Increased expression of ethylene signaling and biosynthetic genes in fus3 mutant, and ethylene-related fus3 phenotypes. (A) RT-PCR of ethylene-related genes in wild-type (WT) and fus3 seeds germinated for 12, 24 and 48 hours in minimal medium (MS). ACTIN7 (ACT7) served as a control. (B) RT-PCR of ethylene-related genes in wild-type and fus3 seeds germinated for 48 hours in MS media or MS media containing 10 μM aminoethoxyvinylglycine (AVG) or 100 μM AgNO3 (Ag). Fold change in gene expression was normalized to ACTIN7, which served as a control. Similar trends were seen in two independent experiments. (C) GFP-EIN3 fluorescence in wild-type and fus3 roots incubated for 48 hours in minimal medium with (+) or without (-) 100 μM AgN03 (Ag). The inset shows the GFP-EIN3 fluorescence in wild-type roots exposed to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Scale bar = 15.2 μm. (D) Images (right panels) and quantification (left panel) of hypocotyls length of 5-day-old fus3 and fus3 ein2-1 seedlings grown in the dark in the air. Scale bar = 0.2 cm. Averages from triplicate experiments ± SD are shown. fus3, n = 24; fus3 ein2-1, n = 21. (E) Quantification of hypocotyls length of 5-day-old wild-type, fus3 and eto1-1 seedlings grown in the dark in MS in the absence (-) or in the presence (+) of the ethylene synthesis inhibitor AVG. The eto1-1 seed, which overaccumulates ethylene gas, was used as a positive control to demonstrate rescue by AVG addition. Averages from triplicate experiments ± SD are shown. Wild type, n = 25; fus3, n = 25; eto1-1, n = 24.