Protoplast isolation, transformation and auxin treatment were performed as described previously [18 (link)]. In brief, protoplasts were isolated from Arabidopsis thaliana by plasmolysis and flotation. For each sample, 105 protoplasts were transformed with 20 μg of plasmid DNA using a PEG1500-based method with a total volume of 120 μL of protoplast/DNA mixture and the addition of 120 μL PEG1500-Ca(NO3)2 solution. Volumes were adjusted to 2 mL with PCA regeneration medium after transformation and protoplasts were incubated in the dark for 24 h. Auxin (indole-3-acetic acid, Sigma, cat. no. 128869) was dissolved in PCA medium and added to a final concentration of 1 μM where appropriate and renilla and firefly bioluminescence was determined after 1 h simultaneously using a BioTec Synergy 4 and Tecan infinite M200 pro plate reader, respectively.
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