4 methylubelliferone

PTR2 and CaMV 35S promoters were cloned and fused to the β-glucuronidase (GUS) reporter gene in pCAMBIA3301 using primers listed in Supplementary Table S1. The resulting constructs were transiently transformed into the WT, abi4, and ABI4OE lines. Four-day-old seedlings grown in darkness were vacuum-infiltrated with Agrobacterium twice for 1 min each (Marion et al. 2008 (link)). The samples were incubated in the dark for 1 day and then grown under white light for 2 days. GUS activities were measured using a spectrofluorometer (LS-55; Perkin-Elmer) with the substrate 4-methylumbelliferyl-β-d-glucuronide (Sigma-Aldrich); 4-methylubelliferone (Sigma-Aldrich) was used for calibration. Protein content was determined using bovine serum albumin as the standard (Jaquinod et al. 2007 ). To perform GUS histochemical staining, germinating seeds were fixed by immersing in 90% (v/v) acetone. The seeds were then washed twice with a solution containing 50 mM sodium phosphate (pH 7), 0.5 mM K₃Fe(CN)₆, and 0.5 mM K₄Fe(CN)₆, and subsequently incubated in a staining solution containing 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (Duchefa) (Lee et al. 2015 (link)).