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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-016-2562-y) contains supplementary material, which is available to authorized users.

Single‐step purification of cyclotides using affinity chromatography

“…These results suggest that the anti‐cyO2 IgG antibodies may be suitable for group separation targeting bracelet cyclotides rather than expecting specificity against cyO2 alone. In a previous study, using a slightly modified setup (2 µL dots and time of SIGMA FAST™ BCIP/NBT development prolonged to 30 min), binding was detected for 6.25 µM concentrations (12.5 pmole cyO2) . In that study, antibodies were used for immunohistochemistry studies of cyclotide occurrence in planta …”

“…Nitrocellulose membranes (Invitrogen, Carlsbad, CA) were prepared as described previously . Briefly, 1 µL of cyclotide aqueous solutions were blotted at decreasing concentrations, that is, 100, 50, 25, 12.5, and 6.25 µM.…”

“…Polyclonal anti-cyO2 antibodies (IgG) were raised in rabbits by Capra Science Antibodies AB ( € Angelholm, Sweden) as described before. [23] Briefly, rabbits were immunized with 500 lg/animal of antigen (50% free cyO2, 50% conjugated to keyhole limpet hemocyanin, KLH) on week 0, followed by repeated immunizations on week 2, 4, 7, and 10. At the end of each treatment, the animals were bled (weeks 6, 9, and 12) and antiserum was collected (10 mL antiserum/rabbit).…”

“…Nitrocellulose membranes (Invitrogen, Carlsbad, CA) were prepared as described previously. [23] Briefly, 1 mL of cyclotide aqueous solutions were blotted at decreasing concentrations, that is, 100, 50, 25, 12.5, and 6.25 mM. The membranes were probed with a 1:1000 dilution of rabbit anti-cyO2 IgG polyclonal antibody (stock solution at concentration of 0.15 mg/mL) for 3 h at room temperature following blocking for 1 h at room temperature using TBS-T blocking buffer: 5% non-fat dry milk in Tris-buffered saline (Bio-Rad, Hercules, CA), 0.05% Tween 20 (Bio-Rad).…”

“…In a previous study, using a slightly modified setup (2 mL dots and time of SIGMA FAST TM BCIP/NBT development prolonged to 30 min), binding was detected for 6.25 mM concentrations (12.5 pmole cyO2). [23] In that study, antibodies were used for immunohistochemistry studies of cyclotide occurrence in planta. [23]…”