Comparative Transcriptomic Analysis of Genes in the 20-Hydroxyecdysone Biosynthesis in the Fern Microsorum scolopendria towards Challenges with Foliar Application of Chitosan.

Microsorum scolopendria is an important medicinal plant that belongs to the Polypodiaceae family. In this study, we analyzed the effects of foliar spraying of chitosan on growth promotion and 20-hydroxyecdysone (20E) production in M. scolopendria. Treatment with chitosan at a concentration of 50 mg/L in both young and mature sterile fronds induced the highest increase in the amount of accumulated 20E. Using RNA sequencing, we identified 3552 differentially expressed genes (DEGs) in response to chitosan treatment. The identified DEGs were associated with 236 metabolic pathways. We identified several DEGs involved in the terpenoid and steroid biosynthetic pathways that might be associated with secondary metabolite 20E biosynthesis. Eight upregulated genes involved in cholesterol and phytosterol biosynthetic pathway, five upregulated genes related to the methylerythritol 4-phosphate (MEP) and mevalonate (MVA) pathways, and several DEGs that are members of cytochrome P450s and ABC transporters were identified. Quantitative real-time RT-PCR confirmed the results of RNA-sequencing. Taken together, we showed that chitosan treatment increased plant dry weight and 20E accumulation in M. scolopendria. RNA-sequencing and DEG analyses revealed key enzymes that might be related to the production of the secondary metabolite 20E in M. scolopendria.

De Novo Transcriptome Assembly of Two Microsorum Fern Species Identifies Enzymes Required for Two Upstream Pathways of Phytoecdysteroids.

Microsorum species produce a high amount of phytoecdysteroids (PEs), which are widely used in traditional medicine in the Pacific islands. The PEs in two different Microsorum species, M. punctatum (MP) and M. scolopendria (MS), were examined using high-performance liquid chromatography (HPLC). In particular, MS produces a high amount of 20-hydroxyecdysone, which is the main active compound in PEs. To identify genes for PE biosynthesis, we generated reference transcriptomes from sterile frond tissues using the NovaSeq 6000 system. De novo transcriptome assembly after deleting contaminants resulted in 57,252 and 54,618 clean transcripts for MP and MS, respectively. The clean Microsorum transcripts for each species were annotated according to gene ontology terms, UniProt pathways, and the clusters of the orthologous group protein database using the MEGAN6 and Sma3s programs. In total, 1852 and 1980 transcription factors were identified for MP and MS, respectively. We obtained transcripts encoding for 38 and 32 enzymes for MP and MS, respectively, potentially involved in mevalonate and sterol biosynthetic pathways, which produce precursors for PE biosynthesis. Phylogenetic analyses revealed many redundant and unique enzymes between the two species. Overall, this study provides two Microsorum reference transcriptomes that might be useful for further studies regarding PE biosynthesis in Microsorum species.

Evolutionary relationships of the ancient fern lineage the adder's tongues (Ophioglossaceae) with description of Sahashia gen. nov.

As an ancient lineage of ferns, Ophioglossaceae are evolutionarily among the most fascinating because they have the highest chromosome count of any known organism as well as the presence of sporophores, subterranean gametophytes, eusporangiate sporangia without annuli, and endophytic fungi. Previous studies have produced conflicting results, identifyingsome lineages with unresolved relationships, and have paid much attention to the subfamily Botrychioideae. But the other species-rich subfamily, Ophioglossoideae, has remained largely understudied and only up to 12 accessions of Ophioglossoideae have been sampled. In this study, DNA sequences of seven plastid markers of 149 accessions (75 in Ophioglossoideae) representing approximately 82 species (approximately 74% of estimated species diversity sensu J. Syst. Evol., 2016, 54, 563) in the family, and two Marattiaceae and two Psilotaceae, are used to infer a phylogeny. Our major results include: (1) Ophioglossaceae are resolved as monophyletic with strong support, and so are all four subfamilies and genera sensu PPG I except Botrypus and Ophioglossum; (2) a new genus Sahashia is segregated from Botrypus so that the monophyly of Botrypus can be retained; (3) the monophyly of Ophioglossum in its current circumscription is uncertain in spite of our large character sampling; (4) there is substantial cryptic speciation in Ophioderma detected by our molecular and morphological study; (5) the recognition of Holubiella is advocated based on its morphology and its sister relationship with Sceptridium; and (6) a novel sister relationship between Botrychium and the JHS clade (Japanobotrychium + (Holubiella + Sceptridium)) is discovered.