J2 hs centrifuge

Sediment samples were extracted as described in Bünning et al. (2021) (link). In brief, 100 g wet sediment was mixed with 250 mL ultrapure water, spiked with 25 ng 13 C 15 N-TNT, shaken for 80 min and sonicated for 15 min. Samples were then centrifuged at 4500 rpm (10 °C) for 15 min (J2-HS centrifuge, Beckman Coulter GmbH, Krefeld, Germany), filtered through a 595 1/2 pleated filter and applied onto SPE-columns using a mild vacuum. Columns were dried for 30 min i.vac., eluted with 4 mL ACN, concentrated to 600 μL and stored at -20 °C in 1.5 mL amber vials.

Following the protocol of Wu et al. (2009) , 2 g of leaf tissue was incubated in 10 mL of enzyme solution (1% [w/v] cellulase from Trichodermaviride [Sigma, St. Louis, Missouri, USA], 0.25% [w/v] pectinase from Rhizopus spp. [Sigma], 0.4 M mannitol, 10 mM CaCl 2 , 20 mM KCl, 0.1% [w/v] bovine serum albumin, and 20 mM MES at pH 5.7) for 1 h in light after placing Time Tape on the upper epidermis of the leaves and removing the lower epidermis of the leaves via Magic Tape. Protoplasts were then pelleted by centrifugation at 73 rcf for 3 min at 4°C using a Beckman J2-HS centrifuge and JS-13.1 rotor. Protoplasts were resuspended in a solution containing 0.4 M mannitol, 15 mM MgCl 2 , and 4 mM MES at pH 5.7. eGFP fluorescence was visualized using protoplasts and leaves from stable eGFP plants with a Leica SP2 confocal microscope. The white light laser was used with 5% laser power and smart gain was adjusted to 100%. A 40× oil-emersion lens was used to visualize protoplasts and a 20× objective lens was used to visualize intact cells from leaf samples. eGFP and chlorophyll were excited using a krypton/argon laser tuned to 488 nm, and eGFP and chlorophyll fluorescence were observed between the wavelengths of 500-520 nm and 660-700 nm, respectively.

Stable eGFP, GUS, and complementation lines were created following a modified procedure (Weigel and Glazebrook, 2002) . A total of 200 μL of transformed A. tumefaciens was used to inoculate 200 mL of LB medium supplemented with antibiotics (30 μg mL -1 gentamycin and 10 μg mL -1 rifampicin for A. tumefaciens helper plasmids and either 100 μg mL -1 spectinomycin for the eGFP and GUS vectors or 50 μg mL -1 kanamycin for the complementation vector). The cultures were grown overnight at 28°C with vigorous shaking, and cells were pelleted in the morning by centrifugation at 7,250 rcf for 10 min at 20°C using a Beckman J2-HS centrifuge and JA-10 rotor. Pelleted cells were resuspended in 400 mL of A. tumefaciens infiltration medium (one-half-strength Murashige and Skoog [MS] medium with Gamborg's vitamins from Caisson Laboratories, 5% [w/v] Suc, 0.044 μM benzylaminopurine suspended in dimethyl sulfoxide, and 50 μL L -1 Silwet L-77 from Lehle Seeds). Stalks with flowers of Arabidopsis plants were dipped in the A. tumefaciens infiltration medium for 40 s and then laid sideways in a flat with a covered dome to recover overnight, incubating in constant light at 21°C (Weigel and Glazebrook, 2002) . Positive transformants were selected on soil by spraying seedlings with a 1:1,000 dilution of BASTA (AgrEvo).