title: Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae).
abstract: Plants from Violaceae produce cyclotides, peptides characterized by a circular peptide backbone and a cystine knot. This signature motif gives stability that can harness a wide spectrum of biological activities, with implications in plant defense and with applications in medicine and biotechnology. In the current work, cyclotide expressing in vitro cultures were established from Viola uliginosa. These cultures are useful models for studying biosynthesis of cyclotides and can also be used in their production. The cyclotide expression pattern is shown to be dependent on exogenous plant growth regulators, both on peptide and gene expression levels. The highest yields of cyclotides were obtained on media containing only a cytokinin and were correlated with storage material accumulation. Exposure to auxins decreased cyclotide production and caused shifting of the biosynthesis pattern to root specific cyclotides. The response to stimuli in terms of cyclotide expression pattern appears to be developmental, and related to polar auxin transportation and the auxin/cytokinin ratio regulating tissue differentiation. By the use of whole transcriptome shotgun sequencing (WTSS) and peptidomics, 20 cyclotide sequences from V. uliginosa (including 12 new) and 12 complete precursor proteins could be identified. The most abundant cyclotides were cycloviolacin O3 (CyO3), CyO8 and CyO13. A suspension culture was obtained that grew exponentially with a doubling time of approximately 3 days. After ten days of growth, the culture provided a yield of more than 4 mg CyO13 per gram dry mass.
authors.affiliation: Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, 9 Gronostajowa Str., 30-387 Cracow, Poland; Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, 751 23 Uppsala, Sweden; W. Szafer Istitute of Botany, Polish Academy of Sciences, 46 Lubicz St., 31-512 Cracow, Poland. Electronic address: blazejslazak@gmail.com.
authors.forename: Blazej
authors.initials: B
authors.lastname: Slazak
authors.affiliation: Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, 751 23 Uppsala, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Jacobsson
authors.affiliation: Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, 9 Gronostajowa Str., 30-387 Cracow, Poland.
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Kuta
authors.affiliation: Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, 751 23 Uppsala, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Göransson
mesh_terms: D000595:Amino Acid Sequence; D047169:Cyclotides / Q000235:genetics* / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D003583:Cytokinins / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010671:Phenylurea Compounds / Q000494:pharmacology; D010937:Plant Growth Regulators / Q000378:metabolism / Q000494:pharmacology; D018515:Plant Leaves / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D013830:Thiadiazoles / Q000494:pharmacology; D046509:Tissue Culture Techniques; D032413:Viola / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D000099240:Asparaginyl Endopeptidase
chemical_list: D047169:Cyclotides; D003546:Cysteine Endopeptidases; D003583:Cytokinins; D010671:Phenylurea Compounds; D010937:Plant Growth Regulators; D013830:Thiadiazoles; D000099240:Asparaginyl Endopeptidase; C527840:cycloviolacin O13; C016785:thidiazuron
keywords: Cyclotides; In vitro culture; Mass spectrometry; Plant growth regulators; Viola uliginosa (Violaceae); Whole transcriptome shotgun sequencing