Polyethylene glycol 4000

Acetic anhydride, bismuth nitrate, and sodium nitrite were obtained from Panreac^® (Barcelona, Spain). Diphenylboric acid-β-ethylamino ester, fast blue B salt, gallic acid, iodine, oleanolic acid, polyethylene glycol-4000, protocatechuic acid, and vanillin were obtained from Sigma-Aldrich^® (Steinheim, Germany). Ethanol was obtained from Aga^® (Prior Velho, Portugal). Glacial acetic acid and sulfuric acid were obtained from Chem-Lab^® (Zedelgem, Belgium). Acetone, Folin–Ciocalteu reagent, hydrochloric acid 37% extra pure, iron trichloride, perchloric acid, potassium iodate GR, sodium carbonate, sodium hydroxide, and TLC cellulose plates (catalog number 105552) were obtained from Merck^® (Darmstadt, Germany). Butanol-1 and methanol were obtained from Fisher Chemicals^® (Leicestershire, UK). Ferrous sulfate was obtained from May and Baker LTD^® (Dagenham, UK). The (+)-Catechin (hydrate) was obtained from Cayman Chemical Company^® (Ann Arbor, MI, USA). Aluminum chloride was obtained from Fluka^® (Buchs, Switzerland). Acetonitrile was obtained from Honeywell Riedel-de Haën™ (Seelze, Germany). Formic acid 99–100% was obtained from VWR Chemicals (Fontenay-sous-Bois, France). All used reagents were of analytical grade.

An EC of Norway spruce (Picea abies L. Karst.), genotype AFO 541, was obtained from AFOCEL (Nangis, France). The cultivation of the EC followed a protocol described by Cvikrová et al. (2016) (link). During proliferation, the EC was cultivated in plastic Petri dishes (10 cm in diameter) on GD medium (Gupta and Durzan, 1986 ) solidified with 0.75% agar and supplemented with sucrose (30 g/L) and the phytohormones 2,4-D (5 μM), kinetin (2 μM), and BAP (2 μM) (all Duchefa, Haarlem, Netherlands). To initiate the maturation phase, cytokinins and auxin in the medium were replaced with abscisic acid (20 μM) and 3.75% polyethylene glycol 4000 (all Sigma-Aldrich, St. Louis, MO, United States). The cultures were kept in darkness at 23 ± 1°C during both proliferation and maturation and were subcultured onto fresh medium once per week.

Overnight cultures were washed twice with PBS and then resuspended in BHI and used for inoculation of day cultures without antibiotics at an OD₆₀₀ of 0.2. After 3.5 h, the OD₆₀₀ was adjusted to 2, and the bacterial cultures were washed twice with PBS and resuspended in 1 ml of BHI without l-cysteine. Then, 1 ml of 20% polyethylene glycol 4000 (Sigma-Aldrich) in BHI without l-cysteine was added so that a final PEG 4000 concentration of 10% was achieved. The cultures were incubated at 37°C shaking for 1 h. Afterward, 1 ml of the PEG 4000-treated samples was centrifuged at 16,000 × g for 1.5 min. A total of 0.9 ml of supernatant was used for trichloroacetic acid (TCA)/acetone protein precipitation. In brief, 100 μl of 100% TCA (wt/vol) (Sigma-Aldrich) was added to the harvested supernatants, followed by incubation at 4°C for 10 min with mixing in between. After centrifugation at 18,000 × g and 4°C for 5 min, the precipitated proteins were washed twice with cold acetone (Merk Millipore) and left to dry at room temperature. Then, the precipitated proteins were resuspended in 40 μl 1× lithium dodecyl sulfate (LDS) buffer (Thermo Fisher). The remaining bacterial cells were resuspended in 100 μl PBS, boiled at 95°C for 10 min, and sonicated afterwards. Thirty microliters of these samples were mixed with 10 μl 4× LDS buffer.

Polyethylene glycol PEG-8000 with a number average molecular weight (Mn) of 8000 Da; polyethylene glycol PEG-10000 with Mn of 10,000 Da; polyethylene glycol 6000, Mn = 6000 Da; polyethylene glycol 4000, Mn = 4000 Da; polyethylene glycol 1000, Mn = 1000 Da, and polyethylene glycol 600, Mn = 600 Da were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Dextran-75 (Lot 119945) with an average molecular weight (Mw) of 75,000 Da by light scattering was purchased from USB Corporation (Cleveland, OH, USA). Ucon 50-HB-5100, Mw = 3930 Da was purchased from Dow-Chemical (Midland, MI, USA). Ficoll 70, Mw ~ 70,000 Da was purchased from GE Healthcare Biosciences AB (Sweden). All polymers were used without further purification.