Zeaxanthin is required for eyespot formation and phototaxis in Euglena gracilis.

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. ß-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, ß-carotene hydroxylase (cytochrome P450 97h1, cyp97h1) mutants accumulated ß-carotene and its hydroxylated products ß-cryptoxanthin and zeaxanthin and formed phototactic eyespot apparatuses, while cyp97h1 cyp97f2 double mutants were deficient in ß-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.

Expression of peat moss VASCULAR RELATED NAC-DOMAIN homologs in Nicotiana benthamiana leaf cells induces ectopic secondary wall formation.

KEY MESSAGE: The homologs of VASCULAR RELATED NAC-DOMAIN in the peat moss Sphagnum palustre were identified and these transcriptional activity as the VNS family was conserved. In angiosperms, xylem vessel element differentiation is governed by the master regulators VASCULAR RELATED NAC-DOMAIN6 (VND6) and VND7, encoding plant-specific NAC transcription factors. Although vessel elements have not been found in bryophytes, differentiation of the water-conducting hydroid cells in the moss Physcomitrella patens is regulated by VND homologs termed VND-NST-SOMBRERO (VNS) genes. VNS genes are conserved in the land plant lineage, but their functions have not been elucidated outside of angiosperms and P. patens. The peat moss Sphagnum palustre, of class Sphagnopsida in the phylum Bryophyta, does not have hydroids and instead uses hyaline cells with thickened, helical-patterned cell walls and pores to store water in the leaves. Here, we performed whole-transcriptome analysis and de novo assembly using next generation sequencing in S. palustre, obtaining sequences for 68,305 genes. Among them, we identified seven VNS-like genes, SpVNS1-A, SpVNS1-B, SpVNS2-A, SpVNS2-B, SpVNS3-A, SpVNS3-B, and SpVNS4-A. Transient expression of these VNS-like genes, with the exception of SpVNS2-A, in Nicotiana benthamiana leaf cells resulted in ectopic thickening of secondary walls. This result suggests that the transcriptional activity observed in other VNS family members is functionally conserved in the VNS homologs of S. palustre.

Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System.

Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution.

Copper mediates auxin signalling to control cell differentiation in the copper moss Scopelophila cataractae.

The copper (Cu) moss Scopelophila cataractae (Mitt.) Broth. is often found in Cu-enriched environments, but it cannot flourish under normal conditions in nature. Excess Cu is toxic to almost all plants, and therefore how this moss species thrives in regions with high Cu concentration remains unknown. In this study, we investigated the effect of Cu on gemma germination and protonemal development in S. cataractae. A high concentration of Cu (up to 800 µM) did not affect gemma germination. In the protonemal stage, a low concentration of Cu promoted protonemal gemma formation, which is the main strategy adopted by S. cataractae to expand its habitat to new locations. Cu-rich conditions promoted auxin accumulation and induced differentiation of chloronema into caulonema cells, whereas it repressed protonemal gemma formation. Under low-Cu conditions, auxin treatment mimicked the effects of high-Cu conditions. Furthermore, Cu-induced caulonema differentiation was severely inhibited in the presence of the auxin antagonist α-(phenylethyl-2-one)-indole-3-acetic acid, or the auxin biosynthesis inhibitor l-kynurenine. These results suggest that S. cataractae flourishes in Cu-rich environments via auxin-regulated cell differentiation. The copper moss might have acquired this mechanism during the evolutionary process to benefit from its advantageous Cu-tolerance ability.

Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora.

Vacuoles are suggested to play crucial roles in plant defense-related cell death. During programmed cell death, previous live cell imaging studies have observed vacuoles to become simpler in structure and have implicated this simplification as a prelude to the vacuole's rupture and consequent lysis of the plasma membrane. Here, we examined dynamics of the vacuole in cell cycle-synchronized tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cells during cell death induced by application of culture filtrates of Erwinia carotovora. The filtrate induced death in about 90% of the cells by 24 h. Prior to cell death, vacuole shape simplified and endoplasmic actin filaments disassembled; however, the vacuoles did not rupture until after plasma membrane integrity was lost. Instead of facilitating rupture, the simplification of vacuole structure might play a role in the retrieval of membrane components needed for defense-related cell death.

High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis.

Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.

Regulation of gemma formation in the copper moss Scopelophila cataractae by environmental copper concentrations.

Considerable attention has recently been focused on the use of hyperaccumulator plants for the phytoremediation of soils contaminated with heavy metals. The moss, Scopelophila cataractae (Mitt.) Broth., is a typical hyperaccumulator that is usually observed only in copper-rich environments and which accumulates high concentrations of copper in its tissues. However, many of the physiological processes and mechanisms for metal hyperaccumulation in S. cataractae remain unknown. To address this issue, we examined the mechanisms regulating gemma formation, which is considered the main strategy by which S. cataractae relocates to new copper-rich areas. From this study we found that treatment of S. cataractae with high concentrations of copper suppressed gemma formation but promoted protonemal growth. The suppressive effect was not observed by treatment with heavy metals other than copper. These results suggest the importance of copper-sensitive asexual reproduction in the unique life strategy of the copper moss, S. cataractae.

Nicotinamide adenine dinucleotide extends the lifespan of Caenorhabditis elegans mediated by sir-2.1 and daf-16.

It is well understood that sir2 (sirtuin), an NAD-dependent deacetylase, is essential for the extension of lifespan under caloric restriction. However, the mechanism underlying activation of sir2 is unclear. Life extension through caloric restriction requires the sir2 ortholog sir-2.1 in nematodes but occurs independently of the forkhead-type transcription factor DAF-16. We aimed here to elucidate the correlation between life extension in nematodes and NAD-dependent activation of sirtuin by analyzing the relationship between NAD and DAF-16. Lifespan was extended when Caenorhabditis elegans were bred using medium containing NAD. An RNA interference experiment revealed that life extension by NAD was sir-2.1 dependent. However, life extension by NAD did not occur in daf-16-RNAi nematodes, suggesting that NAD-dependent longevity requires daf-16. This result suggested that different signaling pathways are involved in life extension resulting from caloric restriction and from NAD addition. Expression of sod-3, a target gene of daf-16, and increased oxidative-stress resistance and adiposity were observed in response to NAD addition, indicating that NAD activated daf-16 in each phenotype. These results suggest that NAD affected lifespan through the activation of SIR-2.1 and DAF-16 along a signaling pathway, namely insulin-like signalling pathway (at least parts of it), different from that associated with caloric restriction.

Highly Efficient CRISPR-Associated Protein 9 Ribonucleoprotein-Based Genome Editing in Euglena gracilis.

Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, and biofuels. However, targeted mutagenesis in this species has been a long-standing challenge. We recently developed a transgene-free, highly efficient, genome editing method for E. gracilis using CRISPR/Cas9 ribonucleoproteins (RNPs). Our method achieved mutagenesis rates of approximately 80% or more through an electroporation-based direct delivery of Cas9 RNPs. Therefore, this method is suitable for basic research and industrial applications, such as the breeding of Euglena. For complete details on the use and execution of this protocol, please refer to Nomura et al. (2019).

Genetic Engineering Strategies for Euglena gracilis and Its Industrial Contribution to Sustainable Development Goals: A Review.

The sustainable development goals (SDGs) adopted at the 2015 United Nations Summit are globally applicable goals designed to help countries realize a sustainable future. To achieve these SDGs, it is necessary to utilize renewable biological resources. In recent years, bioeconomy has been an attractive concept for achieving the SDGs. Microalgae are one of the biological resources that show promise in realizing the "5F"s (food, fiber, feed, fertilizer, and fuel). Among the microalgae, Euglena gracilis has the potential for achieving the "5F"s strategy owing to its unique features, such as production of paramylon, that are lacking in other microalgae. E. gracilis has already been produced on an industrial scale for use as an ingredient in functional foods and cosmetics. In recent years, genetic engineering methods for breeding E. gracilis have been researched and developed to achieve higher yields. In this article, we summarize how microalgae contribute toward achieving the SDGs. We focus on the contribution of E. gracilis to the bioeconomy, including its advantages in industrial use as well as its unique characteristics. In addition, we review genetic engineering-related research trends centered on E. gracilis, including a complete nuclear genome determination project, genome editing technology using the CRISPR-Cas9 system, and the development of a screening method for selecting useful strains. In particular, genome editing in E. gracilis could be a breakthrough for molecular breeding of industrially useful strains because of its high efficiency.

Parental legacy and regulatory novelty in Brachypodium diurnal transcriptomes accompanying their polyploidy.

Polyploidy is a widespread phenomenon in eukaryotes that can lead to phenotypic novelty and has important implications for evolution and diversification. The modification of phenotypes in polyploids relative to their diploid progenitors may be associated with altered gene expression. However, it is largely unknown how interactions between duplicated genes affect their diurnal expression in allopolyploid species. In this study, we explored parental legacy and hybrid novelty in the transcriptomes of an allopolyploid species and its diploid progenitors. We compared the diurnal transcriptomes of representative Brachypodium cytotypes, including the allotetraploid Brachypodium hybridum and its diploid progenitors Brachypodium distachyon and Brachypodium stacei. We also artificially induced an autotetraploid B. distachyon. We identified patterns of homoeolog expression bias (HEB) across Brachypodium cytotypes and time-dependent gain and loss of HEB in B. hybridum. Furthermore, we established that many genes with diurnal expression experienced HEB, while their expression patterns and peak times were correlated between homoeologs in B. hybridum relative to B. distachyon and B. stacei, suggesting diurnal synchronization of homoeolog expression in B. hybridum. Our findings provide insight into the parental legacy and hybrid novelty associated with polyploidy in Brachypodium, and highlight the evolutionary consequences of diurnal transcriptional regulation that accompanied allopolyploidy.

Elongation and desaturation of fatty acids are critical in growth, lipid metabolism and ontogeny of Caenorhabditis elegans.

Recently, it was reported that a deficit in the mouse stearoyl-CoA desaturase 1 gene decreases biosynthesis and accumulation of fatty acid and revitalizes the beta-oxidation of fatty acid. To examine the physiological role of fatty acid desaturase (FAT) and elongase (ELO)-gene transduction in ontogeny, fatty acid accumulation and individual lifespan, we performed bacteria-mediated RNA interference (RNAi) in the nematode Caenorhabditis elegans. Suppression of the expression of FAT-2 gene mRNA caused a drastic decrease in the amount of body fat and defects in egg-hatching. The amount of body fat was markedly decreased, and body size reduced, by down regulation of FAT-6 and FAT-7, whereas lifespan was drastically reduced. RNAi of the FAT-2 gene caused a remarkable increase of the beta-oxidation-related gene expression and the DAF-16 transcriptional activity, whereas, ELO-2 RNAi caused a remarkable decrease in fatty acid biosynthesis-related gene expression. Additionally, RNAi of FAT-6 decreased the mRNA levels of the genes involved in fatty acid synthesis, and FAT-7 RNAi increased the mRNA levels of beta-oxidation system genes. These results indicated that the elongation and desaturation of fatty acids are integral to various phenomena such as ontogeny and lifespan and play important roles in fatty acid accumulation and consumption.

Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses.

RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method ( Nomura et al., 2016 ), indicating that this experimental system could be applied to a wide range of mosses species.

Fat accumulation in Caenorhabditis elegans is mediated by SREBP homolog SBP-1.

Research into the metabolism of fats may reveal potential targets for developing pharmaceutical approaches to obesity and related disorders. Such research may be limited, however, by the cost and time involved in using mammalian subjects or developing suitable cell lines. To determine whether invertebrates could be used to carry out such research more efficiently, we investigated the ability of Caenorhabditis elegans (C. elegans) to accumulate body fat following the consumption of excess calories and the mechanisms it uses to metabolize fat. C. elegans worms were grown on media containing various sugars and monitored for changes in body fat and expression of sbp-1, a homolog of the mammalian transcription factor SREBP-1c, which facilitates fat storage in mammals. The fat content increased markedly in worms exposed to glucose. In situ analysis of gene expression in transgenic worms carrying the GFP-labeled promoter region of sbp-1 revealed that sbp-1 mRNA was strongly expressed in the intestine. An sbp-1 knockdown caused a reduction in body size, fat storage, and egg-laying activity. RT-PCR analysis revealed a considerable decrease in the expression of fatty acid synthetic genes (including elo-2, fat-2, and fat-5) and a considerable increase of starvation-inducible gene acs-2. Normal egg-laying activity and acs-2 expression were restored on exposure to a polyunsaturated fatty acid. These findings suggest that SBP-1 and SREBP regulate the amount and composition of fat and response to starvation in a similar manner. Thus, C. elegans may be an appropriate subject for studying the metabolism of fats.