O- and N-Methyltransferases in benzylisoquinoline alkaloid producing plants.

BACKGROUND: Secondary metabolites such as benzylisoquinoline alkaloids (BIA) have attracted considerable attention because of their pharmacological properties and potential therapeutic applications. Methyltransferases (MTs) can add methyl groups to alkaloid molecules, altering their physicochemical properties and bioactivity, stability, solubility, and recognition by other cellular components. Five types of O-methyltransferases and two types of N-methyltransferases are involved in BIA biosynthesis. OBJECTIVE: Since MTs may be the source for the discovery and development of novel biomedical, agricultural, and industrial compounds, we performed extensive molecular and phylogenetic analyses of O- and N-methyltransferases in BIA-producing plants. METHODS: MTs involved in BIA biosynthesis were isolated from transcriptomes of Berberis koreana and Caulophyllum robustum. We also mined the methyltransferases of Coptis japonica, Papaver somniferum, and Nelumbo nucifera from the National Center for Biotechnology Information protein database. Then, we analyzed the functional motifs and phylogenetic analysis. RESULT: We mined 42 O-methyltransferases and 8 N-methyltransferases from the five BIA-producing plants. Functional motifs for S-adenosyl-L-methionine-dependent methyltransferases were retained in most methyltransferases, except for the three O-methyltransferases from N. nucifera. Phylogenetic analysis revealed that the methyltransferases were grouped into four clades, I, II, III and IV. The clustering patterns in the phylogenetic analysis suggested a monophyletic origin of methyltransferases and gene duplication within species. The coexistence of different O-methyltransferases in the deep branch subclade might support some cases of substrate promiscuity. CONCLUSIONS: Methyltransferases may be a source for the discovery and development of novel biomedical, agricultural, and industrial compounds. Our results contribute to further understanding of their structure and reaction mechanisms, which will require future functional studies.

Isolation and Characterization of the Genes Involved in the Berberine Synthesis Pathway in Asian Blue Cohosh, Caulophyllum robustum.

Caulophyllum robustum, commonly named Asian blue cohosh, is a perennial herb in the family Berberidaceae. It has traditionally been used for folk medicine in China. We isolated berberine from the leaves, stem, roots, and fruits of C. robustum, and this is the first report on berberine in this species. Transcriptome analysis was conducted for the characterization of berberine biosynthesis genes in C. robustum, in which, all the genes for berberine biosynthesis were identified. From 40,094 transcripts, using gene ontology (GO) analysis, 26,750 transcripts were assigned their functions in the categories of biological process, molecular function, and cellular component. In the analysis of genes expressed in different tissues, the numbers of genes in the categories of intrinsic component of membrane and transferase activity were up-regulated in leaves versus stem. The berberine synthesis genes in C. robustum were characterized by phylogenetic analysis with corresponding genes from other berberine-producing species. The co-existence of genes from different plant families in the deepest branch subclade implies that the differentiation of berberine synthesis genes occurred early in the evolution of berberine-producing plants. Furthermore, the copy number increment of the berberine synthesis genes was detected at the species level.

Whole-genome sequencing revealed different demographic histories among the Korean endemic hill pigeon (Columba rupestris), rock pigeon (Columba livia var. domestica) and oriental turtle dove (Streptopelia orientalis).

BACKGROUND: The family Columbidae is known as the pigeon family and contains approximately 351 species and 50 genera. Compared to the wealth of biological and genomic information on these Columba livia var. domesteca, information on Columba rupestris and Streptopelia orientalis has been rather limited. The C. rupestris population size is decreasing in Korea. OBJECTIVES: Whole-genome sequencing and identification of population characterization of each species based genome variation on 9 Korean pigeon and dove samples, namely, six hill pigeon (C. rupestris), one rock pigeon (C. livia var. domestica) and two oriental turtle dove (S. orientalis) samples. RESULTS: The whole genome of 9 genotypes were sequenced and mapped to the C. livia reference genome. Sequence alignment showed over 96% identity in C. rupestris and 94% identity in S. orientalis to the reference genome (GenBank assembly accession: GCA_001887795.1). Sequence variations, including single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), and structural variations, revealed that intergenus (Columba vs. Streptopelia) variations were approximately four times higher than intragenus variations (C. livia vs. C. rupestris). Of the two Columba species, C. livia var. domestica is closer to S. orientalis than C. rupestris. Pairwise sequentially Markovian coalescent (PSMC) demographic history analysis revealed that the three species underwent a common population bottleneck between 105 and 120 Kya; since then, the effective population sizes of the rock pigeon and oriental turtle dove have increased. CONCLUSION: The effective population size of the hill pigeon, an Endangered Species of Grade II in Korea, has increased slowly from the second severe bottleneck that occurred approximately 0.5-1.4 × 104 years ago. Our results showed no relationship between copy number variation in the Norrie disease protein (NDP) regulatory regions and plumage color patterns. We report the first comparative analysis of three pigeon genomes.

Identifying Terpenoid Biosynthesis Genes in Euphorbia maculata via Full-Length cDNA Sequencing.

The annual herb Euphorbia maculata L. produces anti-inflammatory and biologically active substances such as triterpenoids, tannins, and polyphenols, and it is used in traditional Chinese medicine. Of these bioactive compounds, terpenoids, also called isoprenoids, are major secondary metabolites in E. maculata. Full-length cDNA sequencing was carried out to characterize the transcripts of terpenoid biosynthesis reference genes and determine the copy numbers of their isoforms using PacBio SMRT sequencing technology. The Illumina short-read sequencing platform was also employed to identify differentially expressed genes (DEGs) in the secondary metabolite pathways from leaves, roots, and stems. PacBio generated 62 million polymerase reads, resulting in 81,433 high-quality reads. From these high-quality reads, we reconstructed a genome of 20,722 genes, in which 20,246 genes (97.8%) did not have paralogs. About 33% of the identified genes had two or more isoforms. DEG analysis revealed that the expression level differed among gene paralogs in the leaf, stem, and root. Whole sets of paralogs and isoforms were identified in the mevalonic acid (MVA), methylerythritol phosphate (MEP), and terpenoid biosynthesis pathways in the E. maculata L. The nucleotide information will be useful for identifying orthologous genes in other terpenoid-producing medicinal plants.

Gibberellic acid sensitive dwarf encodes an ARPC2 subunit that mediates gibberellic acid biosynthesis, effects to grain yield in rice.

The plant hormone gibberellic acid (GA) is important for plant growth and productivity. Actin-related proteins (ARPs) also play central roles in plant growth, including cell elongation and development. However, the relationships between ARPs and GA signaling and biosynthesis are not fully understood. Here, we isolated OsGASD, encoding an ARP subunit from rice (Oryza sativa), using the Ac/Ds knockout system. The osgasd knockout (Ko) mutation reduced GA3 content in shoots as well as plant growth and height. However, GA application restored the plant height of the osgasd Ko mutant to a height similar to that of the wild type (WT). Rice plants overexpressing OsGASD (Ox) showed increased plant height and grain yield compared to the WT. Transcriptome analysis of flag leaves of OsGASD Ox and osgasd Ko plants revealed that OsGASD regulates cell development and the expression of elongation-related genes. These observations suggest that OsGASD is involved in maintaining GA homeostasis to regulate plant development, thereby affecting rice growth and productivity.

The genome of the freshwater monogonont rotifer Brachionus angularis: Identification of phase I, II, and III detoxification genes and their roles in molecular ecotoxicology.

Brachionus spp. rotifers, which are widely distributed in aquatic environments, have been proposed as model organisms for ecotoxicological studies. Although the genomes of several rotifers belonging to the genus Brachionus have been assembled, the genome for the freshwater rotifer Brachionus angularis remains unknown. In this study, we analyzed the whole-genome sequence of B. angularis, which revealed a total length of 56.5 Mb and 21 contigs. The N50 and the GC content were 5.42 Mb and 23.66%, respectively. A total of 13,952 genes were predicted. Of them, we identified the main detoxification-related gene families, including those for cytochrome P450, glutathione S-transferase (GST), and the ATP-binding cassette transporter. In comparison with other Brachionus rotifers, massive species-specific expansion in GST sigma genes was found in B. angularis. This whole-genome analysis of B. angularis provides a basis for molecular ecotoxicological studies and provides useful biological tools for comparative studies of the evolution of detoxification mechanisms in Brachionus spp.

The genome of the European estuarine calanoid copepod Eurytemora affinis: Potential use in molecular ecotoxicology.

In this study, we sequenced and assembled the genome of a European estuarine calanoid copepod using Oxford Nanopore PromethION and Illumina HiSeq 2500 platforms. The length of the assembled genome was 776.1 Mb with N50 = 474.9 kb (BUSCO 85.9%), and the genome consisted of 2473 contigs. A total of 18,014 genes were annotated and orthologous gene clusters were analyzed in comparison to other copepods. In addition, genome-wide identification of cytochrome P450s, glutathione S-transferases, and ATP-binding cassette transporters in E. affinis was performed to determine gene repertoire of these detoxification-related gene families. Results revealed the presence of species-specific gene inventories, indicating that these gene families have evolved through species-specific gene loss/expansion processes, possibly due to adaptation to different environmental stressors. Our study provides a new inventory of the European estuarine calanoid copepod E. affinis genome with emphasis on phase I, II, and III detoxification systems.

The genome of the marine water flea Diaphanosoma celebensis: Identification of phase I, II, and III detoxification genes and potential applications in marine molecular ecotoxicology.

To assemble the genome of the marine water flea Diaphanosoma celebensis, a sentinel model for marine environmental monitoring, we constructed a high-quality genome using PromethION and HiSeq 2500 platforms. The total length of the assembled genome was 100.08 Mb, with N50 = 2.56 Mb (benchmarking universal single-copy orthologs, 96.9%) and consisted of 179 scaffolds. A total of 15,427 genes were annotated, and orthologous gene clusters in D. celebensis were analyzed and compared with those of the cladocerans Daphnia magna and Daphnia pulex. In addition, phase I, II, and III detoxification gene families of cytochrome P450s, glutathione S-transferases, and ATP-binding cassette were fully identified and revealed lineage-specific gene loss and/or expansion, suggesting that the evolution of detoxification gene families likely modulates fitness and susceptibility in response to environmental stressors. The study improves our understanding of the detoxification-related gene system and should contribute to future studies of molecular ecotoxicology in cladoceran species and their responses to emerging pollutants.

Complete mitochondrial genome of the euryhaline monogonont rotifer Brachionus paranguensis (Rotifera, Brachionidae).

The two complete mitochondrial genomes were sequenced from the euryhaline monogonont rotifer Brachionus paranguensis. The mitochondrial genome sequences were 11,603 bp and 12,901 bp in size, and the gene order of 12 protein-coding genes (PCGs) were identical to those of the marine rotifer Brachionus plicatilis, but the positions of some tRNAs (e.g. tRNA-Ile, tRNA-Leu[TTA], tRNA-Phe, and tRNA-Leu[CTC]) of mitochondrial DNA I were different between B. paranguensis and B. plicatilis mitochondrial genomes. Of 12 PCGs, four genes (ND1, ATPase 6, ND5, and ND3) had incomplete stop codons. Furthermore, the start codon of ND4, ND5, and CO3 genes was ATT, while the start codon of other PCGs was ATG. The base composition of 12 PCGs in B. paranguensis mitochondrial genomes was 26.6% for A, 43.0% for T, 17.65% for C, and 12.75% for G, respectively.

Complete mitochondrial DNA of the marine water flea Diaphanosoma celebensis (Cladocera, Sididae).

The complete mitochondrial genome was sequenced from the marine water flea Diaphanosoma celebensis. The sequenced mitochondrial genome size was 17,060 bp, possessing identical gene order of 13 protein-coding genes (PGCs) to those of the congeneric freshwater species Diaphanosoma dubium in the genus Diaphanosoma. The mitochondrial genome of D. celebensis had 13 PGCs, two rRNAs, and 22 tRNAs. Of 13 PGCs, three genes (CO3, ND3, and ND4) had incomplete stop codons. Furthermore, the stop codons of the remaining ten PGCs were TAA (for CO1, ATP8, ATP6, ND5, ND6, and ND1) and TAG (for NL4L, Cytb, and ND2). The second and third base composition of codon on 9 PCGs on the L strand in D. celebensis mitogenome showed an anti-G bias (11.0% and 15.0%), respectively.

Complete mitochondrial genome of the freshwater monogonont rotifer Brachionus angularis (Rotifera, Brachionidae).

The two complete mitochondrial genomes were sequenced from the freshwater monogonont rotifer Brachionus angularis. The mitochondrial genome sequences were 10,764 bp (mitochondrial DNA I) and 12,238 bp (mitochondrial DNA II) in size, respectively. The gene structure and its orientation of 12 protein-coding genes (PCGs) of complete mitochondrial genomes of B. angularis was identical to those shown in other marine rotifers and the freshwater rotifer Brachionus rubens, but was different from the freshwater rotifer Brachionus calyciflorus. Of 12 PCGs, one gene (ND5) had incomplete stop codon. Furthermore, the start codon for CO1, ND4L, ND5, and CO2 was GTG, while the start codon for ND3 and other PCGs was ATA and ATG, respectively. The base composition of 12 PCGs in B. angularis mitogenome showed 20.4% for A, 47.3% for T, 17.5% for C, and 14.8% for G, respectively. The mitochondrial genome A + T base composition (67.7%) of 12 PCGs was higher than G + C (32.3%), while the complete mitochondrial genome A + T base composition (66.3%) was higher than G + C (33.7%).

Genome-enabled discovery of anthraquinone biosynthesis in Senna tora.

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we reveal the genome sequence of S. tora at the chromosome level with 526 Mb (96%) assembled into 13 chromosomes. Comparison among related plant species shows that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identify a CHS-L gene contributing to the biosynthesis of anthraquinones. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants.

The genome of the marine monogonont rotifer Brachionus rotundiformis and insight into species-specific detoxification components in Brachionus spp.

The monogonont rotifer Brachionus spp. have been widely used for ecotoxicological studies because of their advantages as one of the most suitable laboratory experimental species. In the present study, we obtained and assembled the whole genome sequence of the rotifer Brachionus rotundiformis, consisting of 13,612 annotated genes with 213 scaffolds and 58 Mb in total length. Focusing on ecotoxicological aspects, we conducted a comparative genome analysis on the gene families involved in detoxification, including four to six sulfotransferase gene families, seven uridine 5'-diphospho-glucuronosyltransferase gene families, and 58, 61, or 70 ATP-binding cassette genes in the genus Brachionus including Brachionus koreanus and Brachionus plicatilis. Our results suggest that these gene families have undergone a species- and/or lineage-specific evolution in response to the surrounding environmental pressure. Our genome resource for B. rotundiformis would be highly useful for future ecotoxicological studies and also provides a better understanding on the view of evolutionary mechanism of detoxification in the genus Brachionus spp.

Genome Assembly and Annotation of Soft-Shelled Adlay (Coix lacryma-jobi Variety ma-yuen), a Cereal and Medicinal Crop in the Poaceae Family.

Coix lacryma-jobi, also called adlay or Job's tears, is an annual herbal plant belonging to the Poaceae family that has been cultivated as a cereal and medicinal crop in Asia. Despite its importance, however, genomic resources for better understanding this plant species at the molecular level and informing improved breeding strategies remain limited. To address this, we generated a draft genome of the C. lacryma-jobi variety ma-yuen (soft-shelled adlay) Korean cultivar, Johyun, by de novo assembly, using PacBio and Illumina sequencing data. A total of 3,362 scaffold sequences, 1.28 Gb in length, were assembled, representing 82.1% of the estimated genome size (1.56 Gb). Genome completeness was confirmed by the presence of 91.4% of the BUSCO angiosperm genes and mapping ratio of 98.3% of Illumina paired-end reads. We found that approximately 77.0% of the genome is occupied by repeat sequences, most of which are Gypsy and Copia-type retrotransposons, and evidence-based genome annotation predicts 39,574 protein-coding genes, 85.5% of which were functionally annotated. We further predict that soft-shelled adlay diverged from a common ancestor with sorghum 9.0-11.2 MYA. Transcriptome profiling revealed 3,988 genes that are differentially expressed in seeds relative to other tissues, of which 1,470 genes were strongly up-regulated in seeds and the most enriched Gene Ontology terms were assigned to carbohydrate and protein metabolism. In addition, we identified 76 storage protein genes including 18 seed-specific coixin genes and 13 candidate genes involved in biosynthesis of benzoxazinoids (BXs) including coixol, a unique BX compound found in C. lacryma-jobi species. The characterization of those genes can further our understanding of unique traits of soft-shelled adlay, such as high seed protein content and medicinal compound biosynthesis. Taken together, our genome sequence data will provide a valuable resource for molecular breeding and pharmacological study of this plant species.

An improved genome assembly and annotation of the Antarctic copepod Tigriopus kingsejongensis and comparison of fatty acid metabolism between T. kingsejongensis and the temperate copepod T. japonicus.

Copepods in the genus Tigriopus are widely distributed in the intertidal zone worldwide. To assess differences in fatty acid (FA) metabolism among congeneric species in this genus inhabiting polar and temperate environments, we analyzed and compared FA profiles of the Antarctic copepod Tigriopus kingsejongensis and the temperate copepod T. japonicus. Higher amounts of total FAs were found in the Antarctic copepod T. kingsejongensis than the temperate copepod T. japonicus under administration of the identical amount of Tetraselmis suecica. To determine the genomic basis for this, we identified fatty acid metabolism-related genes in an improved genome of T. kingsejongensis. The total length of the assembled genome was approximately 338 Mb with N50 = 1.473 Mb, 938 scaffolds, and a complete Benchmarking Universal Single-Copy Orthologs value of 95.8%. A total of 25,470 genes were annotated using newly established pipeline. We identified eight elongation of very long-chain fatty acid protein (Elovl) genes and nine fatty acid desaturase (Fad) genes in the genome of T. kingsejongensis. In addition, fatty acid profiling suggested that the duplicated Δ5/6 desaturase gene in T. kingsejongensis is likely to play an essential role in synthesis of different FAs in T. kingsejongensis to those in T. japonicus. However, further experimental research is required to validate our in silico findings. This study provides a better understanding of fatty acid metabolism in the Antarctic copepod T. kingsejongensis.

The reference genome of the selfing fish Kryptolebias hermaphroditus: Identification of phases I and II detoxification genes.

The selfing fish Kryptolebias hermaphroditus has unique reproductive system for self-fertilization, making genetically homozygous offsprings. Here, we report on high density genetic map-based genome assembly for the K. hermaphroditus Panama line (PanRS). The numbers of scaffolds were 5212 and the genome was 683,992,224 bp (N50 = 27.45 Mb). The length of anchored scaffold onto 24 linkage groups was 652,231,070 bp (95.3% of genome) with 0.01% of the gap and 39.33% of GC content and complete Benchmarking Universal Single-Copy Orthologs value was 96.6%. The numbers of annotated genes were 36,756 (average gene length 1368 bp) with the GC content of 54.1%. To examine the difference between the two sister species in the genus Kryptolebias, we compared the genomes of K. hermaphroditus PanRS and Kryptolebias marmoratus PAN line on the composition of transposable elements. To demonstrate applications of genome library, phase I and II detoxification related gene families have been analyzed, and compared the syntenies containing loci of CYP and GST genes on linkage groups. This K. hermaphroditus genome information will be helpful for a better understanding on genome-wide mechanistic view of detoxification and antioxidant-related genes over evolution in the view of fish environmental ecotoxicology.

The genome of the Java medaka (Oryzias javanicus): Potential for its use in marine molecular ecotoxicology.

The Java medaka (Oryzias javanicus) is distributed in tropical brackish water and is considered as an ecotoxicological experimental organism for assessing diverse pollutions and global climate change effects in the ocean. In this study, we sequenced and assembled the genome of O. javanicus using the Oxford Nanopore technique and anchored the scaffolds to the 24 genetic linkage map of a sister species Oryzias melastigma. The assembled genome consisted of 773 scaffolds including 24 LG-based scaffolds, and the estimated genome length was 846.3 Mb (N50 = 19.3 Mb), containing 24,498 genes. As detoxification processes are crucial in aquatic organisms, antioxidant-related genes including glutathione S-transferases, superoxide dismutase, catalase, and glutathione peroxidase were identified in this study. In the genome of O. javanicus, a total of 21 GSTs, 4 SODs, 1 CAT, and 7 GPxs were identified and showed high similarities between sister species O. melastigma and Oryzias latipes. In addition, despite having 8 classes of cytosolic GSTs family, medaka showed no presence of GST pi and sigma classes, which are predominantly found in carp and salmon, but not in neoteleostei. This study adds another set to genome-library of Oryzias spp. and is a useful resource for better understanding of the molecular ecotoxicology.

The genome of the harpacticoid copepod Tigriopus japonicus: Potential for its use in marine molecular ecotoxicology.

The copepod Tigriopus japonicus has been widely used as an experimental species in the field of ecotoxicology. We have sequenced and assembled the whole genome of T. japonicus with comparative analysis of gene families that represent detoxification phases in two additional public genomes of Tigriopus spp., namely, T. californicus and T. kingsejongensis. The total length of the T. japonicus assembled genome was 196.6 Mb with an N50 value of 10.65 Mb and consisted of 339 scaffolds and 25,143 annotated genes. The detoxification gene families encoding cytochrome P450s (CYP450s), glutathione S-transferases (GSTs), and ATP-binding cassette (ABC) proteins in Tigriopus spp. have shown species-dependent diversity in several gene sets, suggesting that these genes have undergone a species-specific expansion to increase their fitness to different marine habitats and environmental pressures. Our study will provide a better understanding of the detoxification system in Tigriopus spp. and will contribute to various areas of research, including ecotoxicology.

The genome of the marine rotifer Brachionus koreanus sheds light on the antioxidative defense system in response to 2-ethyl-phenanthrene and piperonyl butoxide.

BRACHIONUS: spp. (Rotifera: Monogononta) have been introduced as ecotoxicological model-organisms that are widely distributed in aquatic environments. Among the Brachionus spp., the monogonont rotifer Brachionus koreanus has been widely used for ecology, ecotoxicology, and evolution, thus, providing the whole genome data of B. koreanus is important for further understandings of in-depth molecular mechanisms. In this study, the completed assembly and characterization of the B. koreanus genome resulted in a total length of 85.7 Mb with 14,975 annotated genes. The final number of scaffolds was 567 with an N50 value and a GC content of 1.86 Mb and 24.35 %, respectively. Based on the fully constructed genome database, a total of 24 CYPs, 23 GSTs, two SODs, and a single CAT genes were identified and analyzed antioxidant activities (CAT, SOD, and GST), and transcriptional regulation of the entire CYPs, GSTs, SODs, and CAT in response to 2-ethyl-phenanthrene (2-ethyl-PHE) and piperonyl butoxide (PBO), to demonstrate the usefulness of the whole genome library of B. koreanus in response xenobiotic-induced oxidative stress. The assembled B. koreanus genome will provide a better understanding on the molecular ecotoxicology in the view of molecular mechanisms underlying toxicological responses, particularly on xenobiotic detoxification processes in the rotifer B. koreanus.