Transient Luciferase Reporter Assay

For transient reporter assays, constructs with the nopaline synthase (NOS) promoter driving the expression of the 5′UTR of ADH1 fused with the firefly luciferase (Luc) coding region were made using the pGreenll-0800 Luc plasmid (47 (link)), which also contains the 35S promoter driving Renilla luciferase (Ren) expression. The luciferase activity assays were performed following the instructions of the Dual Luciferase Reporter Assay System (Promega). The Luc/Ren ratio of each sample was calculated. For low oxygen treatment, 1:10 (v/v) Oxyrase (Sigma-Aldrich) was added after protoplast transformation and protoplasts were incubated overnight. For the quantification of reporter expression, reverse transcription and quantitative reverse transcription PCR were performed as previously described (44 (link)). The primers for the genes analyzed are listed in table S2.