Specimens of Viola uliginosa Besser were obtained as previously described (Slazak et al. 2015b). Viola odorata L. was collected by Prof. Elżbieta Kuta during the summer of 2014 and then maintained under laboratory conditions with a controlled temperature and photoperiod (Gdańsk University). Arabidopsis thaliana L. Heynh. (ecotype Columbia) and Nicotiana benthamiana (Domin) were cultivated in the laboratory with a controlled temperature and photoperiod.
Cycloviolacin O2 (cyO2) was extracted from dried plant material of V. odorata using a previously reported protocol (Herrmann et al. 2008). Briefly, after three rounds of extraction with fresh 60 % aq. methanol, the extract was partitioned with dichloromethane. Then, to separate cyO2, positively charged molecules were captured from the (three times diluted) aqueous layer of the partitioned extract using solid phase strong cation-exchange extraction.
The plant extracts were fractionated using a Waters 600 HPLC system (Waters Corporation, MA, USA) fitted with a Phenomenex Jupiter C18 column (250 × 21.2 mm i.d., 10 µm, 300Å). Elution was performed using a linear gradient from 10 % acetonitrile (ACN) containing 0.05 % trifluoroacetic acid (TFA) (buffer A) to 60 % ACN containing 0.05 % TFA (buffer B) over 45 min, with a flow rate of 15 ml/min. Fractions were analyzed by ESI–MS (Finnigan LCQ ion trap, Thermo Electron Co., Waltham, MA, USA) in positive ion mode.
Cyclotide-containing fractions were subjected to a second purification step using an ÄKTA basic HPLC system (Amersham Biosciences, Uppsala, Sweden) fitted with a Phenomenex Jupiter C18 column (250 × 10 mm i.d., 5 µm, 300Å). Elution was performed using a linear gradient from 40 to 70 % of buffer B at a flow rate of 4 ml/min. Fractions were analyzed by ESI–MS and the pure fractions were freeze-dried. The purity of the isolated peptide was determined using a nanoAcquity Ultra Performance LC system (Waters Corporation, MA, USA).
CyO3, cyO8, and cyO8 were obtained from V. uliginosa using the method described previously (Slazak et al. 2015a).
Raising of antibodies
Polyclonal anti-cyclotide antibodies were raised in rabbit using standard procedures (Capra Science Antibodies AB, Ängelholm, Sweden). Immunization was performed with a mixture of free cyO2 and cyO2-conjugated keyhole limpet hemocyanin (KLH). Two rabbits were immunized with approx. 500 μg of this mixture per animal over the complete 12-week immunization period, which featured an initial immunization on week 0 followed by immunization boosts on weeks 2, 4, 7, and 10. The rabbits were bled on weeks 6, 9, and 12 (10 mL antiserum/rabbit). Antisera from the two rabbits were then pooled and purified using an affinity column to which 1–2 mg of the antigen was coupled. The eluted antibodies were dissolved in water (0.15 mg/ml) and stored at −20 °C.
Dot blot and Western blot experiments to confirm the antibodies’ specificity
Petioles and leaves of V. odorata,
V. uliginosa, A. thaliana and N. benthamiana were snap frozen in liquid nitrogen immediately after collection and ground to a powder in a mortar. The resulting material was then homogenized in an ice-cold lysate buffer containing 50 µM Tris HCl (Bio-Rad, Hercules, CA, USA), 150 mM NaCl (Sigma-Aldrich, St. Louis, MO), 1 % v/v Triton X (Bio-Rad), 5 mM Dithiothreitol (DTT, Sigma-Aldrich), 5 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich) and 1 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich), pH 7.6. The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected. The protein concentration in the supernatant was determined using a Lowry protein assay with acetone protein precipitation according to a procedure described by Olson and Markwell (2001). Lysates from V. uliginosa and V. odorata each containing 25 µg of total protein and a solution of pure cyO2 containing 2 µg of the peptide were mixed in a ratio of 4:1 with Laemmli Sample Buffer (5×), boiled for 10 min to achieve denaturation, loaded onto a 15 % acrylamide (Bio-Rad) casted gel (15 well, 0.75 mm thickness) along with 3 µl of PageRuler™ prestained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA), and separated by electrophoresis (45 min, 200 V). Additionally, as negative controls, A. thaliana, N. benthamiana and V. uliginosa lysates (20 µg protein each) were separated by electrophoresis along with pure cyO2 (1 µg) using a 16 % acrylamide casted gel (1 h, 200 V). The separated proteins were then transferred onto a nitrocellulose membrane (Whatman Protran BA85, Sigma-Aldrich). Blots prepared with the same methods were stained for whole protein with Ponceau S (Sigma-Aldrich). Membranes for dot blot experiments were prepared by blotting 2 µl of purified bracelet (cyO2, cyO3, cyO8 and cyO13) and Möbius (kB1 and kB2) cyclotide solutions of decreasing concentrations in water (200, 100, 50, 25 and 12.5 pmol of peptides per dot). Furthermore, 2 µg of ovalbumin (Sigma-Aldrich) and bovine serum albumin (BSA, Sigma-Aldrich) were dot blotted for probing. The membranes (from both dot and Western blots) were then blocked with 5 % non-fat dry milk in Tris-buffered saline containing 0.05 % Tween 20 (TBS-T) for 1 h at room temperature before being probed with a 1:1000 or 1:500 dilution in TBS-T (for dot blot and Western blot membranes, respectively) of the rabbit anti-cyO2 polyclonal antibody (stock solution at concentration of 0.15 mg/ml) for 3 h at room temperature. After three washes with TBS-T, the membranes were incubated with alkaline phosphatase (ALP)-conjugated goat anti-Rabbit IgG (H&L) (Agrisera AB, Vännäs, SE) (1:4000 dilution of the purchased stock solution in TBS-T) for 1 h at room temperature. After another three washes, the membrane was incubated for 30 min at room temperature with SIGMA FAST™ BCIP/NBT (Sigma-Aldrich) substrate for development.
Fixation and embedment
Sections/parts of V. odorata, V. uliginosa (leaf blades, petioles, roots), A. thaliana and N. benthamiana (leaf blades, petioles) were fixed in 4 % formaldehyde (freshly prepared from paraformaldehyde) and 0.25 % glutaraldehyde in microtubule stabilizing buffer (MSB) for 4 h at room temperature and then at 4 °C overnight, immediately after cutting. The MSB consisted of 50 mM PIPES (piperazine-N,N′-bis[2-ethanesulfonic acid]), 10 mM EGTA (ethylene glycol-bis[β-aminoethyl ether]N,N,N′,N′-tetraacetic acid), and 1 mM MgCl2, pH 6.8 (Świerczyńska et al. 2013). After fixation and three rinses in MSB, the plant material was dehydrated in a graded ethanol series and infiltrated with Steedman’s wax, i.e., a 9:1 (w/w) mixture of polyethylene glycol 400 distearate and cetyl alcohol (Sigma-Aldrich). After wax polymerization, plant material was sectioned (5 or 10 µm) and stretched on microscope slides coated with Mayer’s egg albumin. The wax was then removed with ethanol. Finally, the slides were rehydrated in an ethanol-PBS series and immersed in 0.05 M NH4Cl (15 min at room temperature) to reduce background fluorescence.
Immunostaining and fluorescence microscopy
Sections were preincubated in PBS with 1 % BSA (Sigma-Aldrich) for 45 min to prevent nonspecific binding, then incubated overnight at 4 °C with a solution of the rabbit anti-cyclotide polyclonal antibody Ab Cyo2 that was prepared by diluting a 0.15 mg/ml stock solution at 1:800 with 1 % BSA in PBS. Sections were subsequently rinsed in PBS and incubated for 4 h in a solution of a goat anti-rabbit secondary antibody conjugated with DyLight™ 549 (AS12 2084, Thermo Fisher Scientific), diluted 1:800 in PBS. In addition, double staining was performed to enable visualization of the cytoplasm, using a rat primary antibody against α-tubulin (Ab6161, Abcam, UK) and a goat anti-rat secondary antibody conjugated with FITC (Ab6840, Abcam). The chromatin of the nuclei was stained with 7 μg/ml 4′,6′-diamidino–2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) in PBS. In negative control experiments, the primary, secondary or both antibodies were omitted. Finally, the sections were cover–slipped using Mowiol medium and viewed with a fully automated upright fluorescent microscope (Leica DM6000 B) equipped with a digital 5 megapixel color microscope camera with an active cooling system (Leica DFC450 C), a selection of lenses (HC PL FLUOTAR 10×/0.30 dry, HCX PL FLUOTAR 40×/0.75 dry and HC PL APO 63×/1,40 oil), and an external light source for fluorescence excitation (Leica EL6000). All of this equipment was controlled using the Leica LAS AF software suite. To verify that staining occurred specifically, a set of control experiments was performed (the procedure without the primary, secondary or both antibodies for the Viola species, and a complete procedure applied to A. thaliana and N. benthamiana). All specimens were viewed/photographed with the following exposure parameters: 40× magnification, RHO cube: 730 ms, gain 1,9 and FIM 55 % (patented Fluorescence Intensity Management—Leica FIM) and DAPI cube: 780 ms, gain 1 and FIM 30 %. The photos were acquired as Z stacks (depth throughout the whole specimen—5 µm or 10 µm) and deconvolved using 10 iterations of a 3D non-blind algorithm (Autoquant™) to maximize spatial resolution unless otherwise noted. The images shown in the following sections are maximum projections of the acquired Z stacks or 3D projections.
Sections of the leaves, petioles, and peduncles of V. uliginosa and V. odorata were fixed in a mixture of 96 % ethanol and glacial acetic acid (3:1; v/v) for 48 h then stored in 70 % ethanol. Handmade transverse sections of organs were double-stained (carmine-iodine green) with 0.4 % alum carmine (Grenacher) for 10 min, rinsed with distilled water until all the free carmine was removed, and then stained with iodine green for 3–4 s (Lillie 1977). The green stain was removed by washing several times in distilled water and the stained sections were mounted in glycerol. This caused the epidermis, parenchyma, and phloem elements to be stained red, while the sclerenchyma and xylem vessels were stained green.