Phosphate buffered saline (pbs)

Rucola (Eruca sativa L.) seeds were washed in Tween 80 1% (Sigma, United States) for 2 min, surface sterilized with sodium hypochlorite 12% (NaOCl, Roth, Germany) for 8 min and washed three times in sterile deionized water for 2 min. Sterilized seeds were placed on Hoagland’s solution (Sigma, United States) with 0.8% agar to germinate 4 days. Rhodococcus strains were pre-grown in TSB. Overnight grown cultures were washed two times in 1x PBS (AppliChem, Germany) and diluted to a concentration of 10⁷ CFUs. Seedlings were inoculated in the prepared bacterial solution of RL1, BG43, and djl6 for 1 h under shaking at 160 rpm at 28°C. Seedlings inoculated in 1x PBS served as negative control. Inoculated seedlings were transferred to an axenic system with 80 ml sterile quartz sand and 20 ml Hoagland’s solution in a sterile Phytatray II (Sigma, United States). Seedlings inoculated with RL1 were additionally transferred on plates with 0.5x Murashige and Skoog Medium (0.5x MS) including vitamins (Duchefa Biochemie, Netherlands); pH was adjusted to 5.7 with 2N KOH. No additional sucrose was added to 0.5x MS. The axenic system was placed in a Phytochamber (Weiss Technik, Modell SGC120PG2, Germany) with 23°C, 55% humidity, day-night-cycle 12 h : 12 h. After seven and 14 days freshly harvested roots were washed in 1x PBS, fixed in 55% EtOH and 1x PBS mix and stored at –20°C until further use.

Cell viability was assessed using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltertazolium Bromide (MTT) (Invitrogen, Carlsbad, CA, USA) assay, and 15,000 cells were seeded into 96-well plates (NUNC, Langenselbold, Germany). After 24 h, cells were treated with increasing doses of AP (5–50 µM) for 24 h. To assess cell viability, 50 μL of MTT lysis solution with a final concentration of 0.5 mg/mL in 1X PBS (AppliChem, Darmstadt, Germany) was first added to each well followed by 30 min incubation at 37 °C. The MTT solution was discarded and 50 μL of DMSO (Sigma-Aldrich, Taufkirchen, Germany) was added to each well. For the readout, a multi-scanner micro-plate reader (VersaMax Microplate Reader, Molecular Diagnostics, CA, USA) was used to measure the absorbance at 570 nm with a background absorbance of 670 nm.

Antibiotic broth medium No. 3 (complex medium) was purchased from Oxoid LTD (Hampshire, Great Britain). Pyruvic acid, sucrose, KH₂PO₄, KOH, and (NH₄)₂SO₄ were purchased from Roth (Karlsruhe, Germany). D-glucose, MgSO₄·7 H₂O, and FeSO₄·7 H₂O were obtained from Merck (Darmstadt, Germany). Trehalose and NaCl were purchased from Fluka (Buchs, Schwitzerland). Ectoine (≥99%) for lactate deydrogenase stress experiments was purchased from bitop AG (Witten, Germany). HydroxyEctoine (≥99%) for lactate dehydrogenase (LDH) stress experiments was isolated from H. elongata strain DSM 2581^T in our laboratories. Freeze-dried LDH (rabbit muscle) and nicotinamide adenine dinucleotide were purchased from Sigma (Steinheim, Germany). Phosphate buffered saline (PBS) was purchased from AppliChem (Darmstadt, Germany). Ectoine (≥99.0%) and hydroxyEctoine (≥98%) for spin lable and Fourier transform infrared (FTIR) experiments were purchased from Sigma (USA). Perdeuterated spin probe Tempone-d₁₆ (4-Oxo-2,2,6,6-tetramethylpiperidine-d₁₆-1-oxyl; Figure 7 inset) was a kind gift of Prof. I. Grigoriev (Institute of Organic Chemistry of the Russian Academy of Sciences, Novosibirsk, Russia).