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Cyclotides are cyclic peptides produced by plants. Due to their insecticidal properties, they are thought to be involved in host defense. Violets produce complex mixtures of cyclotides, that are characteristic for each species and variable in different environments. Herein, we utilized mass spectrometry (LC–MS, MALDI-MS), transcriptomics and biological assays to investigate the diversity, differences in cyclotide expression based on species and different environment, and antimicrobial activity of cyclotides found in violets from the Canary Islands. A wide range of different habitats can be found on these islands, from subtropical forests to dry volcano peaks at high altitudes. The islands are inhabited by the endemic Viola palmensis, V. cheiranthifolia, V. anagae and the common V. odorata. The number of cyclotides produced by a given species varied in plants from different environments. The highest diversity was noted in V. anagae which resides in subtropical forest and the lowest in V. cheiranthifolia from the Teide volcano. Transcriptome sequencing and LC–MS were used to identify 23 cyclotide sequences from V. anagae. Cyclotide extracts exhibited antifungal activities with the lowest minimal inhibitory concentrations noted for V. anagae (15.62 μg/ml against Fusarium culmorum). The analysis of the relative abundance of 30 selected cyclotides revealed patterns characteristic to both species and populations, which can be the result of genetic variability or environmental conditions in different habitats. The current study exemplifies how plants tailor their host defense peptides for various habitats, and the usefulness of cyclotides as markers for chemosystematics.

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C−G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I−IV, II−III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC50 0.64 μM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts., K.S. thanks the Swedish pharmaceutical society (Apotekarsocieteten) for funds provided to purchase reagents for SPPS. K.S., A.R., and P.C. acknowledge SponGES; SponGES has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 679849.