Cyclotides are cyclic plant polypeptides of 27–37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet (Viola odorata L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides’ potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens (Fusarium oxysporum, F. graminearum, F. culmorum, Mycosphaerella fragariae, Botrytis cinerea) and fungi isolated from violets (Colletotrichum utrechtense and Alternaria alternata), with minimal inhibitory concentrations (MICs) ranging from 0.8 μM to 25 μM. Inhibition of phytopathogenic bacteria – Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum – is also observed with MIC = 25–100 μM. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest (ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides’ sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in V. odorata, in which different types of peptides have specific targets that determine their distribution in plant tissues.

Bacteria that thrive in extreme conditions and the bacteriophages that infect them are sources of valuable enzymes resistant to denaturation at high temperatures. Many of these heat-stable proteins are useful for biotechnological applications; nevertheless, none have been utilized as antibacterial agents. Here, we demonstrate the bactericidal potential of Ts2631 endolysin from the extremophilic bacteriophage vB_Tsc2631, which infects Thermus scotoductus, against the alarming multidrug-resistant clinical strains of Acinetobacter baumannii, Pseudomonas aeruginosa and pathogens from the Enterobacteriaceae family. A 2–3.7 log reduction in the bacterial load was observed in antibacterial tests against A. baumannii and P. aeruginosa after 1.5 h. The Ts2631 activity was further enhanced by ethylenediaminetetraacetic acid (EDTA), a metal ion chelator (4.2 log reduction in carbapenem-resistant A. baumannii) and, to a lesser extent, by malic acid and citric acid (2.9 and 3.3 log reductions, respectively). The EDTA/Ts2631 combination reduced all pathogens of the Enterobacteriaceae family, particularly multidrug-resistant Citrobacter braakii, to levels below the detection limit (>6 log); these results indicate that Ts2631 endolysin could be useful to combat Gram-negative pathogens. The investigation of A. baumannii cells treated with Ts2631 endolysin variants under transmission electron and fluorescence microscopy demonstrates that the intrinsic antibacterial activity of Ts2631 endolysin is dependent on the presence of its N-terminal tail.

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.

In the Brassicaceae family, apomictic development is characteristic of the genus Boechera. Hybridization, polyploidy and environmental adaptation that arose during the evolution of Boechera may serve as (epi)genetic regulators of apomictic initiation in this genus. Here we focus on Boechera stricta, a predominantly diploid species that reproduces sexually. However, apomictic development in this species has been reported in several studies, indicating non-obligate sexuality. A progressive investigation of flower development was conducted using three accessions to assess the reproductive system of B. stricta. We employed molecular and cyto-embryological identification using histochemistry, transmission electron microscopy and Nomarski and epifluorescence microscopy. Data from internal transcribed spacer (ITS) and chloroplast haplotype sequencing, in addition to microsatellite variation, confirmed the B. stricta genotype for all lines. Embryological data indicated irregularities in sexual reproduction manifested by heterochronic ovule development, longevity of meiocyte and dyad stages, diverse callose accumulation during meiocyte-to-gametophyte development, and the formation of triads and tetrads in several patterns. The arabinogalactan-related sugar epitope recognized by JIM13 immunolocalized to one or more megaspores. Furthermore, pollen sterility and a high frequency of seed abortion appeared to accompany reproduction of the accession ES512, along with the initiation of parthenogenesis. Data from flow cytometric screening revealed both sexual and apomictic seed formation. These results imply that B. stricta is a species with an underlying ability to initiate apomixis, at least with respect to the lines examined here. The existence of apomixis in an otherwise diploid sexual B. stricta may provide the genomic building blocks for establishing highly penetrant apomictic diploids and hybrid relatives. Our findings demonstrate that apomixis per se is a variable trait upon which natural selection could act.

We recently showed that yellow lupine is highly sensitive to soil water deficits since this stressor disrupts nodule structure and functioning, and at the same time triggers flower separation through abscission zone (AZ) activation in the upper part of the plant. Both processes require specific transformations including cell wall remodeling. However, knowledge about the involvement of particular cell wall elements in nodulation and abscission in agronomically important, nitrogen-fixing crops, especially under stressful conditions, is still scarce. Here, we used immuno-fluorescence techniques to visualize dynamic changes in cell wall compounds taking place in the root nodules and flower AZ of Lupinus luteus following drought. The reaction of nodules and the flower AZ to drought includes the upregulation of extensins, galactans, arabinans, xylogalacturonan, and xyloglucans. Additionally, modifications in the localization of high- and low-methylated homogalacturonans and arabinogalactan proteins were detected in nodules. Collectively, we determined for the first time the drought-associated modification of cell wall components responsible for their remodeling in root nodules and the flower AZ of L. luteus. The involvement of these particular molecules and their possible interaction in response to stress is also deeply discussed herein.