Polymax 2040

Leaf discs were obtained with a 4 mm biopsy punch and incubated overnight in Petri dishes containing milliQ-H₂O at room temperature. Following overnight incubation, three leaf discs were added to glass vials (5 ml) containing 300 µl of milliQ-H₂O. Laminarin was added in a final concentration of 1 mg ml⁻¹ to three glass vials (samples) containing three leaf discs of one plant, and milliQ-H₂O in three separate glass vials containing leaf discs for the same plant served as a negative control. Upon addition of elicitor or water, the glass vials were sealed with septa (Carl Roth GmbH). Samples were incubated for 3 h on a shaker at ~ 20–50 rpm (Heidolph Polymax 2040). Three hours post-incubation, 1 ml of air was retrieved from each samples with a syringe through the rubber cap and injected into a Varian 3300 gas chromatography machine containing an AlO₃ column with length 1 m and 225 °C detector temperature, with 80 °C column and injector temperature. The gases used for the separation of ET from the sample were H₂, N₂, and O₂ at 0.5 MPa each. The amount of ET was calculated based on the standard calculation as developed by Von Kruedener et al. (1995) (link) using the area under the curve (AUC). In total, we measured up to nine samples per plant on three different dates, each date containing up to three samples.

A total of 10 µL of protein sample was mixed with 10 µL of 2× SDS PAGE gel-loading buffer (100 mM of Tris, pH 6.8, 4% w/v SDS, 0.2% w/v of bromophenol blue, 10% v/v of β-mercaptoethanol, and 20% v/v of glycerol). Then, the sample was incubated at 37 °C for 30 min. After incubation, the sample was loaded onto precast SDS-PAGE gel (any kD Mini-PROTEAN, Bio-Rad) and run at 120 V (constant voltage) for 90 min at 4 °C until the dye reached the bottom of the gel. The gel was carefully removed from the cassette and placed in deionized water. The visualization of protein bands in the gel was performed either by a 50 mL solution of Coomassie blue stain (InstantBlue, Abcam ISB1L) incubated for 60 min on an orbital shaking platform (Polymax 2040, Heidolph Instruments GmbH Schwabach, Germany) with subsequent washing in milliQ water or directly visualized in a fluorescent gel imager (Typhoon FLA 9000, GE Health care) in the GFP channel (489 nm exc/508 nm em)