Figure 2.

A, Transcript levels for LeETR1, LeETR2, LeETR3 (NR), and actin in leaf RNA from second generation (R1) transgenic plants. Numbers above lanes refer to the primary transformant number followed by the seedling number. Lanes marked with an asterisk are azygous for the transgene. Each lane was loaded with 40 μg of total RNA from young fully expanded leaves. B, Dot blot of sense (S) and antisense (A) RNA transcripts from LeETR1, LeETR2, and LeETR3 cDNA clones. Probes, hybridization, and washing conditions were the same for A and B. The His kinase domains for LeETR1, LeETR2, and LeETR3 were amplified by PCR and approximately 100 ng of double-stranded DNA labeled by nick translation. Hybridization conditions were at 50°C in 50% (v/v) formamide, 5× SSPE, 5× Denhardt's, 1% (w/v) SDS, and 100 μg mL⁻¹ sheared salmon sperm DNA. The final wash of the blots was in 0.1× SSPE and 0.1% (w/v) SDS at 55°C. Blots were exposed to x-ray film with intensifying screen at −70°C overnight for actin and the dot blots and from 4 to 7 d for the ethylene receptor blots for R1 transgenic plants.