Cyclotide host-defense tailored for species and environments in violets from the Canary Islands.

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.

Effects of Viola tricolor Flower Hydroethanolic Extract on Lung Inflammation in a Mouse Model of Chronic Asthma.

Asthma is a chronic inflammatory disease of the lungs driven by T cell activation. Viola tricolor L. as a traditional medical herb could suppress activated T lymphocytes and has been used empirically for asthma remedy. In the present study, we investigated the anti-inflammatory effect of Viola tricolor and its underlying mechanism on asthma characteristics induced by ovalbumin (OVA) in mice. BALB/c mice were randomly divided into six groups: normal control, Ovalbumin (OVA) control, OVA mice treated with Viola tricolor (50, 100 and 200 mg/kg) and dexamethasone (3 mg/kg). All mice except normal controls were sensitized and challenged with OVA. Asthmatic mice were treated orally in the last 7 days of OVA challenge. The total and differential leukocyte counts, Interleukin (IL)-4 and interferon (IFN)-γ levels in bronchoalveolar lavage fluid (BALF) were determined. H and E staining for lung inflammation was performed. Viola tricolor treatment at 200 mg/kg significantly decreased IL-4 level but did not considerably affect the IFN-γ level. Therefore, it effectively reduced asthma characteristics including infiltration of leukocytes particularly eosinophil and peribronchial inflammation as compared to dexamethasone. However, Viola tricolor at 100 mg/kg had the most prominent inhibitory effect on the IL-4 level and also markedly increased IFN-γ level. As result, it prevented further reduction of inflammatory parameters in this group compared to the Viola tricolor-treated group at 200 mg/kg. Our study demonstrated that Viola tricolor has anti-inflammatory effects via inhibition of T-helper type 2 (Th2) cytokine production and validated its empirical usage in traditional medicine.

Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense.

MAIN CONCLUSION: The distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique's resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense-relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.

A transcriptional profile of metallophyte Viola baoshanensis involved in general and species-specific cadmium-defense mechanisms.

Viola baoshanensis Shu, Liu et Lan is a newly identified metallophyte, and its defensive strategies against heavy metals are still unclear. In the present study, we firstly constructed a root cDNA library of the plant subjected to 300muM Cd for 48h by using suppression subtractive hybridization (SSH), and 43 unique cDNA fragments were further isolated from the library. Sequence homology analysis showed that half of the identified genes were involved in general stress defense, such as antioxidative enzymes, protein degradation and stress signal transduction. After RT-PCR and RACE analysis, a Cd-responsive gene Vb40 was identified, which could deduce a novel cysteine-rich mini-protein. Meanwhile, five cyclotide precursor genes (VbCP1-VbCP5) were also identified. The Vb40 and the VbCP1-VbCP5 were further investigated by yeast expression analysis, and they could improve copper (Cu) tolerance in hosted yeast, indicating that these species-specific genes possibly functioned in V. baoshanensis heavy metals tolerance. Our results suggested that heavy metal tolerance in V. baoshanensis relied on both general and species-specific defense.