Diversity of extracellular vesicles derived from calli, cell culture and apoplastic fluid of tobacco

Plant material

To initiate the formation of callus of Nicotiana tabacum, seeds (purchased at SemenaOnline; Jeneč; Czech Republic) were surface-sterilized and placed on a Murashige-Skoog (MS) medium previously described for tobacco callus induction with some modifications³⁵. This medium was enriched with saccharose (30 g/l), plant agar (8 g/l; Duchefa Biochemie, B. V; Haarlem; Netherlands), 1-Naphthaleneacetic acid (NAA; 1.2 mg/l; Duchefa Biochemie, B. V; Haarlem; Netherlands), and 6-Benzylaminopurine (BAP; 1.2 mg/l; Duchefa Biochemie, B. V; Haarlem; Netherlands). The pH of the medium was adjusted to a range of 5.6–5.8, and it was subjected to sterilization in an autoclave at 121 °C for a duration of 30 min. Cultivation of callus cultures took place within a controlled environment in a grow box (Garden High Pro Probox Propagator XL; Grow technologies; Prague; Czech Republic), maintaining a temperature of 25 °C under illumination with 16/8 photoperiod. The formation of callus typically became evident within a span of 3 to 4 weeks, after which the callus was routinely transferred to fresh medium every 6 weeks.

The BY-2 suspension culture of Nicotiana tabacum was generously provided by the Institute of Experimental Botany of the Czech Academy of Science. These BY-2 cultures were cultivated under illumination with 16/8 photoperiod and in the absence of light, maintaining a constant temperature of 26 °C on a shaker set at 105 RPM. To ensure their continued growth and vitality, cells were regularly transferred into fresh growth medium on a weekly basis. The growth medium itself was prepared, using Murashige-Skoog basal salt mixture at a concentration of 4.3 g/l, supplemented with KH₂PO₄ (0.2 g/l), saccharose (30 g/l), inositol (100 mg/l), thiamine (1 mg/l), and 2,4-dichlorophenoxyacetic acid (2,4-D; 0.2 mg/l). Carefully maintaining the pH of the medium within the range of 5.6 to 5.8, the entire mixture was subsequently sterilized in an autoclave at 121 °C for a duration of 30 min. All chemicals for media preparation mentioned above were purchased at Duchefa Biochemie, B. V; Haarlem; Netherlands.

About five Nicotiana tabacum seeds were sown in plastic pots measuring 7 × 7 cm, filled with gardening soil enriched with hummus. Plants were cultivated within a plant growth box set at a constant temperature of 25 °C (± °1C). The grow box was equipped with full spectrum LED lighting and operated on a photoperiod of 16 h of light followed by 8 h of darkness. To sustain their growth, the plants received watering every third day. After 28 days from planting, they were harvested, at size about 10 to 15 cm which is the optimal size for the isolation of apoplastic fluid.

PEVs isolation

Collecting plant material

The process of ultracentrifugation was chosen as a method for pEVs isolation. The preparation of plant material for pEVs isolation varies according to the source material. As a universal parameter for isolating pEVs from all three types of plant material, the weight was standardized by using 10 g of each material, which was then subjected to the following analysis.

Exactly 10 g of tobacco callus, six weeks after passage, was weighed and placed into a Falcon tube with 10 ml of 1X PBS buffer. Due to the friable nature of tobacco callus, the callus could be divided into small pieces by shaking the tube for one minute. Subsequently, large particles were removed using plastic kitchen sieve (with the mesh size 1 × 1 mm) and the suspension was transferred to a centrifugation tube and subjected to centrifugation.

Similarly, 10 g of tobacco suspension culture was collected and prepared for the subsequent centrifugation steps. BY-2 suspension cultures were collected 7 days after the transfer to fresh media.

Finally, 10 g of young tobacco plants (typically 3–6 flowers) were harvested and the apoplastic fluid isolation protocol by Rutter et al. was followed³⁰. Briefly, tobacco plants were carefully removed from a soil, cleaned by distilled water and placed into a metal French press filled half way with 1X PBS. French press with tobacco plants and buffer was placed into a glass exsicator and a vacuum (300–400 mbar) was applied for one minute and 20 s, facilitating the entry of the surrounding buffer into the plant’s apoplast by opening its pores. Plants were transferred into a tool made of 50 ml plastic tube with inserted 30 ml syringe, and centrifuged at 700 × g for 25 min (Beckman Coulter’s OPTIMA XPN 90 ultracentrifuge with rotor 70-Ti; Indianapolis; United States), resulting in the release of apoplastic fluid from the plants through the syringe into the 50 ml plastic tube. Collected apoplastic fluid was subjected to centrifugation steps.

Ultracentrifugation

The samples were initially centrifuged at 2000 × g for 20 min at a temperature of 4 °C. The supernatant obtained was collected and underwent a subsequent centrifugation at 10,000 × g for 30 min. This step was carried out using Beckman Coulter’s Avanti JXN-26 high-speed centrifuge with rotor JA-25.50 (Indianapolis; United States), and it was repeated twice to enhance sample purity. The supernatant from these rounds was collected and subjected to another centrifugation at 100,000 × g for 1 h at 4 °C. This step, performed using Beckman Coulter’s OPTIMA XPN 90 ultracentrifuge with rotor 70-Ti (Indianapolis; United States), was also repeated twice³⁶. The resulting pellet was resuspended in 500 μl of 1X PBS and stored at -20 °C for subsequent analysis.

In response to difficulties encountered in resuspending the pellet, the samples were centrifuged at 5,000 × g for 5 min to eliminate large clusters of the pellet remaining in the samples.

Nanoparticle tracking analysis

Particles within the samples were analyzed using NanoSight NS3000 (Malvern Paralytical; Malvern, United Kingdom), and videos were recorded and processed using the NTA software. Roughly 300–400 μl of each sample was introduced into the flow-cell top-plate chamber. A laser beam with a wavelength (λ) of 562 nm illuminated the chamber from below, causing the particles in the solution to scatter light. Each sample underwent three separate analyses, each lasting 30 s, while the ambient temperature remained below 25 °C. The results were evaluated with the assistance of the Malvern software.

Loading of plant extracellular vesicles

siRNA (MISSION® siRNA Fluorescent Universal Negative Control #1, Cyanine 5; Sigma-Aldrich; Saint Louis; United States) with a fluorescent label (cy5) was selected for loading into pEVs. In each variation, the initial concentration of siRNA used for loading was 11.07 µg/ml.

In our initial set of experiments, the loading of siRNA into callus-derived pEVs, utilizing different approaches of sonication, incubation, electroporation, and lipofection was examined.

Sonication was performed using a probe sonicator (Bandelin Sonopuls mini20 homogenizer; Berlin; Germany). Sonication samples were prepared by combining 1 × 10⁹ of pEVs, 11.07 µg/ml of labeled siRNA, and samples were adjusted to a volume of 100 µl using 1X PBS. Four variations of sonication were conducted: a) 20% amplitude, four cycles (1 cycle includes sonication 30 s on, 30 s off for three minutes followed by 2 min of cooling); b) 20% amplitude, six cycles; c) 10% amplitude, four cycles; and d) 10% amplitude, six cycles^24,37. Following sonication, the samples were subjected to a 30-min incubation at 37 °C to facilitate the reconnection of disrupted membranes. After incubation period, the removal of free siRNA was achieved through ultracentrifugation steps at 100,000 × g for one hour, 4 °C. The supernatant was discarded, and the resulting pellet was resuspended in 1X PBS, followed by a repetition of the ultracentrifugation step. Upon completion of the centrifugation, the pellet was resuspended in 50 µl of 1X PBS, and the samples were immediately utilized for NTA analysis and fluorescence measurement.

Loading via incubation, based on a procedure by Didiot et al.³⁸, was conducted in two different variations, each involving four different incubation durations, before the removal of free siRNA. The first variation involved straightforward incubation without the addition of facilitating substances. Samples were prepared by mixing 1 × 10⁹ pEVs with 11.07 µg/ml of siRNA, adjusting the volume to 100 µl using 1X PBS. Samples were incubated at 37 °C for four different time durations, including 1 h, 4 h, 6 h, and 24 h. Following the incubation, the samples were cleaned of free siRNA as described above. In the second variation of incubation, saponin was added as it has been previously used for loading mammalian extracellular vesicles, demonstrating its ability to create membrane pores within EVs by interacting with membrane cholesterol and facilitating cargo loading^39,40. The samples were prepared identically, with the addition of saponin to achieve a final concentration of 0.1 mg/ml. Saponin-supplemented samples were incubated at 37 °C for four different time periods, including 1 h, 4 h, 6 h, and 24 h. The samples underwent the removal of free siRNA as previously described.

The electroporation was performed using Nucleofector™ 2b (Lonza; Allendale, United States), choosing a gentle program designed for HeLa cells with high viability. Samples were prepared by mixing 1 × 10⁹ pEVs, 11.07 µg/ml of labeled siRNA, and adjusting the sample volume to 100 µl. After the electroporation, the samples were subjected to incubation at 37 °C for four different time intervals: 1 h, 4 h, 6 h, and 24 h to recover the membranes. Subsequently, the samples were cleansed of free siRNA using ultracentrifugation as described earlier.

For lipofection, the ultracentrifugation pellets containing 1 × 10⁹ pEVs were used. The transfection solution was prepared by combining two mixtures in a 1:1 ratio: a) a mixture of Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen; Carlsbad, United States) and Opti-MEM (Thermo Fisher Scientific; Waltham; United States) in a 1:17 ratio; b) 11.07 µg/ml of siRNA and Opti-MEM (Thermo Fisher Scientific; Waltham; United States) in a 1:6 ratio. The final solution was incubated at room temperature for 5 min. Subsequently, 100 µl of the mixture was added to each pEVs pellet. Complete samples were then incubated at 37 °C for four different time intervals: 1 h, 4 h, 6 h, and 24 h. Following the incubation, the samples were purified of free siRNA using ultracentrifugation as described above.

Following the outcomes of loading experiments conducted on extracellular vesicles derived from callus, we opted for two loading techniques to assess the loading efficiency of pEVs obtained from tobacco callus, suspension culture, and apoplastic fluid.

The first of the selected methods was sonication, following the same sample preparation procedure mentioned earlier. The sonication parameters were as follows: an amplitude of 20%, with four cycles (1 cycle includes sonication 30 s on, 30 s off for three minutes followed by 2 min of cooling). After sonication, the samples were subjected to incubation at 37 °C for 30 min. After this incubation period, the removal of free siRNA was accomplished through a two series of ultracentrifugation steps at 100,000 × g for one hour at 4 °C. The supernatant was then discarded, and the resulting pellet was reconstituted in 1X PBS. The centrifugation pellet was resuspended in 50 µl of 1X PBS, and the samples were ready for Nanoparticle Tracking Analysis (NTA) and fluorescence measurements.

The second selected method was incubation. Samples were prepared by mixing 1 × 10⁹ pEVs, 11.07 µg/ml of labeled siRNA, and 1X PBS to reach a final volume of 100 µl. These samples were incubated for three different time periods: 1 h, 6 h, and 24 h, all at 37 °C. After each respective incubation period, the removal of free siRNA was achieved through a two series of ultracentrifugation steps at 100,000 × g for one hour at 4 °C. Subsequently, the supernatant was discarded, and the resulting pellet was reconstituted in 50 µl of 1X PBS and it was used for further analysis.

Detection of loaded cy5-siRNA

For detection of the presence of labeled siRNA in plant vesicles after loading, a fluorescence microplate reader (GloMax Discover Microplate Reader; Promega; Walldorf; Germany) was used. The volume of 50 µl of each sample of pEVs washed out of free siRNA were transferred into microplate and the fluorescence intensity was measured (excitation = 627 nm, emission = 660–720 nm). Using the same procedure, the calibration curve of siRNA was prepared to allow the calculation of the concentration of pEVs-loaded siRNA.

LC–MS/MS analysis

LC–MS/MS analysis was performed to verify the presence of nicotine and anabasine within pEVs derived from three different sources of tobacco. Exactly 1 × 10¹⁰ of pEVs were taken from isolated samples from callus, suspension cultures and apoplastic fluid in triplicates. The samples were diluted several times. Separation was carried out using an Agilent 1290 Infinity II UHPLC system (Agilent Technologies; Santa Clara; United States). Chromatographic separation was achieved using a Luna Omega PS C18 analytical column 2.1 × 100 mm, 3 µm particle size from Phenomenex (Torrance; California; USA) at a flow rate of 0.4 ml/min. The mobile phases consisted of (A) H₂O with 0.5 mM NH₄F, pH 10 and (B) acetonitrile. The gradient was 95% A at 0 min, 70% A at 7.0% A at 9 min to 12 min, 95% A at 12.1 min. The post time was 4.9 min with 95% A and the stop time 17 min. Injection volume was 20 μl. The HPLC system was coupled to an Agilent G6495A Triple Quadrupole mass spectrometer equipped with an Agilent Jet Stream electrospray ionization source. Agilent MassHunter Acquisition software was used for data acquisition, and Agilent MassHunter Workstation software was used for data analysis.

Statistical analysis

Statistical analysis and graphs preparation were performed using Prism GraphPad Software. T-test was used to analyze the significance of differences between various samples. Statistical significance was accepted if p < 0.05.

All experiments were performed in accordance with relevant guidelines and regulations.

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• Source: https://www.nature.com/articles/s41598-024-81940-8