Ethnopharmacology of the club moss subfamily Huperzioideae (Lycopodiaceae, Lycopodiophyta): A phylogenetic and chemosystematic perspective.

ETHNOPHARMACOLOGICAL RELEVANCE: The most speciose subfamily Huperzioideae (Lycopodiaceae, Lycopodiophyta) contains about 276 species, and some (ca. 20 species) have traditionally been used for the treatment of e.g., dementia, rheumatism and traumatic injury. Ethnopharmacological studies have also contributed to the development of huperzine A as a drug lead, a compound first isolated from the club moss Huperzia serrata (Thunb. ex Murray) Trevis. AIM OF THE REVIEW: This review, with a phylogenetic and chemosystematic perspective, intends to highlight plant identification challenges in these taxa with examples from club moss phytochemical and ethnopharmacological studies, as these lead to data inconsistency and confusion. We suggest that future studies should include more details on plant identification including for example plant specimen images and DNA barcoding data. An integrative approach combining DNA barcoding and chemical fingerprinting is also introduced. MATERIALS AND METHODS: Literature concerning ethnopharmacology and chemosystematics of Huperzioideae club mosses was searched from databases, e.g. PubMed, Web of Science, SciFinder, etc. Plant names were retrieved from original publications, and compared with up-to-date taxonomic and phylogenetic status. Ethnobotanical uses and herbal preparations were summarized. Production of certain pharmaceutically interesting compounds, such as the alkaloid huperzine A, was explored in a phylogenetic context. RESULTS: Most traditionally used club mosses are associated with psychoactivity, followed by medicinal uses against rheumatism and traumatic injury. Herbs are often prepared as infusions, decoctions or tinctures, and this implies importance of water- or aqueous-alcohol-soluble substances, such as alkaloids. Most ethnopharmacological papers on club mosses need to update or correct plant names according to recent taxonomic nomenclature, and there are still a number of unidentified species with traditional use. Advanced LC-MS chemical profiling techniques, enable distinction of genotypes of the same species as well as annotation of potential chemotaxonomic markers. In combination with DNA barcoding, chemosystematics could also help us select plant taxa with higher pharmaceutical potential. Caution should be taken when interpreting bioassay results, in terms of compounds or extract preparation and bioassay standardization. CONCLUSION: Huperzioideae club mosses have interesting pharmaceutical potential supported by ethnopharmacological investigations. Bioprospecting of these plants should be preceded by careful plant identification to produce consistent and reproducible data. We expect that DNA barcoding and LC-MS-based chemical fingerprinting could facilitate and improve ethnopharmaceutical studies in selection of club moss taxa.

[Cloning and bioinformatics analysis of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase（PcHDR1）gene in Phlegmarirus carinatus].

The open reading frame of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) was cloned from Phlegmarirus carinatus by RT-PCR method and the sequence was analyzed by bioinformatics tools. After searching the transcriptome dataset of P. carinatus, one unique sequence encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase was discovered. The primers were designed according to the cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from the dataset. And then, the open reading frame (ORF) of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, named as PcHDR1 (GenBank Accession number：JQ957845), was cloned by RT-PCR strategy with the template of mixed RNA extracted from roots, stem and leaf of P. carinatus. The bioinformatic analysis of this gene and its corresponding protein was performed. The ORF of PcHDR1 consisted of 1 437 base pairs (bp), encoding one polypeptide with 478 amino acids. The sequence comparison showed that PcHDR1 is closest with GbHDR (Ginkgo biloba),and the sequence homology was up to 78%. Bioinformatics prediction and analysis indicated that PcHDR1 protein contained a conserved domain of LytB, without transmembrane region and signal peptides. This study cloned and analyzed 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from P. carinatus. The result will provide a foundation for exploring the function of PcHDR1 involved in terpene biosynthesis in P. carinatus plants.

Metabolic, genomic, and biochemical analyses of glandular trichomes from the wild tomato species Lycopersicon hirsutum identify a key enzyme in the biosynthesis of methylketones.

Medium-length methylketones (C7-C15) are highly effective in protecting plants from numerous pests. We used a biochemical genomics approach to elucidate the pathway leading to synthesis of methylketones in the glandular trichomes of the wild tomato Lycopersicon hirsutum f glabratum (accession PI126449). A comparison of gland EST databases from accession PI126449 and a second L. hirsutum accession, LA1777, whose glands do not contain methylketones, showed that the expression of genes for fatty acid biosynthesis is elevated in PI126449 glands, suggesting de novo biosynthesis of methylketones. A cDNA abundant in the PI126449 gland EST database but rare in the LA1777 database was similar in sequence to plant esterases. This cDNA, designated Methylketone Synthase 1 (MKS1), was expressed in Escherichia coli and the purified protein used to catalyze in vitro reactions in which C12, C14, and C16 beta-ketoacyl-acyl-carrier-proteins (intermediates in fatty acid biosynthesis) were hydrolyzed and decarboxylated to give C11, C13, and C15 methylketones, respectively. Although MKS1 does not contain a classical transit peptide, in vitro import assays showed that it was targeted to the stroma of plastids, where fatty acid biosynthesis occurs. Levels of MKS1 transcript, protein, and enzymatic activity were correlated with levels of methylketones and gland density in a variety of tomato accessions and in different plant organs.

Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants.

Borate ester cross-linking of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II) is required for the growth and development of angiosperms and gymnosperms. Here, we report that the amounts of borate cross-linked RG-II present in the sporophyte primary walls of members of the most primitive extant vascular plant groups (Lycopsida, Filicopsida, Equisetopsida, and Psilopsida) are comparable with the amounts of RG-II in the primary walls of angiosperms. By contrast, the gametophyte generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide. The glycosyl sequence of RG-II is conserved in vascular plants, but these RG-IIs are not identical because the non-reducing L-rhamnosyl residue present on the aceric acid-containing side chain of RG-II of all previously studied plants is replaced by a 3-O-methyl rhamnosyl residue in the RG-IIs isolated from Lycopodium tristachyum, Ceratopteris thalictroides, Platycerium bifurcatum, and Psilotum nudum. Our data indicate that the amount of RG-II incorporated into the walls of plants increased during the evolution of vascular plants from their bryophyte-like ancestors. Thus, the acquisition of a boron-dependent growth habit may be correlated with the ability of vascular plants to maintain upright growth and to form lignified secondary walls. The conserved structures of pteridophyte, lycophyte, and angiosperm RG-IIs suggests that the genes and proteins responsible for the biosynthesis of this polysaccharide appeared early in land plant evolution and that RG-II has a fundamental role in wall structure.