The life cycle of cyclotides: biosynthesis and turnover in plant cells
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ORIGINAL ARTICLE
The life cycle of cyclotides: biosynthesis and turnover in plant cells Blazej Slazak1,2 · Tobias Haugmo1 · Bogna Badyra2 · Ulf Göransson1 Received: 19 February 2020 / Accepted: 10 July 2020 © The Author(s) 2020
Abstract Key message Turnover rates have implications for understanding cyclotide biology and improving plant cell culturebased production systems. Abstract Cyclotides are a family of polypeptides recognized for a broad spectrum of bioactivities. The cyclic, cystine knot structural motif imparts these peptides with resistance to temperature, chemicals and proteolysis. Cyclotides are found widely distributed across the Violaceae and in five other plant families, where their presumed biological role is host defense. Violets produce mixtures of different cyclotides that vary depending on the organ, tissue or influence of environmental factors. In the present study, we investigated the biosynthesis and turnover of cyclotides in plant cells. Viola uliginosa suspension cultures were grown in media where all nitrogen containing salts were replaced with their 15N counterparts. This approach combined with LC–MS analysis allowed to separately observe the production of 15N-labelled peptides and decomposition of 14N cyclotides present in the cells when switching the media. Additionally, we investigated changes in cyclotide content in V. odorata germinating seeds. In the suspension cultures, the degradation rates varied for individual cyclotides and the highest was noted for cyO13. Rapid increase in production of 15N peptides was observed until day 19 and subsequently, a plateau of production, indicating an equilibrium between biosynthesis and turnover. The developing seedling appeared to consume cyclotides present in the seed endosperm. We show that degradation processes shape the cyclotide pattern present in different tissues and environments. The results indicate that individual cyclotides play different roles—some in defense and others as storage proteins. The turnover of cyclotides should be accounted to improve cell culture production systems. Keywords Cyclic peptides · 15N labelling · Plant cell suspension culture · Viola · Mass spectrometry
Introduction Cyclotides are a family of plant peptides with unique features. As the name suggests, peptides belonging to this family have a macrocyclic amino acid backbone, which is completed through a peptide bond between the N- and C-termini. Communicated by Neal Stewart. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00299-020-02569-1) contains supplementary material, which is available to authorized users. * Blazej Slazak [email protected] 1
Pharmacognosy, Department of Medicinal Chemistry, Biomedical Centre (BMC), Uppsala University, Box 574, 751 23 Uppsala, Sweden
W. Szafer Institute of Botany, Polish Academy of Sciences, 46 Lubicz St., 31‑512 Cracow, Poland
2
The molecule is further stabilized by three disulfide bonds, which create what is referred to as the cyclic cystine knot (CCK
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