Figure 1.

Stability control of ARR2. (A) Effect of ARR2 overexpression (ARR2-ox) on the root length. Seeds were sown and grown on vertically positioned plates with half-strength Murashige and Skoog growth (MS/2) media. After seven days of growth, seedlings were photographed and root length was measured using ImageJ. Data are presented and mean ± SD (n≥12 ). ***, p<0.001 (B) Shoot induction assay. Plants grown on MS/2 media for 7 d under dark conditions were exposed to light for 4 d to strengthen etiolated hypocotyls. Hypocotyls were then excised and placed on MS/2 media containing 1 µM NAA with or without 0.1 µM 2iP. Explants incubated on shoot induction media for 40 d were transferred to a new plate for photography. 2iP, N6-(2-isopentenyl)adenine. (C) Seven-day-old ARR2-ox #3 seedlings were treated for 2, 4 or 16 hours with 200 µM of the protein synthesis inhibitor cycloheximide (CHX) or 100 µM of the proteasome inhibitor MG132 as described.³ Immunoblotting analyses were done as described.³ Proteins were resolved on a 6% acrylamide SDS/PAGE gel. After probing with anti-ARR2 antibodies, membranes were re-probed with anti-HSP90 sera to demonstrate equal loading. Mean ± SD (n≥3 ) of the relative signal intensity (RL) is shown below the blot. (D) Seven-day-old ARR2-ox #3 seedlings were treated for 4 hours with 1 µM or 10 µM of the cytokinin BA. Immunoblotting was done as described in C. (E) Seven-day-old ARR2-ox #3 seedlings were treated for 4 hours with 1 µM of BA in the absence or presence of 100 µM MG132 or 200 µM CHX. (F) Seven-day-old transgenic plants overexpressing ARR1 (ARR1-ox) were treated for 4 hours with 1 µM or 5 µM BA. Proteins were resolved on 7.5% acrylamide SDS/PAGE gels. After probing with anti-ARR1 antibodies, membranes were re-probed with anti-α tubulin (TUA) antibodies.