of the cells. The fact suggests that the nutrient-rich cells have a higher threshold for switching the flagellar motion from straightforward swimming to rotation under a strong light.

At least four peroxiredoxins that are coupled with the thioredoxin (Trx) system have been shown to play a key role in redox metabolism in the unicellular phytoflagellate Euglena gracilis. In order to clarify Trx-mediated redox regulation in this alga, we herein identified three NADPH-dependent thioredoxin reductases (NTRs) using a homologous search and characterized their enzymatic properties and physiological roles. Each Euglena NTR protein belonged to the small, large, and NTRC types, and were named EgNTR1, EgNTR2, and EgNTRC, respectively. EgNTR2 was phylogenetically different from the known NTRs in eukaryotic algae. EgNTR1 was predicted to be localized in mitochondria, EgNTR2 in the cytosol, and EgNTRC in plastids. The catalytic efficiency of EgNTR2 for NADPH was 30-46-fold higher than those of EgNTR1 and truncated form of EgNTRC, suggested that large type EgNTR2 reduced Trx more efficiently. The silencing of EgNTR2 gene expression resulted in significant growth inhibition and cell hypertrophy in Euglena cells. These results suggest that EgNTRs function in each cellular compartment and are physiologically important, particularly in the cytosol.