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. 2005 May;17(5):1360–1375. doi: 10.1105/tpc.105.031716

Figure 5.

Figure 5.

Phenotypic 5mARF17 Plants Accumulate Increased ARF17 mRNA.

(A) RNA gel blot analysis of 7 μg total RNA prepared from 16-d-old T2 seedlings with a 3′ ARF17 probe. The positions of full-length ARF17 mRNA and ARF17 3′ cleavage product are noted on the right. The 25S rRNA is shown as a loading control. The blot was rehybridized with an ACTIN2 probe, and normalized values of ARF17 full-length mRNA to ACTIN2 mRNA (with Col-0 levels set at 1.0) and ratios of ARF17 full-length mRNA to 3′ cleavage product are indicated.

(B) RNA gel blot analysis of 10 μg total RNA prepared from rosette leaves of 32-d-old T2 plants with a 3′ ARF17 probe. The absence (−) or presence of a mild (+) or severe (++) rosette leaf phenotype is indicated. The positions of full-length ARF17 mRNA and ARF17 3′ cleavage products are noted at the right. Normalization was as in (A).

(C) RNA gel blot analysis of 5 μg total RNA prepared from Col-0 and 5mARF17 root (r), rosette leaf (rl), stem (st), cauline leaf (cl), buds and inflorescence meristems (b), flower (f), silique and embryonic tissues (si), and 12-d-old seedling (se) tissues with a 3′ ARF17 probe. The positions of full-length ARF17 mRNA and ARF17 3′ cleavage product are noted at the right. Two uncharacterized ARF17-hybridizing RNAs, one longer and one shorter than the full-length ARF17 transcript, accumulated in siliques of Col-0 and 5mARF17 plants. Ratios of ARF17 full-length mRNA to 3′ cleavage product are indicated, along with the relative levels of ARF17 mRNA in 5mARF17 plants compared with wild-type Col-0 plants in each tissue (5mARF17/Col-0 ARF17).

(D) Expression of 5mARF17 mRNA in untransformed (untxf) and T2 transgenic plants that were individual progeny of the indicated primary transformant. Rosette leaf RNA was reverse transcribed, and the resulting cDNA was PCR-amplified to completion, then digested with ApaLI, which cuts the 5mARF17 amplicon but not the ARF17 amplicon or heteroduplex molecules resulting from annealing ARF17 and 5mARF17 strands. Agarose gel separation and ethidium bromide staining revealed the full-length PCR product (330 bp) and an ApaLI digestion fragment (250 bp). Each lane is an analysis of an individual plant, and the presence (+) or absence (−) of a rosette leaf phenotype in that plant is indicated. DNA gel blot analysis (data not shown) was used to quantitate the 330- and 250-bp DNAs, and the amount of 5mARF17 relative to ARF17 after correcting for heteroduplex formation is shown below each lane.