Luc mix

The vectors containing GUS driven by different promoters were transformed into rice protoplasts with or without SIP1. After incubation at 25°C for 12 h, the protoplasts were lysed with lysis buffer (25 mM Tris‐HCl, pH 7.8, 1 mM DTT, 10% glycerol and 1% Triton X‐100). After centrifugation, 100 μl of supernatant was mixed with 900 μl of fluorescent β‐galactosidase (MUG) substrate mix (10 mM Tris‐HCl, pH 8.0, containing 1 mM MUG and 2 mM MgCl₂). After incubation at 37°C for 30 min, the reaction was stopped with 40 mM Na₂CO₃. GUS activity was measured using a fluorometer (HITACHI, U‐281; Thermo Fisher, Fluoroskan Ascent FL, Waltham, MA, USA) with 365 nm excitation wavelength and 456 nm emission wavelength. The luciferase (LUC) activity was measured using the GloMax 96 Luminometer system (Promega, E6501) with LUC mix (Promega, E1980).

We conducted dual-LUC assays in N. benthamiana protoplasts as previously described [85 (link), 86 (link)]. N. benthamiana plants were grown in an incubator kept under a light regime of 16 hours day/8 hours night at 22°C. The 2378-bp promoter region of the THM1 gene was amplified and inserted into the vector pGREEN0800 (named pGREEN0800-pTHM1). We amplified the CDSs of H and HL and inserted them into the vector of pXSN-Flag (named Flag-H and Flag-HL). The full-length CDS of JAZ2 was amplified and inserted into the vector of pXSN-HA (named HA-JAZ2). The vectors of Flag-H and Flag-HL were used as effectors. HA-JAZ2 was used as the plus vector. pGREEN0800-pTHM1 was used as the reporter. Vector transformation of N. benthamiana protoplasts was performed as previously described [82 (link)]. LUC mix (Promega) was used to measure LUC activity with a luminometer (Cytation 5 Imaging Reader). We performed three biological replicates for each experiment. Error bars represent the SD of three biological replicates.